Rahat 

Nazar · Noushina Iqbal
Nafees A. Khan Editors

Salicylic
Acid: A
Multifaceted
Hormone
Salicylic Acid: A Multifaceted Hormone
Rahat Nazar  •  Noushina Iqbal
Nafees A. Khan
Editors

Salicylic Acid:
A Multifaceted Hormone
Editors
Rahat Nazar Noushina Iqbal
Department of Botany Department of Botany
Jamia Hamdard University Jamia Hamdard University
New Delhi, Delhi, India New Delhi, India

Nafees A. Khan
Department of Botany
Aligarh Muslim University
Aligarh, Uttar Pradesh, India

ISBN 978-981-10-6067-0    ISBN 978-981-10-6068-7 (eBook)

https://doi.org/10.1007/978-981-10-6068-7

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Preface

The rapid increase in industrialization and urbanization during the past few decades
has posed various unpredicted disturbances in the environment resulting in stressful
conditions. Plants are constantly exposed to changes in environmental conditions.
When these changes are rapid and extreme, plants generally perceive them as
stresses which are of two types, biotic and abiotic, depending on the source of stress.
A broad range of abiotic stresses includes osmotic stress caused by salinity, drought,
and low and high temperatures, as well as ionic, nutrient, or metal stresses. The
responses to abiotic stresses are not the linear pathways but are the complicated
integrated circuits involving the interaction of additional cofactors and/or signaling
molecules to coordinate a specified response to a given stimulus.
Plant hormones regulate a number of signaling networks involving developmen-
tal processes and plant response to environmental stresses. Phytohormones, the
chemical messengers, play a vital role in the resistance of plants to the changing
environments by regulating physiological and molecular processes. Their signaling
pathways are not isolated but rather interconnected with a complex regulatory net-
work involving various defense signaling pathways. To understand how plants coor-
dinate multiple hormonal components in response to various developmental and
environmental cues is a major challenge for the future. The role of phytohormones
under abiotic stress is critical in modulating physiological responses that will even-
tually lead to adaptation to an unfavorable environment. Among the recognized
major classes of phytohormones, attention has largely been focused on salicylic
acid (SA). In recent years, salicylic acid has been the focus of intense research due
to its function as an endogenous signal mediating local and systemic plant defense
responses against pathogens and also because it participates in the regulation of
physiological processes and plant resistance to biotic and abiotic stress. Salicylic
acid regulates photosynthetic events, nutrient metabolism, osmotic relations, and
defense mechanisms in plants growing under optimal and changing environmental
conditions.
This book primarily deals with the importance of SA in regulating plant growth
and development under stress conditions along with its interaction with other hor-
mones or molecules in controlling the process. The editors and contributing authors
hope that this book will include a practical update on the current knowledge of
abiotic stress tolerance and lead to new discussions and explore the mechanisms
responsible for the perception and signal transduction of salicylic acid under control

v
vi Preface

and stress conditions and the efforts to use the informative tools for the improve-
ment of crop plants in the era of global climatic change.
The chapters of the book deal with the importance of salicylic acid and/or its
structural analogs in response to some biotic and abiotic challenges in relation to
their effect in the antioxidative metabolism in plants. It also emphasizes on the
recent understanding to underpin the interaction of defense regulators, such as salic-
ylates, jasmonates, hydrogen peroxide, nitric acid, and abscisic acid, with growth
phytohormones, viz., auxins, cytokinins, gibberellic acid, and ethylene, in correla-
tion with disease development in different plant-microbe interactions. The percep-
tion and transduction of a signal from salicylic acid induced defense response under
biotic and abiotic stress conditions are also studied. It deals with the current knowl-
edge of the role of SA on plant growth and development, and explores the identifica-
tion of potential targets for the modulation of salicylic acid signal pathways in
response to plant stress tolerance. Critical evaluation of and cross-talks in salicylic
acid signaling pathways under optimal and stressful conditions is also discussed. It
also gives an insight to the genetic and molecular aspects of plant resistance to stress
through recent advancements and the role of salicylic acid in stress resistance. The
mechanism to induce thermotolerance in plants by SA interaction is also studied.
This book presents an overview of stresses on crop plants and effects of SA on dif-
ferent stresses on plant physiology and stress agronomy, as well as the synergies
between types of stresses. In addition, an understanding on the mechanisms under-
lying between SA and nutrient signal transduction pathways in plants for abiotic
stress tolerance is also covered.
The book covers interesting topics dealing with the role of SA and the mechanis-
tic approaches for abiotic stress tolerance to pave the path for agricultural scientists
and breeders to develop high-yielding sustainable transgenic crops.
We extend our gratitude to all those who have contributed in making this book
possible. Also, we would like to apologize unreservedly for any mistake or failure
to acknowledge fully.

New Delhi, India Rahat Nazar

Aligarh, India  Noushina Iqbal
 Nafees A. Khan
Acknowledgments

First and foremost, we would like to thank the Almighty without whose blessings
the task would be impossible. We thankfully acknowledge the DS Kothari postdoc-
toral fellowship for providing the research grant. We also acknowledge the aca-
demic staff of Jamia Hamdard, New Delhi, and AMU, Aligarh, for their support.
We are also thankful to our families for their continuous support and inspiration.
And lastly, we thank Springer for providing us the chance to widen the audience for
our work.

vii
Contents

1 Role of Salicylic Acid in the Control of General Plant Growth,

Development, and Productivity��������������������������������������������������������������    1
Cesar J. Tucuch-Haas, Jesica V. Pérez-Balam,
Karen B. Díaz-Magaña, José Manuel Castillo-Chuc,
María G. Dzib-Ek, Gabriel Alcántar-González,
Silvia Vergara-Yoisura, and Alfonso Larqué-Saavedra
2 On the Role of Salicylic Acid in Plant Responses
to Environmental Stresses ����������������������������������������������������������������������   17
José A. Hernández, Pedro Diaz-Vivancos, Gregorio Barba-Espín,
and María José Clemente-Moreno
3 Use of Salicylic Acid and Related Compounds to Improve
the Abiotic Stress Tolerance of Plants: Practical Aspects��������������������   35
Tibor Janda, Magda Pál, Éva Darkó, and Gabriella Szalai
4 Emerging Trends in Physiological and Biochemical Responses
of Salicylic Acid����������������������������������������������������������������������������������������   47
Neha Handa, Sukhmeen Kaur Kohli, Ravdeep Kaur,
Kanika Khanna, Palak Bakshi, Ashwani Kumar Thukral,
Saroj Arora, Puja Ohri, Bilal Ahmed Mir, and Renu Bhardwaj
5 Wheat Germ Agglutinin and Dehydrins as ABA-Regulated
Components of SA-Induced Cadmium Resistance
in Wheat Plants����������������������������������������������������������������������������������������   77
F.M. Shakirova, M.V. Bezrukova, Ch.R. Allagulova,
D.R. Maslennikova, and A.R. Lubyanova
6 Salicylic Acid-Mediated Defence Signalling in Respect
to Its Perception, Alteration and Transduction������������������������������������   97
Kusum Verma and S.B. Agrawal
7 Role of Salicylic Acid in Heavy Metal Stress Tolerance: Insight
into Underlying Mechanism��������������������������������������������������������������������  123
Sukhmeen Kaur Kohli, Neha Handa, Ravdeep Kaur, Vinod Kumar,
Kanika Khanna, Palak Bakshi, Ravinder Singh, Saroj Arora,
Rupinder Kaur, and Renu Bhardwaj
ix
x Contents

8 Heat Stress Tolerance in Plants: Action of Salicylic Acid��������������������  145

Rahat Nazar, Noushina Iqbal, and Shahid Umar
9 Salicylic Acid: Molecular Basis of Stress Resistance in Plants������������  163
Uğur Uzuner, Aykut Sağlam, and Asım Kadıoğlu
10 Interaction of Salicylic Acid with Plant Hormones in Plants Under
Abiotic Stress��������������������������������������������������������������������������������������������  201
Shagun Bali, Poonam, Vandana Gautam, Parminder Kaur,
Kanika Khanna, Rupinder Kaur, Adarsh Pal Vig, Puja Ohri,
and Renu Bhradwaj
11 Salicylic Acid and Nutrients Interplay in Abiotic Stress Tolerance��������  221
Tasir S. Per, Mehar Fatma, Mohd. Asgher, Sofi Javied,
and Nafees A. Khan

Index������������������������������������������������������������������������������������������������������������������  239
Contributors

S.B. Agrawal  Laboratory of Air Pollution and Global Climate Change, Center for
Advanced Studies, Department of Botany, Banaras Hindu University, Varanasi,
Uttar Pradesh, India
Gabriel  Alcántar-González  Edafología, Colegio de Postgraduados, Texcoco,
Estado de México, Mexico
Ch.R. Allagulova  Institute of Biochemistry and Genetics, Ufa Scientific Centre,
Russian Academy of Sciences, Ufa, Russia
Saroj Arora  Department of Botanical and Environmental Sciences, Guru Nanak
Dev University, Amritsar, Punjab, India
Mohd.  Asgher  Plant Physiology and Biochemistry Division, Department of
Botany, Aligarh Muslim University, Aligarh, UP, India
Molecular and Conservation Lab, School of Biosciences and Biotechnology, Baba
Ghulam Shah Badshah University, Rajouri, India
Palak Bakshi  Department of Botanical and Environmental Sciences, Guru Nanak
Dev University, Amritsar, Punjab, India
Shagun Bali  Department of Botanical and Environmental Sciences, Guru Nanak
Dev University, Amritsar, Punjab, India
Gregorio  Barba-Espín  Department of Plant and Environmental Sciences,
University of Copenhagen, Copenhagen, Denmark
M.V.  Bezrukova  Institute of Biochemistry and Genetics, Ufa Scientific Centre,
Russian Academy of Sciences, Ufa, Russia
Renu  Bhardwaj  Department of Botanical and Environmental Sciences, Guru
Nanak Dev University, Amritsar, Punjab, India
José  Manuel  Castillo-Chuc  Recursos Naturales, Centro de Investigación
Científica de Yucatán, Mérida, Yucatán, Mexico
María José Clemente-Moreno  Department of Biology, University of the Balearic
Islands, Palma, Spain

xi
xii Contributors

Éva  Darkó  Agricultural Institute, Centre for Agricultural Research, Hungarian

Academy of Sciences, Martonvásár, Hungary
Karen B. Díaz-Magaña  Recursos Naturales, Centro de Investigación Científica de
Yucatán, Mérida, Yucatán, Mexico
Pedro  Diaz-Vivancos  Fruit Tree Biotechnology Group, Department of Plant
Breeding, CEBAS-CSIC, Murcia, Spain
María  G.  Dzib-Ek  Recursos Naturales, Centro de Investigación Científica de
Yucatán, Mérida, Yucatán, Mexico
Mehar  Fatma  Plant Physiology and Biochemistry Division, Department of
Botany, Aligarh Muslim University, Aligarh, UP, India
Plant Molecular Biology, School of life Sciences, Jawaharlal Nehru University,
New Delhi, Delhi, India
Vandana  Gautam  Department of Botanical and Environmental Sciences, Guru
Nanak Dev University, Amritsar, Punjab, India
Neha Handa  Department of Botanical and Environmental Sciences, Guru Nanak
Dev University, Amritsar, Punjab, India
José  A.  Hernández  Fruit Tree Biotechnology Group, Department of Plant
Breeding, CEBAS-CSIC, Murcia, Spain
Noushina  Iqbal  Department of Botany, Jamia Hamdard University, New Delhi,
India
Tibor Janda  Agricultural Institute, Centre for Agricultural Research, Hungarian
Academy of Sciences, Martonvásár, Hungary
Sofi Javied  Plant Physiology and Biochemistry Division, Department of Botany,
Aligarh Muslim University, Aligarh, UP, India
Asım Kadıoğlu  Biology, Karadeniz Technical University, Trabzon, Turkey
Parminder  Kaur  Department of Botanical and Environmental Sciences, Guru
Nanak Dev University, Amritsar, Punjab, India
Ravdeep Kaur  Department of Botanical and Environmental Sciences, Guru Nanak
Dev University, Amritsar, Punjab, India
Rupinder Kaur  Biotechnology Department, DAV College, Amritsar, India
Nafees  A.  Khan  Plant Physiology and Biochemistry Division, Department of
Botany, Aligarh Muslim University, Aligarh, UP, India
Kanika  Khanna  Department of Botanical and Environmental Sciences, Guru
Nanak Dev University, Amritsar, Punjab, India
Sukhmeen  Kaur  Kohli  Department of Botanical and Environmental Sciences,
Guru Nanak Dev University, Amritsar, Punjab, India
Contributors xiii

Vinod Kumar  Department of Botany, DAV University, Jalandhar, India

Alfonso Larqué-Saavedra  Recursos Naturales, Centro de Investigación Científica
de Yucatán, Mérida, Yucatán, Mexico
A.R.  Lubyanova  Institute of Biochemistry and Genetics, Ufa Scientific Centre,
Russian Academy of Sciences, Ufa, Russia
D.R. Maslennikova  Institute of Biochemistry and Genetics, Ufa Scientific Centre,
Russian Academy of Sciences, Ufa, Russia
Bilal  Ahmed  Mir  Department of Botany, School of Life Sciences, Satellite
Campus Kargil, University of Kashmir, India
Rahat Nazar  Department of Botany, Jamia Hamdard University, New Delhi, India
Puja Ohri  Department of Zoology, Guru Nanak Dev University, Amritsar, Punjab,
India
Magda  Pál  Agricultural Institute, Centre for Agricultural Research, Hungarian
Academy of Sciences, Martonvásár, Hungary
Tasir S. Per  Plant Physiology and Biochemistry Division, Department of Botany,
Aligarh Muslim University, Aligarh, UP, India
Govt Degree College Doda, Doda, Jammu and Kashmir, India
Jesica V. Pérez-Balam  Recursos Naturales, Centro de Investigación Científica de
Yucatán, Mérida, Yucatán, Mexico
Poonam  Department of Botanical and Environmental Sciences, Guru Nanak Dev
University, Amritsar, Punjab, India
Aykut Sağlam  Molecular Biology and Genetics, Karadeniz Technical University,
Trabzon, Turkey
F.M.  Shakirova  Institute of Biochemistry and Genetics, Ufa Scientific Centre,
Russian Academy of Sciences, Ufa, Russia
Ravinder  Singh  Department of Botanical and Environmental Sciences, Guru
Nanak Dev University, Amritsar, Punjab, India
Gabriella  Szalai  Agricultural Institute, Centre for Agricultural Research,
Hungarian Academy of Sciences, Martonvásár, Hungary
Ashwani Kumar Thukral  Department of Botanical and Environmental Sciences,
Guru Nanak Dev University, Amritsar, India
Cesar J. Tucuch-Haas  Edafología, Colegio de Postgraduados, Texcoco, Estado de
México, Mexico
Shahid  Umar  Department of Botany, Jamia Hamdard University, New Delhi,
India
xiv Contributors

Uğur Uzuner  Molecular Biology and Genetics, Karadeniz Technical University,

Trabzon, Turkey
Silvia Vergara-Yoisura  Recursos Naturales, Centro de Investigación Científica de
Yucatán, Mérida, Yucatán, Mexico
Kusum Verma  Laboratory of Air Pollution and Global Climate Change, Center
for Advanced Studies, Department of Botany, Banaras Hindu University, Varanasi,
Uttar Pradesh, India
Adarsh  Pal  Vig  Department of Botanical and Environmental Sciences, Guru
Nanak Dev University, Amritsar, Punjab, India
About the Editors

Dr.  Rahat  Nazar received her master’s degree and PhD from Aligarh Muslim
University, Aligarh. She did her post doc at Jamia Hamdard University, New Delhi,
India. She worked as JRF and SRF in DST- and UGC-funded projects during her
research. She has published many research papers in leading journals of the world
with a high impact factor. She has also edited two books and authored one book. Dr.
Nazar is a life member of the Indian Society for Plant Physiology (ISPP) and the
National Environmental Science Academy (NESA), New Delhi, India. She has been
awarded with Junior Scientist Award (2010) by NESA. Dr. Nazar’s is actively
engaged in studying the role of phytohormones and mineral nutrition with special
emphasis on photosynthetic efficiency and nutrient-use efficiency under optimal
and abiotic stress conditions.

Dr.  Noushina  Iqbal has done her Ph.D. at Aligarh and then postdoc at Jamia
Hamdard, New Delhi, India. She has many good publications in journals of national
and international repute with high impact and has written five chapters and pre-
sented four papers in conferences. She has edited two books and authored one book.
She is an eminent researcher and has worked as a project fellow in a UPCST-funded
research project and received UGC-BSR and CSIR-SRF fellowship during her
research. She is a member of the Indian Society for Plant Physiology (ISPP). Dr.
Iqbal works on the role of phytohormones, osmolytes and nutritional factors par-
ticularly nitrogen and sulphur in combating abiotic stress.

Nafees A. Khan, a professor of plant physiology, is affiliated with AMU, Aligarh

(India). Prof. Khan obtained his bachelor’s, master’s and doctoral degrees at AMU,
Aligarh, and also earned his D.Sc. at the same university. In addition to innovative
teaching, he is fond of researching and publishing and has to his credit more than
170 significant peer-reviewed scientific papers and a dozen of edited volumes on
important aspects including plant stress physiology, abiotic stress tolerance, phyto-
hormones, S and N nutrition, nutrient-use efficiency and source-sink relations. A
member of academic bodies of universities, Prof. Khan is also a life member of

xv
xvi About the Editors

major professional societies including the Indian Society for Plant Physiology
(ISPP) and National Environmental Science Academy (NESA), New Delhi, India,
and the Indian Botanical Society. He also worked as subject expert for national and
international bodies and was honoured with the position of editor-in-chief of the
Journal of Functional and Environmental Botany (2011–2013). Since 2013, he is
serving as an executive editor of the Journal of Functional and Environmental
Botany. In recognition of his contributions, Prof. Khan was elected as a fellow of the
‘Indian Society for Plant Physiology’ and the ‘Indian Botanical Society’ and was
also awarded with the UGC Research Award (2002); Distinguished Scientist Award
(2015) by VIFRA International Foundation (India); Group Leader, DBT-BUILDER
Programme (2013–2016), DBT, Government of India, New Delhi; and Scientist of
the Year Award (2005) and Eminent Scientist of the Year Award (2006) by NESA,
New Delhi, India.
Role of Salicylic Acid in the Control
of General Plant Growth, Development, 1
and Productivity

Cesar J. Tucuch-Haas, Jesica V. Pérez-Balam,
Karen B. Díaz-­Magaña, José Manuel Castillo-Chuc,
María G. Dzib-Ek, Gabriel Alcántar-González,
Silvia Vergara-Yoisura, and Alfonso Larqué-Saavedra

Abstract
Applications of low concentrations of salicylic acid (SA) to the shoots of seed-
lings of horticultural plants such as habanero pepper (Capsicum chinense) or to
perennial trees such as the Ramon (Brosimum alicastrum) significantly increase
their growth, development and productivity.
In chili pepper it was found that the positive effect of SA on root growth is
correlated with an increased uptake of macro nutrients and micronutrients which
are allocated in the plant tissues. Data have shown that plant tissues treated with
SA had significantly higher levels of macronutrients. Accumulation of nitrogen,
phosphorus and potassium was higher in fruits (116%, 110% and 97%), leaves
(45.5%, 39.4% and 29.1%), roots (52.6%, 17% and 29.4%), and stems (5.0%,
39.4% and 28.3%) with respect to the control plants. The levels of other nutri-
ents, such as copper, zinc, manganese, boron, calcium, magnesium and iron,
were also higher.
The application of 1 μM SA to shoots of trees, affected the root length. The
control plants had 42 cm, and those of the treated plants 65.5 cm, equivalent to
an increase of 55.7%. Fresh weight of the root was 158.3% higher in the treated
plants and the dry weight increased by 160.7%. Increases were also observed in
stem length (46%), stem diameter (25.9%), fresh weight (78.3%), and dry weight

C.J. Tucuch-Haas • G. Alcántar-González

Edafología, Colegio de Postgraduados, Carretera Federal México–Texcoco km 36.5
Montecillo, Texcoco, Estado de México C.P. 56230, Mexico
e-mail: [email protected]; [email protected]
J.V. Pérez-Balam • K.B. Díaz-Magaña • J.M. Castillo-Chuc • M.G. Dzib-Ek
S. Vergara-­Yoisura • A. Larqué-Saavedra (*)
Recursos Naturales, Centro de Investigación Científica de Yucatán, Calle 43 No. 130,
por 32 y 34, Chuburná de Hidalgo, Mérida, Yucatán C.P. 97205, Mexico
e-mail: [email protected]; [email protected]; [email protected];
[email protected]; [email protected]; [email protected]

© Springer Nature Singapore Pte Ltd. 2017 1

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_1
2 C.J. Tucuch-Haas et al.

(89%), in comparison with the control. The number of leaves presented in treated
plants averaged 12.6, whereas the control plants showed an average of 9 leaves
with a lower leaf area.

Keywords
Salicylic acid • Root • Growth regulation

1.1 Introduction

As examples of the results obtained from the research carried out by our working
group on the effect of applications of salicylic acid (SA) on plant growth, develop-
ment, and productivity over a period of 40 years, three basic contributions can be
highlighted. The first is the publication, in 1978, in which it was proposed that the
application of aspirin to bean plantlets had an effect on plant water status, a fact that
was confirmed in specific bioassays conducted with bean explants and with stomata,
using the bioassay of Commelina communis epidermis strips (Larqué-Saavedra
1978, 1979). These results were widely commented on in the international press,
and it was emphasized that aspirin could be used to save crops under conditions of
stress from the impact of droughts (Times, UK 1978). This aspect, i.e., that salicy-
lates had the potential to participate in the physiology of plants subjected to stress,
was subsequently demonstrated by Shimakawa and collaborators in 2012.
The second observation was published in 1998 (Gutiérrez-Coronado et al. 1998)
in which it was reported that the application of salicylates to intact soya plantlets
favored growth in the radical system of the plants. This discovery was accompanied
by a third contribution which referred to the fact that concentrations in the order of
micromoles, nanomoles, and femtomoles were sufficient to stimulate root growth
and differentiation of secondary roots (Echeverria et  al. 2007; San Miguel et  al.
2003; Larqué-Saavedra et al. 2010), a fact that was confirmed using the bioassay of
transformed roots of Catharanthus roseus. This discovery can be considered of sig-
nificant importance for science given that it demonstrated the high sensitivity and
capacity of response of the plant tissues to the application of low concentrations of
salicylates which were reported in bioassays with animal tissues.
Based on these three observations, a number of experiments have been carried
out in these laboratories, in which we have been able to confirm that SA plays a role
in the control of plant growth, development, and productivity (Larqué-Saavedra and
Martin-Mex 2007; Martin-Mex et al. 2013).
Salicylic acid (SA) is a phenolic compound produced by plants and possesses
growth-regulating functions as well as the capacity to mediate in responses to patho-
gens (Delaney 2010; Hayat et al. 2007; Hayat et al. 2013; Rivas-San Vicente and
Plasencia 2011; Yusuf et al. 2012). Since 1979, endogenous levels have been associ-
ated with physiological processes which include the induction of flowering in
Lemna (Cleland and Tanaka 1979, Shimakawa et al. 2012) and the induction of heat
production in plants such as the lily (Raskin 1992).
1  Role of Salicylic Acid in the Control of General Plant Growth, Development… 3

Numerous physiological effects resulting from the applications of SA have been

reported. One effect of particular interest refers to its capacity to stimulate rooting
in explants of Phaseolus, Acer, and Vigna (Basu et al. 1969; Li and Li 1995) and
growth of the radical system in species such as soya or habanero pepper (Gutiérrez-­
Coronado et al. 1998; Deef 2007) or to stimulate the length and differentiation of
secondary roots of Catharanthus roseus (Echeverría et al. 2007).
There are also a number of reports dealing with its capacity to increase protec-
tion against damage caused by ions such as boron, arsenic, lead, and cadmium
(Choudhury and Panda 2004; Chen et al. 2007; El-Feky et al. 2014), to favor the
accumulation of ions such as magnesium, calcium, and potassium (El Tayeb and
Ahmed 2010) and to reduce the adverse effects caused by salinity and high tempera-
tures (Pirasteh-Anosheh et al. 2014), drought (Habibi 2012; Farzane et al. 2014),
and cold (Mutlu et al. 2013). The effects estimated support the proposal that this
molecule can be considered an elicitor, which is a biotic inductor that triggers mea-
surable physiological and morphological processes (Zhao et al. 2005). It is possible
that the receptors and mechanisms of action stimulated by the application of this
growth regulator could be similar to those reported in 1989 (Larqué-Saavedra and
Rodríguez 1989).
It has also been reported that the application of SA produces alterations in pro-
cesses such as the stimulation of nitrate reductase activity (Fariduddin et al. 2003),
the increase in photosynthetic activity, electron transport, and chlorophyll (Arfan
et al. 2007; Sánchez-Chávez et al. 2011). It has been published that SA also increases
seedling height and vigor (Anwar et al. 2013) and that it affects processes relating
to the bioproductivity of cultivated plants (Hayat et al. 2007, 2012).
The effect of SA applications in woody perennial species, however, has been
poorly studied; thus the present research study was carried out to measure the effect
of SA on the development of a tree from the Mexican tropics denominated Brosimum
alicastrum (common name, Ramon); this species has drawn much attention due its
high potential as a food source and its role in mitigating climate change (National
Academy of Science 1975; Pardo-Tejeda et al. 1976).

1.2  tudies of SA on Horticultural Plants: Capsicum

S
chinense

1.2.1 Salicylic Acid on Root and Shoot Growth

It is already known that a larger, more vigorous root system will contribute to better
crops and horticultural plants. Our results are consistent with previous publications
which have reported that 1 μM or less is sufficient to favor root growth. In Pinus
patula, for instance, concentrations of 1.0 and 0.01 μM increased root growth in
33% and 30%, respectively, (San Miguel et al. 2003), while in Chrysanthemum a
concentration of 0.01 μM SA increased dry root weight significantly (Villanueva
et al. 2009). Dry root weight was also favored by the application of 1.0 μM or less
in tobacco and cotton (Gutiérrez-Coronado et al. 1998).
4 C.J. Tucuch-Haas et al.

Table 1.1  Effect of spraying 1  μM of salicylic acid on habanero pepper plantlets (Capsicum
­chinense) in different morphological variables
Concentrations of salicylic acid
Morphological variable Control 1 μM
Height (cm) 18.44 ± 0.43 c 24.51 ± 0.18 a
Stem diameter (cm) 0.28 ± 0.05 b 0.34 ± 0.05 a
Leaf area (cm2) 150.4 ± 13.3 b 204.3 ± 7.68 a
Fresh weight of the aerial part (g) 2.85 ± 0.15 b 3.65 ± 0.14 a
Dry weight of the aerial part (g) 0.37 ± 0.03 b 0.45 ± 0.02 a
Fresh weight of the root (g) 0.49 ± 0.04 b 0.69 ± 0.04 a
Dry weight of the root (g) 0.05 ± 0.004 b 0.08 ± 0.005 a
The values are the means with their respective standard errors. Different letters in the same line
represent different statistics (Tukey, α = 0.05). n = 25 (Data adapted from preliminary research
work to be published)

One observation that was particularly relevant in studies of salicylate applica-

tions to habanero pepper plants (Capsicum chinense) was the evidence indicating
that the plantlets which were sprayed with this compound showed much greater
vigor in comparison with the control plants. It is reported that applications of 1 μM
salicylic acid (SA) to seedling shoots significantly increased growth and fresh and
dry weight of roots, stems, leaves, and fruits of this plant species at harvest time
(Table 1.1).

1.2.2 The Uptake of Nutrients

Experiments were conducted to determine if the proposed positive effect of SA on

root growth could be correlated with an increase in the uptake of macronutrients and
micronutrients; to achieve this, these elements were measured in the plant tissues of
treated plants.
The results showed that the tissues of plants treated with SA had significantly
higher levels of macronutrients. Accumulation of nitrogen, phosphorus, and potas-
sium was higher in fruits (116%, 110%, and 97%), leaves (45.5%, 39.4%, and
29.1%), roots (52.6%, 17%, and 29.4%), and stems (5.0%, 39.4%, and 28.3%),
respectively, in comparison with the control plants. The levels of other nutrients,
such as copper, zinc, manganese, boron, calcium, magnesium, and iron, were also
higher in the majority of the plant tissues treated with SA (Table 1.2, Figs. 1.1 and
1.2).

1.2.3 Effect on Flowering

The effect of SA on flower induction was analyzed by means of an experiment in

which plantlets of habanero pepper (Capsicum chinense) were sprayed with 1 μM
of this growth regulator. The results demonstrated that 50% of the treated plants
1  Role of Salicylic Acid in the Control of General Plant Growth, Development… 5

Table 1.2  Effect of spray applications of 1  μM of salicylic acid to habanero pepper plantlets
(Capsicum chinense) on the content of macronutrients in the different organs harvested 128 days
after application
N P K Ca Mg
Tissue Treatment Mg planta−1
Fruit Control 150.18 b 12.41 b 101.12 b 33.81 a 11.22 b
1 μM de AS 325.72 a 26.13 a 199.37 a 34.83 a 19.85 a
Leaf Control 440.93 b 26.01 b 108.79 b 417.10 b 78.44 b
1 μM de AS 641.94 a 36.27 a 140.46 a 616.33 a 131.27 a
Stem Control 512.14 b 16.09 b 142.62 b 311.55 b 111.95 a
1 μM de AS 561.04 a 20.97 a 183.12 a 437.62 a 191.36 b
Root Control 1353.53 b 90.04 b 475.03 b 1819.98 a 303.65 a
1 μM de AS 2065.70 a 105.88 a 615.12 a 1861.48 a 382.28 a
Values with the same letter are not significant (Tukey p = 0.05). Each value is the mean of five
individuals (Data adapted from preliminary research work to be published)

5.0 7.0 16.0 250.0

a b
4.5 a a
Content of Fe in leaves (Mg plant1)

Content of Fe in Stems (Mg plant1)

14.0

Content of Fe in Roots (Mg plant1)

6.0
Content of Fe in fruits (Mg plant1)

4.0 200.0
12.0
3.5 5.0

3.0 10.0 150.0

b 4.0 b
2.5 8.0 b

2.0 3.0 100.0

6.0
b
1.5 2.0
4.0
1.0 50.0
1.0 2.0
0.5
0.0 0.0 0.0 0.0
Control 1µMAS Control 1µMAS Control 1µMAS Control 1µMAS

Fig. 1.1  Effect of spray application of 1 μM of salicylic acid on the iron content in the root and
leaf. Stem and fruit of habanero pepper plants. Values with the same letter are not significant
(Tukey p = 0.05). Each value is the mean of five individuals ± s.e. (Data adapted from preliminary
research work to be published)

initiated flowering 25 days after spraying, while the control plants presented flower-
ing after 45  days (Fig.  1.3). Moreover, at 55  days after spraying, flowering was
observed in 97 of the treated plants and in 82 of the control plants. The number of
flowers per plant observed at 80 days, in the plants sprayed with SA, was 112, while
the control plants presented only 66 flowers per plant (Fig. 1.4).

1.2.4 Effect on Fruit Formation

Once the effect of SA on the flowering process was demonstrated, fructification of

the plants treated with this growth regulator was evaluated. The results show that the
plants sprayed with 1 μM SA formed 342 fruits, while the control plants formed
6 C.J. Tucuch-Haas et al.

0.60 1.80
a
a 1.60 a
0.50
1.40

Content in leaves (Mg plant1)

Content in fruits (Mg plant1)

0.40 1.20 b

1.00
0.30 Control b Control
b
0.80
1µMAS 1µMAS
0.20 a 0.60
a b a
0.40
0.10 a a b b
0.20 a a

0.00 0.00
Cu Zn Mn B Cu Zn Mn B

1.80 3.50 a

1.60 a
3.00

Content in Roots (Mg plant1)

Content in Stems (Mg plant1)

1.40
a 2.50
1.20 a
1.00 2.00
Control b Control
a
0.80 1.50
1µMAS b b 1µMAS
0.60 a a
1.00
0.40 a a a a
a a 0.50
0.20

0.00 0.00
Cu Zn Mn B Cu Zn Mn B

Fig. 1.2  Content of copper (Cu), zinc (Zn), manganese (Mn), and boron (B) in root, leaf, stem,
and fruit of habanero pepper plants sprayed with 1 μM of salicylic acid. Values with the same letter
are not significant (Tukey p = 0.05). Each value is the mean of five individuals (Data adapted from
preliminary research work to be published)

Fig. 1.3  Days to flowering of the habanero pepper plants treated with 1 μM of SA. The dotted line
indicates 50% of the individuals with flowers. The values represent means with their standard
error. An ANOVA (α = 0.05) was performed; significant differences are identified with an asterisk
n = 20 (Data adapted from preliminary research work to be published)
1  Role of Salicylic Acid in the Control of General Plant Growth, Development… 7

Fig. 1.4  Number of flowers in plants sprayed with 1 μM of SA. The values represent means with
their standard error. ANOVAS (α = 0.05), significant differences are indicated with an asterisk.
n = 20 (Data adapted from preliminary research work to be published)

only 244. In other words, the treated plants produced 40% more fruits in compari-
son with the control. Measurements of fruit form showed that the apical diameter
and the medial region showed no significant differences (Table 1.3).

1.3  tudies on the Effect of SA on the Growth

S
and Development of Woody Perennial Plants

The effect of SA applications in woody perennial plant species has been poorly
studied; thus the present research study was carried out to measure the effect of SA
application on the growth and development of Brosimum alicastrum (common
name, Ramon), a tree of the Moraceae family from the Mexican tropics. This plant
species has drawn much attention due to the environmental services it offers to miti-
gate climate change and its high potential as a food source (National Academy of
Science 1975; Pardo-Tejeda et al. 1976).
First, a series of experiments were carried out to measure the effect of sprayed
applications of this hormone on the root length. A specific bioassay was set up.
Seedbeds consisting of PVC tubes 23 cm high and 4 cm in diameter were filled with
perlite substrate. One seed was planted in each PVC tube, and these were placed in
8 C.J. Tucuch-Haas et al.

Table 1.3  Effect of spray Variable Treatments

applications of 1 μM salicylic Witness AS_1x10-6
acid to habanero pepper
NFP 244.8 ± 28.6b 342 ± 52.9a
plantlets (Capsicum chinense)
on different estimators of the AWF 3.6 ± 0.34a 3.7 ± 0.20a
fruit. The mean of 20 WFP 770.0 ± 45.1b 1032.7 ± 100.2a
repetitions ± s.e. is presented NFP Number of fruits per plant, AWF Average
weight per fruit, WFP Average weight of fruits
per plant. Means with the same letter between
each line are statistically equal (Tukey, α = 0.05
for NFP, PPF; Duncan, α = 0.05 for PFP) (Data
adapted from preliminary research work to be
published)

a randomized experimental block designed with six replicates per treatment. The
seedbeds were kept in a growth room at a temperature between 28 and 30 °C with
daily watering and a photoperiod of 12 h of light and 12 h of darkness.
The SA solution was spray applied to the shoots of 13-day-old Ramon seedlings
on ten occasions. When the seedling roots from the different treatments were seen
to protrude from the base of the PVC tube, the seedlings were harvested in order to
measure the root and stem lengths of the seedlings. For the measurement of the
biomass weight, after the roots were harvested and measured, they were dried in an
oven at 40 °C, for a 12 h period.
The results showed that SA treatment significantly increased the length of the
roots of Brosimum (Fig. 1.5). This value was, on average, 4.3 cm longer than that of
the control treatment. Fresh weight of the root also increased significantly in com-
parison with the control. Dry weight of the roots, however, was not significant,
although the pattern does indicate that the treatment with SA showed a higher value.
SA treatment also favored stem height of Brosimum seedlings with 4.2 cm higher
than that of the control (Fig. 1.6). From these results, we can infer that spraying SA
on the leaves of Brosimum will stimulate root growth while increasing the capacity
to absorb water and nutrients which will facilitate a more successful development,
particularly in adverse conditions, such as drought.
With the results obtained, it was found that the sprayed application of 1 μM SA
to the leaves of Brosimum alicastrum stimulates growth of the roots by a length of
4.3 cm, in comparison with the control. The treatment also increased fresh weight
by 0.3 g with respect to the control, and stem height showed an increase of 4.2 cm
over the control. Taking into consideration these results, one can infer that sprayed
applications of salicylic acid to the leaves of Brosimum alicastrum seedlings stimu-
late growth of the roots and stems, which could possibly increase their capacity to
absorb water.
Further work was carried out to estimate the effect of SA on plantlets of this
perennial tropical tree. An experiment was conducted with potted plants, cultivated
under shade netting in the open. Six weeks after planting, when the seedlings
reached an average height of 13.8 cm and presented the first two complete leaves,
1  Role of Salicylic Acid in the Control of General Plant Growth, Development… 9

A
30.0
b
25.0
a
Length (cm) 20.0

15.0

10.0

5.0

0.0
B
1.2
b
Fresh weight (g)

1.0
a
0.8
0.6
0.4
0.2
0.0

C
0.4
a
0.35
0.3 a
Dry weight (g)

0.25
0.2
0.15
0.1
0.05
0
Control water SA 1.0mM
Treatment

Fig. 1.5  Effect of spray applications of 1 μM of salicylic acid to Brosimum alicastrum seedlings
on the length (a), fresh weight (b), and dry weight (c) of roots. Average of six repetitions ± stan-
dard error is shown. Similar letters indicate no significant difference (Fisher, p  =  0.05) (Data
adapted from preliminary research work to be published)

they were sprayed with 1 μM SA or water on the canopy, twice a week for a period
of 3 weeks, after sunrise. The concentration of 1.0 μM of SA was evaluated as it was
considered to be the optimal, based on the results obtained from previous experi-
ments carried out in these laboratories (Larqué-Saavedra and Martin-Mex 2007;
Martin-Mex et al. 2013).
10 C.J. Tucuch-Haas et al.

30.0
b
23.1
25.0
a
18.9
20.0
Length (cm)

15.0

10.0

5.0

0.0
Water control SA 1.0mM
Treatment

Fig. 1.6  Effect of spray applications of 1 μM of salicylic acid to Brosimum alicastrum seedlings
on the length of the stems. Data are the mean value of six replicate samples ± s.e. Similar letters
indicate no significant differences (Data adapted from preliminary research work to be published)

a a b 20
70
a a
60 a
15
Number of leaves

50 a a
a
Height (cm)

a a
40
b b 10
30 b b a a
b a
b b
20 b
5 b
a a a
10
0 0
Spraying 1 2 3 4 5 6 Spraying 1 2 3 4 5 6
Readings Readings
CONTROL
1 mM AS

Fig. 1.7  Effect of spray application of 1 μM of SA on the pattern of height (a) and leaf exposure
(b) in Brosimum alicastrum seedlings after initiation of treatment (AIT). Each point is the average
of six plantlets ± standard error. Same letters indicate no significant difference (Tukey, p ≤ 0.05)
(Data adapted from preliminary research work to be published)

After 8  months, six plants were harvested to measure the length of root and
canopy, as well as fresh and dry weight. The different parts of the plants were dried
in an oven at 60 °C, until constant weight. Stem diameter was measured 5 cm above
the vital node.
The results showed that the application of SA increases the growth pattern and
rate of leaf exposure in comparison with the control. These differences were main-
tained and amplified, as can be appreciated in Fig.  1.7; thus, after 8  months, the
increase observed, due to the effect of SA, was 46% in height and 40% more leaves.
1  Role of Salicylic Acid in the Control of General Plant Growth, Development… 11

Similarly, the spraying of 1 μM of SA on the canopy of Ramon plantlets stimu-

lates root length significantly, in comparison with the control (Fig. 1.8). In fact, at
the end of the test, root length of the control plants was 42.0 cm, and the treated
plants registered 65.5  cm, equivalent to an increase of 55.7%, as a result of SA
application. Fresh weight and dry weight of the root were also increased signifi-
cantly, compared to those of the control. Fresh weight was 158.3% higher in the
plants treated with SA, and dry weight increased by 160.1%.
The treatment with SA also favored stem length, with the treated plants present-
ing 60.2 cm, while the control was 41.2 cm, an increase equivalent to 46% in com-
parison with the control. Moreover, the effect of SA resulted in a stem diameter of
4.46 mm, and that of the control was 3.54 mm, equivalent to an increase of 25.9 %.
The total fresh weight of the shoot was 78.3% greater in treated plants in compari-
son with the control, and the dry weight increased by 89.7% (Fig. 1.9).
The number of leaves was also affected by SA spraying. Treated plantlets
increased the number of leaves by 40% over the control, presenting an average of
12.6 leaves with a foliar area of 387 cm2, while the control had 9 with a foliar area
of 231 cm2. Fresh weight of leaves for the control was an average of 3.5 g, while that
of the treated plants was 5.6 g, equivalent to an increase of 61.9%. Dry weight of
control leaves was 1.37 g, and that of the treated plants was 2.22 g, equivalent to an
increase of 62% (Fig. 1.10).

A B
b b
80 8
Fresh weight of root (g)

60 6
Root length (cm)

40 4
a

20 2

0 0
CONTROL 1 mM AS
CONTROL 1 mM AS

C
3
b
Dry weight of root (g)

a
1

0
CONTROL 1 mM AS

Fig. 1.8  Effect of spray application of 1 μM of SA to the shoots of seedlings on length (a), fresh
weight (b), and dry weight (c) of the roots of Brosimum alicastrum plantlets, 8 months after the
application of the growth regulator. Each block is the mean of six repetitions ± standard error.
Same letters indicate no significant difference (Tukey, p ≤ 0.05) (Data adapted from preliminary
research work to be published)
12 C.J. Tucuch-Haas et al.

A B
80 6
b b

Stem diameter (mm)

5
Stem Length (cm)

60 a
4
a
40 3
2
20
1
0 0
CONTROL 1 mM AS CONTROL 1 mM AS

C D
4
Fresh weight of the canopy (g)

Dry weight of the canopy (g)

8 b
b
6 3

a
4 2 a

2 1

0 0
CONTROL 1 mM AS CONTROL 1 mM AS

Fig. 1.9  Effect of spray application of 1 μM of salicylic acid to the shoots of seedlings on stem
length (a), stem diameter (b), fresh weight (c), and dry weight (d) of Brosimum alicastrum plant-
lets, 8 months after the application of the growth regulator. Each block is the mean of six replicate
samples ± standard error. Same letters indicate no significant difference (Tukey, p ≤ 0.05) (Data
adapted from preliminary research work to be published)

1.4 Discussion

The positive effects reported indicate that spray applications of SA favor plant
growth, development, and productivity of Capsicum chinense, a horticultural plant,
as well as Brosimum alicastrum, a woody plant species of the Mexican tropics.
These data are consistent with previous reports for horticultural species (Larqué-­
Saavedra and Martín-Mex 2007; Martín-Mex et  al. 2013) and the report by San
Miguel and collaborators (2003) on Pinus patula plantlets, in which growth of the
aerial part and roots was favored.
One of the effects of particular interest, reported in the present study, is the effect
of SA on the development of roots of intact plants of Ramon, which concurs with
reports on the stimulation of rooting in explants and the stimulation of growth and
the elongation of the radical system, as well as the differentiation of secondary roots
of species of horticultural importance such as soya, habanero pepper, and
Catharanthus roseus (Gutiérrez-Coronado et al. 1998; Deef 2007; Basu et al. 1969;
Li and Li 1995).
1  Role of Salicylic Acid in the Control of General Plant Growth, Development… 13

A B
600
b b
15

Foliar Area (cm2)

Number of leaves

a 400
10 a

200
5

0 0
CONTROL 1 mM AS CONTROL 1 mM AS

C D

10 4
Fresh weight of the leaf (g)

Dry weight of the leaf (g)

b b
8 3
6 a
a 2
4
1
2

0 0
CONTROL 1 mM AS CONTROL 1 mM AS

Fig. 1.10  Effect of spray application of 1 μM of salicylic acid to the shoots of seedlings on the
number of leaves (a), foliar area (b), fresh weight (c), and dry weight (d) of the leaves of Brosimum
alicastrum plantlets, 8 months after the application of the growth regulator. The mean of six repeti-
tions is shown ± standard error. Same letters indicate no significant difference (Tukey, P ≤ 0.05)
(Data adapted from preliminary research work to be published)

The increase in root size reported in the present study most likely favored the
capacity of these plants to absorb water and nutrients from the soil which, once
transported to the aerial part of the plant, allowed the seedlings to be more success-
ful in their development by favoring a faster growth rate as well as the accumulation
of greater biomass, which coincides with a report stating that SA increases vigor
and height of the plantlets (Anwar et al. 2013).
The positive effect of SA on woody tropical trees reported in the present study
should be verified as soon as possible due to its potential for use in the establishment
of commercial plantations, given the importance of its capacity to produce large
quantities of seeds that could be used for animal and human consumption or for the
repopulation of open natural areas which require restoration, where Brosimum could
be planted for use in confronting adverse conditions such as those propitiated by
climate change.
14 C.J. Tucuch-Haas et al.

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On the Role of Salicylic Acid in Plant
Responses to Environmental Stresses 2
José A. Hernández, Pedro Diaz-Vivancos, Gregorio Barba-­
Espín, and María José Clemente-Moreno

Abstract
Salicylic acid (SA) is a plant hormone more commonly known by its role in
human medicine than in the field of plant physiology. However, in the last two
decades, SA has been described as an important signalling molecule in plants
regulating growth, development and response to a wide number of biotic and
abiotic stresses. Indeed, actually, it is well known that SA is a key signalling
molecule involved in systemic acquired resistance (SAR), and recent works
reported a role for SA in the response to salt or drought stresses.
The precise mode of the stress hormone SA action is unclear, although it has
been shown to interact in a complex manner with the antioxidative metabolism,
modulating cellular redox homeostasis and leading to changes in transcription
factor activities and defence gene activation. In this sense, SA activates defence
signalling pathway(s) through non-expressor of PR-protein 1 (NPR1), which is
one of the few known redox-regulated proteins in plants.
Different synthetic chemicals are able to mimic the ability of SA to activate
resistance to various stresses, both biotic and abiotic, in plants with agronomic
interest. Among these chemicals, 2,6-dichloroisonicotinic acid (INA) and benzo-
thiadiazole (BTH) are the most widely studied compounds due to its ability to
induce SAR.

J.A. Hernández (*) • P. Diaz-Vivancos

Fruit Tree Biotechnology Group, Department of Plant Breeding, CEBAS-CSIC,
Campus Universitario de Espinardo, Murcia, P.O. Box 164, 30100, Spain
e-mail: [email protected]
G. Barba-Espín
Department of Plant and Environmental Sciences, University of Copenhagen,
Copenhagen, Denmark
M.J. Clemente-Moreno
Department of Biology, University of the Balearic Islands, Palma, Spain

© Springer Nature Singapore Pte Ltd. 2017 17

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_2
18 J.A. Hernández et al.

In this chapter we present the role of SA and/or some of its structural ana-
logues in the response to some biotic and abiotic challenges in relation to their
effect in the antioxidative metabolism in plants.

Keywords
Antioxidative metabolism • Biotic stress • Drought stress • Oxidative stress •
Salicylic acid analogues • Salt stress

2.1 Introduction

Salicylic acid (SA) is a phenolic plant hormone widely distributed in plants

although with basal levels differing among species. It plays an important role in the
regulation of multitude of physiological processes such as seed germination, veg-
etative growth, photosynthesis, respiration, thermogenesis, flower formation, seed
production or senescence. Effect of SA on these processes can be direct or indirect,
because SA is implicated also in the synthesis and/or signalling regulation of other
plant hormones. Indeed hormonal relations and especially the antagonisms between
SA and abscisic acid (ABA) or jasmonic acid (JA) are currently highly studied and
discussed. However, SA is mainly known for its central role in plant pathogen
interaction, and during the last two decades, hundreds of papers regarding its
implication in the plant response to biotic stress have been published. Under biotic
stress conditions, SA fulfils a key function as an endogenous signal mediating
in local defence responses and SAR, as well as contributing to maintain cellular
redox homeostasis through the regulation of antioxidant enzyme activity. In addi-
tion, SA is required for pathogenesis-related (PR) gene expression (Goellner and
Conrath 2008), and increases in the amount of endogenous SA levels are correlated
with expression of PR genes and development of SAR. In addition, exogenous SA
application, as well as its functional analogues or derivatives, induces PR gene
expression and increases resistance to diseases. In this sense, some synthetic chem-
icals are able to mimic the ability of SA to activate resistance to biotic and abiotic
stresses in plants of agronomic interest. Among these chemicals 6-dichloroisonico-
tinic acid (INA) and benzothiadiazole (BTH) are the most widely studied com-
pounds by its ability to induce SAR.
On the other hand, recently works point out an important role for SA in response
to abiotic stresses such as drought, chilling or saline stress (Takatsuji and Jiang
2014). However, the role of SA in these abiotic situations is even less unravelled,
and several contradictory data and antagonisms are reported. Indeed, in the bibliog-
raphy we can find that the effects of exogenous SA applications on physiological
parameters differ depending on the concentration applied and plant species tested.
For example, the effect of SA in salt-stressed plants seems to depend on different
factors, including the SA concentrations used, the plant species, how the SA treat-
ment is applied, the physiological state of the plant during application as well as the
level of salinity and the exposure time to NaCl. Unlike the response to salt stress,
2  On the Role of Salicylic Acid in Plant Responses to Environmental Stresses 19

the effect of SA on water stress seems to be clearer, and an evident effect of SA

improving the response to drought stress has been reported by different authors.
However, despite the great progress made during the last decades, the molecular
and biochemical mechanisms behind these responses are not deeply well known
yet. Although several bingeing proteins with very high affinity for SA such as
tobacco SABP2 have been found, and some key components in the network of
SA response such as the transcriptional factor NPR1 have been described, the SA
receptor has been not identified yet, and SA downstream signalling is not fully
understood (Manohar et al. 2015). In this chapter we present the role of SA and
some of its structural analogues in the response to some biotic and abiotic chal-
lenges throughout its interaction with the antioxidative metabolism in plants. In this
sense, a complex interplay between SA and reactive oxygen species (ROS) in the
regulation of defence genes has been also described, and several papers supported
the idea that H2O2 can be a mediator in the SA-dependent induction of PR genes
(Garretón et al. 2002). Regarding the evidences of role of SA as regulator of plant
growth and development, the reader is referred to other chapter or reviews on this
subject (Rivas-SanVicente and Plasencia 2011).

2.2 SA and ROS Interplay

Although most studies on signal interaction have focused on phytohormone interac-

tions, it is largely described that ROS are used by plants as signalling molecules
during development and stress situations. Activation of an ROS burst is a common
response to both biotic and abiotic stresses (Miller et al. 2009). To provide an appro-
priate defence response to diverse stress stimuli in different physiological stages
and tissues, the defence signalling must be conducted under a complex and strongly
regulated network within an accurate physiological context. Different studies indi-
cate that plants are able to coordinate signals from diverse signalling pathways and
to prioritise among them and that a combination of stresses could lead to unique
gene expression profiles (Xu and Brosché 2014). Moreover, ROS are also used by
plants as second messengers in signal transduction cascades in a variety of pro-
cesses, being their accumulation crucial for plant development as well as defence.
Thus, ROS production and scavenging are intimately linked, and the balance
between them together with other signalling pathways such as those mediated by
SA will determine defence signalling output (Fig. 2.1).
Early in the 1990s, SA level and ROS metabolism were found to be closely con-
nected, describing that SA increases correlated with increases in ROS production.
However, recent studies point out novel antagonistic interplay between SA and ROS
signalling which could explain the high degree of responsiveness of plant to differ-
ent situations and biotic and abiotic challenges (Chen et al. 1993; Xu and Brosché
2014). For example, recently it has been described in several different mutants that
the constitutive activation of defence by SA signalling interferes with the plant’s
ability to properly respond to an ROS signal from the apoplast after ozone stress.
Treatment of plants with ozone generates a burst of apoplastic ROS, but in plant
20 J.A. Hernández et al.

Fig. 2.1  Interplay SA-ROS in plant stress response

with high SA levels and constitutive defence activation, it is attenuated and vice
versa, so the authors conclude that there could exist an attenuation of apoplastic
ROS signalling by SA at the level of gene expression. Thus, these recent results
strongly support the idea that a coordinated network integrating diverse signalling
pathways such as those mediated by SA and ROS pathways plays a key role in envi-
ronmental stress responses (Xu and Brosché 2014) (Fig. 2.1).
On the other hand, it has been also reported that there is a decrease of some anti-
oxidant enzymes such as ascorbate peroxidase (APX) and catalase (CAT) under
stress situations. In this sense, it has been shown that SA and some of its analogues
inhibit the activity of CAT and APX enhancing the accumulation of H2O2 that can
perturb the cellular redox state. In contrast with CAT or APX, the guaiacol peroxi-
dases, which participate in the cross-linking of cell wall components, are not inhib-
ited by SA (Durner and Klessig 1996; Apel and Hirt 2004; Mittler et  al. 2004;
Garretón et al. 2002). More recently, biochemical screens for SA-binding proteins
resulted in the identification of multiple enzymes, such as CAT, APX, the E2 subunit
of α-ketoglutarate dehydrogenase and glutathione S-transferases, inhibited upon
binding to SA (Fu and Dong 2013). However, the inhibition of CAT by SA does not
always occur, especially in abiotic stress responses. For example, SA-accumulating
Arabidopsis lines presented higher CAT levels than wild-type line (Mateo et  al.
2006).

2.3 SA Role in Biotic Stress and SAR

Early last century, several studies showed that when a plant was infected by a patho-
gen, some systemic defence mechanisms were activated involving an increased
resistance against subsequent pathogen attacks. Hypersensitive response (HR) is
one early response associated with necrotic lesions at the site of pathogen entry,
ROS accumulation and activation of defence-related genes that (among others)
2  On the Role of Salicylic Acid in Plant Responses to Environmental Stresses 21

encode several families of PR proteins. After that, increased levels of PR gene

expression are observed in non-inoculated tissues, and the development of SAR, as
a broad resistance to different pathogens, is underway (Conrath et al. 2001; Gary
and Goodman 2004; Vlot et al. 2009). Nevertheless, a systemic resistance implies
the existence of a signal that can be transmitted through tissues. In this sense, an
intensive research has focused in the search of the key in the activation of response’s
defence against pathogens and the establishment of SAR.
Although elicitors from various extracts of plants and microorganisms, or other
compounds such as β-aminobutyric acid, have been described as activators or resis-
tance inductors (Oostendorp et al. 2001), several early studies carried out in cucum-
ber, Arabidopsis and tobacco showed that SAR induction was dependent on SA
accumulation, and it was also correlated with PR gene expression (Yalpani et al.
1991; Durrant and Dong 2004). SA was described as endogenous signal in the resis-
tance response at first time in 1979 in tobacco when White (1979) observed that
acetyl salicylic acid (aspirin) induced resistance to tobacco mosaic virus (TMV),
increasing PR protein accumulation and reducing lesion numbers. Subsequently,
Malamy et al. (1990) observed that the endogenous salicylic acid levels in resistant
but not susceptible cultivars increased in infected and uninfected leaves after TMV
inoculation. Moreover, prior to the establishment of SAR, SA levels increase in both
inoculated and non-inoculated systemic tissues (Kessmann et al. 1994; Sticher et al.
1997). In addition, different studies showed that both high endogenous levels of SA
correlated with enhanced resistance to pathogen infection and transgenic plants
defective for SA biosynthesis, or in which SA was removed quickly, developed a
greater susceptibility to diseases and were unable to induce SAR (Bowling et al.
1994; Ryals et al. 1996). In both cases disease resistance and PR expression could
be restored by treatment with exogenous SA or synthetic analogues, being this issue
discussed below.
On the other hand, various studies suggested that, after a pathogen attack,
changes in the concentration of SA have an effect on the maintenance of the redox
state of the cell, probably by regulating the expression of genes encoding antioxi-
dants (Rao and Davis 1999; Vanacker et al. 2000). It has been described that biotic
stress situations increase the ROS production which could act as second messengers
mediating SA pathways for expression of defence genes (Yoshioka et  al. 2008;
Torres 2010) (Fig. 2.1). In this sense, it has been also reported that high concentra-
tions of SA can act uncoupling oxidative phosphorylation and hence the respiration
chain, stimulating ROS generation in mitochondria and also inducing the alternative
respiratory pathway (Moore et al. 2002). Moreover, antioxidants such as glutathione
(GSH) can block the expression of PR genes induced by exogenous application of
H2O2 and other pro-oxidants (May et  al. 1998). However, GSH levels increased
significantly in soy cells after incubation for 2  days with SA or its analogues
(Knörzer et al. 1999). In addition, it has been described that GSH could regulate the
expression of SA-dependent genes via NPR1, after exposure to the pathogen
(Urbanek and Müller 2006). NPR1 protein is a transcriptional factor whose location
or activity was influenced by the redox state of the cell (Mou et al. 2003). In response
to avirulent pathogen treatments (or other inducers of SAR), the npr1 mutant (also
22 J.A. Hernández et al.

known as nim1 or sai1) accumulated SA like the wild type but was unable to develop
SAR and express PR genes. However, overexpression of the NPR1 protein leads to
constitutive expression of PR genes in the absence of inducers, suggesting that
NPR1 is a positive regulator of SAR required for the translation of the signal accu-
mulation of SA and expression of resistance genes (Cao et  al. 1994; Mou et  al.
2003). In this sense, it has been suggested that the conformation of NPR1 is sensi-
tive to cellular redox changes. In the absence of SA, NPR1 is localised in the cyto-
plasm as oligomer, whereas SA accumulation induces redox changes leading to the
monomerization of NPR1, probably by intermolecular disulphide bond reduction,
allowing it to be transported to the nucleus. Then NPR1 in the nucleus promotes the
binding of transcription factors to SA-responsive promoters, regulating the expres-
sion of PR genes (Mou et al. 2003; Deprés et al. 2003). The inhibition of the reduc-
tion of NPR1 and therefore its monomerization lead to a decrease in the expression
of PR genes. Diverse data indicate that SA interactions with antioxidative enzymes,
such as CAT and APX, modify the redox state enough to promote NPR1 reduction
to monomers and their entry into the nucleus. However, the molecular mechanisms
behind SA-induced responses and its link with ROS metabolism are still not com-
pletely understood.
Other novel mechanisms by which NPR1 mediates SA responses are being cur-
rently studied, and also NPR1-independent pathways are being reported, suggesting
that other yet unknown proteins could be important in SA signalling (Robert-­
Seilaniantz et  al. 2011). For additional information about the studies that estab-
lished the SA as endogenous signal in SAR, we refer the reader to reviews on this
topic such as those by Vlot et  al. (2009), Robert-Seilaniantz et  al. (2011) and
Takatsuji and Jiang (2014).

2.4 SA Analogues

Systemic acquired resistance (SAR) is a highly desirable form of resistance that

protects against a broad spectrum of pathogens. Thus, following the identification of
SA as an essential endogenous signal for SAR, it started a period of intense search
to identify synthetic chemicals able to mimic the ability of SA to activate resistance
to various stresses, both biotic and abiotic, in plants with agronomic interest. During
the two last decades, considerable progress have been done, and a high number of
chemical signals, SA and non-SA-related, contributing to SAR, have been isolated
and characterised (Walters et al. 2013). Generally, these chemical resistance induc-
ers do not directly affect the pathogens, so they are less likely to lead to resistance
in the pathogens, a problem that often arises with fungicides and bactericides.
Currently, some of these chemicals are produced commercially and broadly used in
agriculture as chemical defence inducers (also known as ‘plant activators’).
However, this resistance induction is normally not complete, because it depends on
the genotype and environment factors. In this sense, we can find early references in
the literature describing differences among species. For example, in wheat PR1
genes are induced by pathogen infection but not by SAR chemical inducers (Molina
et  al. 1999), while in corn both pathogenic and chemical inducers increase PR1
2  On the Role of Salicylic Acid in Plant Responses to Environmental Stresses 23

expression (Morris et  al. 1998). In addition, this response could be due to direct
activation of defences, or by a priming effect on cells, resulting in an ‘enhanced
status’ defences to face the next pathogen attack (Goellner and Conrath 2008).
In any case, these chemicals act on the SA pathway in plants, inducing expres-
sion of PR genes and leading to partial resistance against viral, bacterial and fungal
pathogens (Friedrich et al. 1996). Moreover, different works evidence that BTH and
INA may activate SA signalling downstream of SA accumulation (functional ana-
logues), while other compounds may induce SAR stimulating SA accumulation
(chemical inducers). In this part, we will focus on the most studied SA functional
analogue, BTH and its role related with ROS metabolism. For more information
about other SAR inductors (SA or non-SA-related) and its action mechanisms, we
suggest to readers the following reviews published recently (Walter et  al. 2013;
Aranega-Bou et al. 2014; Gao et al. 2014; Bektas and Eulgem 2015).
The first synthetic SA analogue described to induce defence was the
2,6-­dichloroisonicotinic acid and its methyl ester (both referred to as INA) (Metraux
et al. 1991; Kessmann et al. 1994; Malamy et al. 1996). INA was reported to mimic
several proposed biochemical and physiological effects of SA, such as inhibition of
CAT and APX activity, the cellular H2O2 accumulation and induction of PR gene
expression (Chen et al. 1993; Conrath et al. 1995; Durner and Klessig 1996). INA
has been shown as an effective resistance inducer against major fungal and bacterial
pathogens in various crops under both greenhouse and field conditions. Moreover,
INA was completely systemic and did not require the accumulation of SA for the
activation of SAR response (Metraux et al. 1991). Although derivatives of the INA
were not marketed as agrochemicals due to its phytotoxic effect on some crops, INA
is still used as an important tool to investigate the mechanisms, whereby chemical
compounds induce SAR (Oostendorp et al. 2001; Bektas and Eulgem 2015).
Some years later, another synthetic chemical, benzo (1,2,3) thiadiazole (BTH)
and derivatives (Kunz et al. 1997), was reported as an inducer of defence responses
against a broad spectrum of diseases in various crops, especially against fungal
infections, helping to reduce the penetration rate (Görlach et al. 1996; Benhamou
1996; Tally et  al. 1999; Gary and Goodman 2004; Darras et  al. 2006) but also
against bacterial and viral infections (Friedrich et  al. 1996; Lawton et  al. 1996;
Anfoka 2000; Hafez et  al. 2004). In contrast to INA, BTH was sufficiently well
tolerated by most crops. Therefore, the benzo (1,2,3) thiadiazole-7-carbothioic acid
S-methyl ester (BTH or acibenzolar-S-methyl or ASM) was marketed as the first
effective synthetic activator of SAR for practical agronomic use under the names
BIONR, ACTIGARDR and BOOSTR.
BTH is the SA functional analogue better known and studied. Recent data sug-
gested that BTH is converted into acibenzolar by SABP2 protein (Bektas and
Eulgem 2015). In this work, when BTH was sprayed on SABP2-silenced tobacco
plants, they failed to induce PR1 protein expression and SAR. On the other hand,
when the same transgenic plants were treated with acibenzolar, SAR was fully
induced (Bektas and Eulgem 2015). Nevertheless, information regarding the bio-
chemical action mechanism of BTH remains unclear, and the protective effect
depends on both crops and concentration. While some reduction in growth was
reported in herbaceous plants such as cauliflower or pea plants (Godard et al. 1999;
24 J.A. Hernández et al.

Clemente-Moreno et al. 2010), a positive effect on the growth of peach plantlets

under in vitro conditions has been described (Clemente-Moreno et al. 2012).
Similar to SA, the exogenous application of BTH leads to the expression of PR
genes and the change in the activity of some antioxidant enzymes, triggering the
accumulation of ROS by the inhibition of the two main H2O2 scavenger enzymes,
CAT and APX (Wendehenne et  al. 1998). In bean plants, treatment with BTH
resulted in resistance against Uromyces appendiculatus (rust), and parallel to this
resistance, increased levels of apoplastic H2O2 and peroxidase (POX) activity were
recorded, which led to the strengthening of the cell walls (Iriti and Faoro 2003).
Furthermore, treatment with BTH increased apoplastic PR proteins, as PR2 (gluca-
nases) and PR3 (chitinase), which could contribute to the inhibition of pathogen
spread (Iriti and Faoro 2003). Similar response has been described in in vitro peach
explants, in which treatment with 10 μM BTH increased endogenous levels of H2O2
(Clemente-Moreno et al. 2012). Moreover, pretreatment with low concentrations of
BTH induces expression of the enzyme phenyl ammonium lyase (PAL) in
Arabidopsis, increasing its mRNA levels after an infection with Pseudomonas
syringae pv tomato DC3000 (Kohler et al. 2002).
Regarding the activation of antioxidant defences, BTH treatment of soybean
cells resulted in increased glutathione reductase (GR), monodehydroascorbate
reductase (MDHAR) and glutathione S-transferase (GST) activities, as well as
higher ascorbate and glutathione content (Knörzer et  al. 1999). Liu et  al. (2005)
reported increases of POX, superoxide dismutase (SOD) and ascorbic acid content
by BTH treatment in peach fruit. In Plum pox virus (PPV)-infected peach plants,
BTH treatment produced an increase in GST and a reduction in dehydroascorbate
reductase (DHAR) and CAT activities (Clemente-Moreno et al. 2013). Moreover, a
possible BTH protection against the PPV-induced oxidative damage to the photo-
synthetic machinery was described (Clemente-Moreno et al. 2013). A positive effect
of BTH on glutathione peroxidase (GPX), GST and GR had been also observed in
pea, soybean cells and apple (Knözer et  al. 1999; Clemente-Moreno et  al. 2010;
Sklodowska et  al. 2011). In apple, BTH treatment increased the GST and GPX
activities up to 70% and 30%, respectively, but reduced APX activity and
α-tocopherol concentration (Sklodowska et  al. 2010). However, these authors
describe also an increased lipid peroxidation levels in BTH-treated plants, suggest-
ing a possible elicitation of pro-oxidant responses by BTH (Sklodowska et al. 2010).
It was demonstrated that treatment of cells with BTH produced an inhibition of the
NADH: ubiquinone oxidoreductase in complex I of the mitochondrial transport
chain (Van der Merwe and Dubery 2006). This enzyme is the most important in the
oxidation of NADH under normal conditions, and it is also the major source of ROS
generation in mitochondria (Moller 2001). This response was dependent on the con-
centration of BTH used and was greater than that produced by SA. However, the
spectrum of protection appears to be specific for each crop. In this sense, BTH
activates resistance to late blight (Phytophthora infestans) in tomato but not in
potato (Tally et al. 1999). In pea plants, BTH treatment prior to PPV inoculation
partially reduced the number of leaves showing symptoms, and in noninfected pea
plants, BTH treatment increased APX, MDHAR, POX and glucose-6-phosphate
dehydrogenase (G6PDH) activities (Clemente-Moreno et al. 2010).
2  On the Role of Salicylic Acid in Plant Responses to Environmental Stresses 25

2.5 Role of SA in NaCl-Stressed Plants

The role of SA in the response of plants under salinity conditions has still to be
unravelled. Different authors studied the effect of exogenous SA treatments in the
response to NaCl stress in different plant systems, and its effect seems to be depen-
dent of the SA concentrations used, the plant species, the application mode of the
treatment, the physiological state of the plant during the application as well as the
level of salinity and the exposure time to NaCl (Table 2.1).
Some authors described that exogenous SA treatments improved plant growth
under saline stress (Szepesi 2006; He and Zhu 2008; Bastam et al. 2013; Liu et al.
2014) as well as the seed germination process in the presence of NaCl (Rajjou et al.
2006; Lee et al. 2010). The pretreatment of tomato plants with low SA concentra-
tions (10−4  M) improved the acclimation of tomato plants to 100  mM NaCl in
hydroponic cultures. SA pretreatment improved the photosynthetic efficiency,
enhanced APX and guaiacol peroxidase activity in roots and induced an accumula-
tion of polyamines (Szepesi 2006).
The foliar SA application alleviated the NaCl-induced damage in tomato plants
(He and Zhu 2008). In this work, the pretreatment was carried out by foliar spray of
1 mM SA in 10-day-old seedlings, and NaCl treatment (100 mM) was analysed at 7
and 14 days. As expected, plant growth was strongly reduced in salt-stressed plants,
but this reduction was less pronounced in SA-treated plants. These authors observed
a SA alleviation of the NaCl-induced oxidative stress as indicated by lower levels of
lipid peroxidation and H2O2 accumulation as well as the enhancement of the antioxi-
dant capacity of tomato plants by increases in CAT, APX, DHAR and ASC and GSH
contents (He and Zhu 2008). Moreover, it has been described that increased SA lev-
els by pathogen infection also promote NaCl resistance. For example, root colonisa-
tion by the fungus Piriformospora indica triggers systemic resistance to fungal
diseases and confers enhanced tolerance to salt stress in barley (Waller et al. 2005).
SA plays a role in seed germination under stress conditions, but a variability of
results has been reported, and both SA-induced inhibition and promotion of seed ger-
mination have been reported. In this way, SA inhibits seed germination in a concen-
tration-dependent manner in pea, maize, Arabidopsis and barley (Guan and Scandalios
1995; Borsani et al. 2001; Xie et al. 2007; Barba-Espín et al. 2011), whereas other
authors showed that SA promotes germination under saline conditions by reducing
the NaCl-induced oxidative damage (Rajjou et al. 2006; Lee et al. 2010).
Rajjou et al. (2006) described that SA improved the germination of the wild-type
Arabidopsis (Ler) and the NahG transgenic line which overexpresses a bacterial salic-
ylate hydroxylase gene that transforms SA to catechol (Delaney et  al. 1995). The
germination of wild-type Arabidopsis (Col-0) seeds was significantly delayed by
NaCl, and this inhibition was even more noticeable in the presence of SA levels higher
than 100  μM, whereas lower SA concentrations, close to physiological levels
(1–10 μM), reduced the inhibitory effect of NaCl during the germination (Lee et al.
2010). These authors suggested that high SA concentrations can have a toxic effect on
plant growth and development possibly due to its reported effect inducing ROS accu-
mulation (Rao et al. 1997), whereas physiological SA levels are able to counteract the
26 J.A. Hernández et al.

Table 2.1  Described effects of exogenous salicylic acid (SA) treatments in plants subjected to
salt or drought stress
Stress and plant SA Antioxidative
species concentration Phenotype effect metabolism References
100 mM NaCl 10−4 M Improved Increased APX and Szepesi
Tomato hydroponic acclimation GPX (2006)
culture
100 mM NaCl 1 mM SA, Improved plant Increased ascorbate, He and Zhu
Tomato foliar growth GSH, CAT, APX, (2008)
application DHAR
150 mM 1–10 μM Germination Decreased H2O2 Lee et al.
Arabidopsis MS agar plates promotion generation (2010)
70 mM NaCl 25–100 μM, Reduction of plant Increased ascorbate, Barba-Espín
Pea plants foliar growth GSH, CAT, SOD et al. (2011)
application Decreased APX and
GR
30–90 mM 0.5–1 mM, Improved plant Reduced electrolyte Bastam
NaCl foliar growth leakage, increased et al. (2013)
application Increased PN chlorophyll contents
Pistachio
100 mM NaCl 0.1 mM, foliar Improved plant Reduced ROS Liu et al.
Cotton application growth accumulation and (2014)
Increased PN lipid peroxidation
Increased CAT
15% peg 0.5 mM, Increased sensitivity Increased electrolyte Németh
Maize, wheat hydroponic to drought leakage and reduced et al. (2002)
culture photosynthesis
Drought stress 1 μM, foliar Delayed symptoms Decreased H2O2 Saruhan
Maize application Improved plant contents and lipid et al. (2012)
growth peroxidation
Increased GSH,
CAT, GR, MDHAR,
DHAR
Drought stress 500 μM, soil Improved plant Increased SOD, CAT Habibi
culture growth (2012)
Barley Photosynthesis
protection
Improved WUE
Water stress 2 mM (in Improved plant Increased CAT, SOD Ying et al.
Red bayberry watering) growth and proline (2013)
15% peg-6000 0.5 mM, Alleviate plant Increased ascorbate, Kang et al.
hydroponic growth inhibition GSH (2013)
culture Gene induction
Wheat
(GST1, GST2, GR,
MDHAR)

inhibitory effect of NaCl in the germination process by lowering the salt-­induced

H2O2 accumulation in germinating seeds (Lee et al. 2010). These results contrast to
those previously reported by Borsani et al. (2001) that found that SA enhanced the
deleterious effect of NaCl or drought stress in Arabidopsis seedlings by increasing the
2  On the Role of Salicylic Acid in Plant Responses to Environmental Stresses 27

rate of ROS generation in photosynthetic tissues. Wild-type Arabidopsis (Ler) seeds

were unable to germinate in the presence of 100 mM NaCl, whereas the SA-deficient
transgenic NahG Arabidopsis line was able to germinate under the same conditions.
According to Lee et al. (2010), the ability of the NahG seeds to germinate under NaCl
stress can be due to the antioxidant activity of catechol.
SA could be also related to plant acclimation to saline conditions. NaCl-adapted
tomato cells contained a lower concentration of SA than unadapted cells (Molina
et al. 2002). The adaptation process to NaCl was also related with a higher antioxi-
dative capacity because salt-adapted cells also contained higher basal levels of APX
and GR activities (Molina et al. 2002). Barba-Espín et al. (2011) observed that SA
negatively affects the response of pea plants to NaCl stress. In this work, pea seeds
and seedlings were treated with different SA levels (25, 50 and 100  μM). In the
absence of NaCl, 100 μM SA significantly reduced plant growth, being the effect
more evident in roots than in shoots. SA treatment had an effect on the antioxidative
machinery of pea plants. For example, in the absence of NaCl, 100 μM SA increased
APX and catalase activities, whereas in the presence of NaCl, a decrease in APX as
well as increases in SOD and GST activities took place, being this response corre-
lated with an accumulation of H2O2 in these plants (Barba-Espín et al. 2011). Low
SA levels produced the induction of the PR-1b gene in leaves from NaCl-stressed
pea plants. These authors suggested that the induction of PR-1b gene could be an
adaptive response in order to prevent a possible opportunistic fungal or bacterial
infection in a weakness situation (Barba-Espín et al. 2011) (Fig. 2.2).
In a more recent work, Bastam et al. (2013) reported that the exogenous applica-
tion of SA improved the tolerance of pistachio seedlings to NaCl stress (up to

Fig. 2.2  Biochemical and

molecular changes in
NaCl-stressed pea plants
treated with SA. In
NaCl-stressed pea plants,
SA increased H2O2
contents and correlated
with the activation of SOD
(H2O2-generating enzyme)
and the reduction in APX
and GR (involved in
H2O2-detoxification).
Furthermore, SA induced
the PR-1b gene in
salt-damaged leaves,
probably to prevent a
possible opportunistic
fungal or bacterial
infection (Barba-Espin
et al. 2011)
28 J.A. Hernández et al.

90 mM NaCl). The SA-treated plants showed lower NaCl-induced injured symp-
toms, a better growth rate, higher chlorophyll contents and photosynthetic capacity
than the non-treated plants. In this case, authors used SA concentrations ranging
from 0 to 1 mM, and the treatments were applied by foliar spray. The foliar applica-
tion of 0.1 mM SA also improved the growth of cotton seedlings in the presence of
100 mM NaCl. The SA-treated plants displayed better growth and photosynthetic
rates and showed low ROS accumulation (O2.- and H2O2) and lipid peroxidation that
correlated with a significant enhancement of CAT activity (Liu et al. 2014).

2.6 SA and Response to Drought Stress

The effect of SA on water stress is more homogeneous than its effect on salt stress,
and some early reports showed that the SA treatment could improve the response to
drought stress (Munne-Bosch and Penuelas 2003; Bechtold et  al. 2010; Khokon
et al. 2011; Ying et al. 2013; Miura et al. 2013) (Table 2.1). Exogenous SA applica-
tion induced drought tolerance in red bayberry plants, wheat seedlings, barley plants
and pea plants (Ying et al. 2013; Singh and Usha 2003; Habibi 2012; Miura et al.
2013). In general, the improved drought response induced by SA is associated with
an increase or maintenance of plant growth, PN, Rubisco activity and the antioxida-
tive capacity (Table 2.1). SA-treated bayberry plants displayed better RWC (relative
water content), photosynthetic rates as well as higher CAT and SOD activity and
proline contents than non-treated plants (Ying et al. 2013). In addition, SA attenu-
ated the drought-induced oxidative stress as recorded by a decrease in some oxida-
tive stress parameters such as lipid peroxidation and electrolyte leakage, suggesting
that SA can partially protect the membrane integrity. SA increased Rubisco and
SOD activities as well as chlorophyll contents in drought-stressed wheat seedlings
(Singh and Usha 2003). The improvement of SA on drought tolerance of barley
plants was associated with an increase in the antioxidative defences and the mainte-
nance of photosynthesis under water stress conditions (Habibi 2012). However, in
spite of the effect of SA in gas exchange parameters, plants displayed a decrease in
shoot biomass (Miura et al. 2013). Similar result was described also in pea plants
treated with 100  μM SA, with a reduction in gs without effects in PN, therefore
increasing WUE (Barba-Espín et al. 2011). Miura et al. (2013) also observed that
drought stress induced the expression of PR-1 and PR-2, two typical SA-inducible
genes, suggesting that SA accumulation may be required for drought tolerance.
In addition, plant genotypes containing high SA contents also showed a higher
degree of drought tolerance. The Arabidopsis genotype C24 contained a SA level
near fivefold higher than control genotype and showed a higher drought tolerance
but also showed biotrophic pathogen resistance and tolerance to ozone (Bechtold
et al. 2010). The treatment of wheat seedlings with 0.5 mM SA alleviated the growth
inhibition induced by drought. This response was linked to the increase in ASC and
GSH as well as the increase in the transcription of GST1, GST2, GR and MDHAR
genes (Kang et  al. 2013). As SA increased the antioxidant performance and
decreased lipid peroxidation levels in different plant species, it has been suggested
2  On the Role of Salicylic Acid in Plant Responses to Environmental Stresses 29

that SA may act as an ROS scavenger (Kang et al. 2013). SA treatment increased the
ASC-GSH cycle enzymes along with SOD and CAT in two maize cultivars, show-
ing different sensibility to water stress, after 10 days of withholding water, suggest-
ing that ASC-GSH cycle can act to remove the H2O2 generated during the early
phase of water stress (Saruhan et al. 2012).
Although different authors reported that SA treatment improves the response in
water-stressed plants, as described by different authors, also an increase in sensitiv-
ity to PEG-induced drought has been described in maize plants (Németh et  al.
2002). It seems that the manner of SA application has a great influence on their
effects. These authors added 0.5 mM SA in the hydroponic solutions in the presence
of 15% PEG, and an increase in electrolyte leakage as well as in PN was produced
(Németh et  al. 2002). These results were supported by the data observed in the
transgenic NahG Arabidopsis line, in response to abiotic stress (Borsani et al. 2001).
These authors described that the NahG line was better able to resist the oxidative
damage generated by salt and osmotic stress than the wild-type plants.
The C24 Arabidopsis genotype responded to drought stress by decreasing the
stomatal conductance (Bechtold et al. 2010). Similar results were observed in the
SA-accumulating Arabidopsis mutants’ siz1, cpr5 and acd6 that sowed reduced sto-
matal aperture and drought tolerance (Miura et al. 2013). In fact, it has been reported
that SA reduced the stomatal conductance in a dose-dependent manner in different
plant species, including Vicia faba (Mori et al. 2001), Commelina communis (Lee
1998) and Arabidopsis (Khokon et al. 2011). The SA-induced stomatal closure is
dependent on ROS generation, because the application of antioxidant enzymes such
as catalase and SOD suppressed the stomatal closure. In addition, the stomatal clo-
sure induced by SA was completely suppressed by the action of salicylhydroxamic
acid (SHAM), a cell wall peroxidase inhibitor, but not by DPI (a NADPH oxidase
inhibitor). These results suggested that SA induced stomatal closure by means of
the ROS generated by cell wall peroxidases (Mori et al. 2001; Khokon et al. 2011;
Miura et  al. 2013). In the effect of SA mediating the stomatal closure, also
extracellular-­free Ca2+ seems to be involved because the use of Ca2+-chelators, as
EGTA, reduced the SA-induced stomatal closure (Khokon et al. 2011). However,
the treatment of two maize cultivars with 1 μM SA by foliar spraying reversed the
drought-induced stomatal closure (Saruhan et al. 2012).
Therefore, it can be suggested that the induction of drought tolerance by exoge-
nous SA application may have a significant practical application in agriculture, hor-
ticulture and forestry. In this sense, although we still have a long way to go to
decipher the networks behind the control response to stress, the implications of SA
and ROS and the antioxidative metabolism in response to stress, understanding the
performance of SA regulation network could be key to provide to agriculture an
appropriate improvement of plant defence responses to face to any stress conditions
and coming environmental changes.

Acknowledgements  PDV acknowledges the CSIC and the Spanish Ministry of Economy and
Competitiveness for his ‘Ramon y Cajal’ research contract, cofinanced by FEDER funds. MJCM
acknowledges the Spanish Ministry of Economy and Competitiveness for her ‘Juan de la Cierva’
research contract.
30 J.A. Hernández et al.

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Use of Salicylic Acid and Related
Compounds to Improve the Abiotic 3
Stress Tolerance of Plants: Practical
Aspects

Tibor Janda, Magda Pál, Éva Darkó, and Gabriella Szalai

Abstract
Due to the urgent and pressing need in the twenty-first century to develop sus-
tainable ways to safeguard crop yields in a way requiring low costs and fewer
chemicals, the demand has been increasing in recent years for natural, biologi-
cally active materials which can be used to improve the nutritional and agro-
nomic traits of agriculturally important food and feed crops. A wide range of
experiments has been performed to find solutions which are applicable in
environment-­safe farming. One promising compound is salicylic acid, which has
been proved to play a role in abiotic and biotic stress response mechanisms. The
present chapter gives a short overview of selected results, focusing mainly on the
practical aspects of its use and possible challenges for future research.

Keywords
Field experiments • Foliar application • Priming • Salicylic acid • Seed soaking
• Yield

3.1 Introduction

Improving the stress tolerance of cultivated plants and thus increasing crop yields
and nutritional values in environmentally friendly ways is a crucial task in agricul-
ture. At present, classical breeding or the production of transgenic plants is gener-
ally used to improve the genetically determined defence potential of crop plants.
However, the former is time-consuming, while the latter is subject to many legal

T. Janda (*) • M. Pál • É. Darkó • G. Szalai

Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences,
2462 Martonvásár, Brunszvik u. 2, Hungary
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2017 35

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_3
36 T. Janda et al.

restrictions. Certain agro-technological methods, such as crop rotation, mixed crop-

ping systems, improved fertilisation techniques, etc., may contribute to the minimi-
sation of yield losses under the given environmental conditions. The need to find
efficient natural solutions for activating of defence responses and improving stress
tolerance of agriculturally important plants is becoming increasingly urgent. Since
food security is an important challenge in the twenty-first century, the improvement
of natural stress tolerance is an excellent way to increase crop yields at low cost with
fewer chemicals. The use of biostimulants in agriculture has substantially increased
in recent decades (Calvo et al. 2014; Brown and Saa 2015). The exogenous applica-
tion of naturally occurring biologically active compounds could be an alternative
approach for improving crop productivity under changing environmental condi-
tions. The replacement of synthetic chemicals by natural secondary plant com-
pounds could be an excellent option from the economic and environmental points of
view. These compounds may include various plant growth regulators and signalling
molecules. Acclimation processes can be intensified through the application of nat-
urally occurring biologically active compounds.

3.2 Role of Salicylic Acid in Plants

Salicylic acid (SA), chemically ortho-hydroxybenzoic acid, belongs to the plant

phenolic group. Historical records show that salicylates originating from plant
materials have been used for medical purposes for thousands of years, for example,
by the ancient Egyptians, Greeks and American Indians. Willow bark was one of the
best known sources of pain killers and fever-reducing medicine. Its active extract is
called salicin, after the Latin name of willow trees, Salix. The commercial produc-
tion of SA started in Germany in 1874 (Raskin 1992). Aspirin, chemically acetyl
SA, was introduced by the Bayer company. This product was more suitable for
human consumption and is still one of the most widely used pain killers all over the
world. Recent medical research also demonstrated its anticancer properties (Schroer
and Rauch 2013).
However, plants do not synthetize SA or its chemically related compounds sim-
ply for human medical purposes. SA can be found in various plant species at a wide
range of endogenous levels. However, its role has only been recognised in the last
few decades, and the exact mode of action is still not fully understood. While the
majority of plants, for example, tobacco, wheat or maize, usually contain SA in a
concentration of well below 1 μg/g fresh weight under optimum growth conditions,
in others, for example, rice leaves or the floral parts of thermogenic plants, it may
reach levels of 30–40 μg/g fresh weight g or even more.
There are two main routes of SA synthesis in plants. First it can be synthesised
via the phenylalanine pathway, in which phenylalanine is converted into cinnamic
acid by phenylalanine ammonia lyase. This enzyme is a key regulator of the phen-
ylpropanoid pathway and can be induced by various biotic or abiotic stressors (Chen
et al. 2009). Cinnamic acid can either be hydroxylated to form ortho-­hydroxycinnamic
acid followed by oxidation of the side chain or initially oxidised to give benzoic
3  Use of Salicylic Acid and Related Compounds to Improve the Abiotic Stress… 37

acid as an immediate precursor, which is then hydroxylated in the ortho position

(Alibert et  al. 1972). The phenylalanine pathway also serves as a rich source of
metabolites in plants, such as flavonoids, coumarins and lignans. The other main
route for SA biosynthesis takes place in the chloroplasts, where SA is synthesised
from chorismate via isochorismate in processes catalysed by the isochorismate syn-
thase and isochorismate pyruvate lyase enzymes (Wildermuth et  al. 2001;
Wildermuth 2006). The role of the shikimate pathway in SA synthesis was also
demonstrated in young pea plants (Szalai et al. 2011).
In connection with stress, SA was first shown to be a critical component for the
development of both local and systemic resistance acting through the activity of one
of the well-known regulators of plant immunity, NPR1 (Cao et al. 1994; Delaney
et al. 1995; Després et al. 2003; Wang et al. 2006). NPR1 and its orthologues, NPR3
and NPR4, act as receptors of SA (Fu et al. 2012). Besides its role in protection
against pathogen attacks, SA has also been shown to be a promising compound for
increasing plant productivity, especially under unfavourable environmental condi-
tions. Drought is one of the most important limiting factors worldwide, and accord-
ing to the predicted climate change and global warming, its negative effects are
likely to become more severe in many agronomically important areas. One of the
very first observations was that aspirin treatment reduced transpiration in bean and
led to stomatal closure in Commelina communis L. plants (Larque-Saavedra 1978,
1979). Similar photosynthesis-regulating effects have since been demonstrated in
various plant species (recently reviewed by Janda et al. 2014), suggesting that SA or
related compounds could be used to protect plants under drought conditions. In
addition to abscisic acid, SA may also be involved in the regulation of drought
responses in plants. A dramatic increase in endogenous SA levels has been reported
in various plant species, such as the evergreen Phillyrea angustifolia (Munne-Bosch
and Penuelas 2003) and barley (Bandurska and Stroinski 2005). Furthermore,
drought also induced the PR1 and PR2 genes, which are typical SA-inducible genes
(Miura et  al. 2013). The application of a low concentration of SA may increase
drought tolerance as demonstrated in bean, tomato (Senaratna et  al. 2000) and
wheat (Hamada 2001) plants, while a high concentration usually reduces it (Miura
and Tada 2014). Besides water stress, the tolerance of plants to several other types
of stress has also been found to be improved by SA (Horváth et al. 2007; Hayat et al.
2010; Khan et al. 2015). These stressors included high (Dat et al. 1998) and low
temperatures (Janda et al. 1999; Szalai et al. 2000; Sasheva et al. 2013), high salin-
ity (El-Tayeb 2005; Szepesi et al. 2009) and heavy metal contamination (Krantev
et al. 2008; Popova et al. 2009). However, in spite of the vast amount of promising
results achieved with SA and related compounds in improving the stress tolerance
of crops, several factors need to be taken into account. First of all, the majority of
the research was carried out under controlled environmental conditions, where usu-
ally only a few parameters were changed. In the field, especially under extreme
climate conditions, the results may be very different. Secondly, the majority of the
experiments cannot be adapted to practical field conditions. For example, the results
of in vitro or hydroponic experiments are far from being suitable for practical appli-
cation. Thirdly, since high concentrations of SA may be harmful (inducing
38 T. Janda et al.

Table 3.1  Changes in certain stress response factors after different kinds of treatment with SA in
maize

oxidative stress), the response of plants to SA is finely controlled in order to reduce

its damaging effects as much as possible (Janda et  al. 2014). In SA-insensitive
plants such as rice, SA is not an effective secondary signal for the activation of
defence genes and induced resistance (Yang et  al. 2004). Interaction with other
compounds may also modify the effects of exogenous SA (Ghazijahani et al. 2014).
It has recently been shown that both different forms of SA (acid or Na-salt) and dif-
ferent modes of application (hydroponic, seed soaking, etc.) may induce different
response mechanisms. The exogenous application of SA at different times and con-
centrations may induce acclimation processes in different ways (Table 3.1).
This means that generalisations about the stress-protective mechanisms induced
by exogenous SA in different experimental systems are misleading (Gondor et al.
2016a, b). Therefore, research using SA in practical agriculture, especially under
field conditions, will be focused on below.

3.3 Use of SA in Practical Agriculture

Several methods of application, for example, seed priming, when seeds are soaked
in SA prior to sowing, adding SA to the hydroponic solution and irrigating or spray-
ing plants with SA solution, have been shown to protect various plant species against
abiotic stress factors. From the practical point of view, not all of these application
methods can be used under field conditions, especially for cereals grown on large
areas. Hydroponic cultures are only suitable for use in greenhouses. Although can-
opy spraying may have undesirable environmental impacts and it may also be rather
expensive, in some cases it may be beneficial. The foliar application of SA under
greenhouse conditions promoted the growth and yield of red amaranth. It also
enhanced chlorophyll biosynthesis and increased the levels of certain bioactive
compounds, such as betacyanins and total polyphenol, and increased the antioxidant
3  Use of Salicylic Acid and Related Compounds to Improve the Abiotic Stress… 39

activity (Khandaker et  al. 2011). Similarly, spraying with SA alleviated the salt-­
induced decrease in photosynthetic activity in mung bean by stimulating the anti-
oxidant metabolism and inducing the activity of the nitrate reductase and
ATP-sulphurylase enzymes (Nazar et  al. 2011). The exogenous application of
methyl jasmonate and methyl salicylate to rice also increased the enzymatic activity
and gene transcription of phenylalanine ammonia lyase and cinnamate-4-­
hydroxylase, two key enzymes in the phenylpropanoid pathway. These changes led
to the accumulation of phenolics while also enhancing the allelopathic potential of
rice plants (Bi et al. 2007).
Priming seed before sowing is also a promising way to improve the yield of
crops. Compared with other techniques, pre-soaking seeds in an appropriate chemi-
cal solution may have several advantages. It needs a lower amount of chemicals than
spraying, so it is relatively cheap. The dose applied can also be controlled better
than in the case of sprayed plants. A previous comparative study also suggested that
seed priming was more effective than foliar application in improving the productiv-
ity of maize plants (Ahmad et al. 2014). Recent results showed that priming seeds
with SA effectively improved the performance of cereals such as rice (Ruan et al.
2002) and maize (Rehman et al. 2015; Mahboob et al. 2015; Szalai et al. 2016).
Seed priming with SA improved the grain yield and the morphological, biochemical
and yield-related attributes of maize, including the plant population, leaf area index,
100-grain weight and antioxidant activities (Ahmad et al. 2014). It was also shown
that soaking maize seeds in SA solution increased the level of superoxide dismutase
activity in the leaves of young seedlings (Krantev et al. 2008) and also significantly
increased the number of grains per cob, grain yield and harvest index parameters
after both optimum and late sowing dates (Rehman et al. 2015). The higher grain
yields obtained after the exogenous application of SA were assumed to be due to
membrane stabilisation and improved antioxidant activity, which could help to
maintain higher photosynthesis rates even at low temperatures. This could result in
more grains per cob and increased grain weight, ultimately leading to higher grain
yield. Recent seed priming experiments on maize plants showed that the application
of SA was more effective at early spring sowing dates in Hungary (Szalai et  al.
2016), where the continental climate conditions may dramatically increase the
probability of chilling injuries. Changes in polyamine contents have also been
reported after seed priming in maize (Szalai et al. 2016). Polyamines can be found
in all living cells and are generally assumed to act as protective compounds playing
a role in stress acclimation processes. Directly or indirectly they are able to influ-
ence several physiological processes, including photosynthesis, the antioxidant sys-
tem and ion channels. The major polyamines, putrescine, spermidine and spermine,
can be converted into each other in the polyamine cycle (Pál et al. 2015). Since the
chilling tolerance of maize was also found to improve after priming seeds with
putrescine (Cao et  al. 2008), this suggests the possible role of polyamines in
SA-induced stress responses.
40 T. Janda et al.

3.4 Future Perspectives

Several recent reports indicated that SA and several other naturally occurring bio-
logically active compounds could be promising for the improvement of stress toler-
ance and consequently crop yields under unfavourable environmental conditions.
However, despite the vast amount of promising experiments under controlled or
field conditions, there are still several open questions that must be answered before
any kind of naturally occurring biologically active materials, including SA, can be
recommended in a responsible manner for practical use, especially under field con-
ditions. Since these compounds are part of a tightly regulated acclimation system,
further research is needed to optimise their use and effectiveness and to avoid unde-
sirable side-effects.
Among others, the following aspects should be addressed:

–– At the basic scientific level:

1. Improvement of knowledge about the mode of action of naturally occurring
biologically active materials. Very little information is available on their
uptake and transport mechanisms, as well as on the regulation of complete
signalling cascades.
2. Little is known about the influence of environmental conditions on the mode
of action of these types of molecules after exogenous application. Specific
experiments should be designed under controlled environmental conditions to
reveal the interactions between various environmental factors (light, tempera-
ture) and the main stress factors. Parallel experiments should be carried out in
various regions with different climates and soil conditions in order to be able
to make generalisations about the effects of the symptoms of naturally occur-
ring biologically active compounds. Earlier data suggest that the effectiveness
and mode of action of certain stress-related compounds depend greatly on
both their concentration and on the given environmental conditions (apart
from stressors). The most important factors are temperature and light. The
effects of temperature on acclimation processes in plants have been exten-
sively studied, but recent studies revealed that light influences acclimation
mechanisms not only directly via the well-known photosynthetic processes
but also by influencing various signalling routes (Janda et  al. 2014). This
approach is very important, because it will provide additional information
about the possible negative effects of certain environmental conditions.
–– At the applied technological level:
1. Recent results also indicate that the mode of action of these compounds can-
not be generalised, because it greatly depends on the way in which they are
applied. In order to increase the range of farm-level crop management strate-
gies and tools that use naturally occurring biologically active materials to
respond in an optimal way to varying levels of water and nutrient supplies,
experiments should also be designed to compare different application modes
(e.g. seed coating, spraying).
3  Use of Salicylic Acid and Related Compounds to Improve the Abiotic Stress… 41

2. Although the effects of SA have been investigated under various stress condi-
tions in various model or crop plants, little research has been done on the
interactions between SA and nutritional stresses. Among the essential ele-
ments nitrogen has the greatest limiting effect on plant productivity (Hirel
et al. 2007). Nutrient supplies below or above the optimum levels may have a
negative effect on crop productivity. The optimum level of nutrients depends
on the genotype and the environmental factors. Among the macronutrients, N
is the most mobile element in the soil, so the use of large amounts of N fertil-
iser may contaminate the groundwater. Moreover, P and K fertilisation in the
ploughed layer may cause uneven root growth in the top soil layer, hindering
water uptake from deeper soil horizons. SA is also required to assemble the
normal root microbiome and influence the colonisation of the roots by spe-
cific bacterial families. The development of procedures for the application of
SA or other naturally occurring biologically active materials and the elabora-
tion of subsoil fertilisation for potassium and phosphorus can be carried out
in at least two different ways. One is the use of PK mineral fertilisation, and
the other is the application of N-poor but PK-rich nonhazardous waste-based
materials to selected crops.
3. The effectiveness of the processes may also depend on the genetic back-
ground, even between individual genotypes within the same species.
Therefore, one of the main objectives of future research should be to investi-
gate the effects of genotypic variation on the effectiveness of biologically
active compounds in enhancing drought tolerance at different nutrient supply
levels. This would help to identify the best combinations of genotypes and
management practices for increasing the water and nutrient use efficiency of
crops.
4. Integration of the use of naturally occurring biologically active material into
breeding programmes.
It seems that naturally occurring biologically active compounds such as SA could
be used as an alternative, environmentally sound approach to improving the toler-
ance of plants to various abiotic and biotic stressors and to enhancing their defence
potential. In the long run, they could be exploited for crop protection as farm chemi-
cals to mitigate stress-induced injury. The implementation of environmentally
friendly fertilisation is impossible without reducing fertiliser rates and increasing
nutrient utilisation. Improvements in the technology will be needed to achieve these
aims. Biologically active ingredients are not considered to be fertilisers, but they
increase the adaptability of the plants and contribute to improved nutrient uptake
and utilisation. Their use needs to be combined with systematic phenotypic analysis
to monitor environment-dependent changes in plant development. The effect of
naturally occurring biologically active compounds on physiological and agronomi-
cal parameters and their interaction with various environmental factors and applica-
tion modes must also be determined using a wide range of genetic materials
including crops with diverse genetic backgrounds. Future work should aim to dis-
cover how the genetic background determines the mechanisms that make some
genotypes tolerant and others sensitive to stress and how it is related to the mode of
42 T. Janda et al.

action of exogenously applied naturally occurring biologically active compounds.

The external application of naturally occurring biologically active compounds may
not only be useful for increasing stress tolerance but also for the identification of
genotypes with functional and effective gene regulation cascades. In the process of
breeding, enhanced adaptation to increasingly severe stress is of major importance.
The efficient selection of tolerant genotypes from segregating populations is, how-
ever, greatly hindered by the seasonally changing patterns of various stress factors
under field conditions and by the limited space and high costs of artificial testing
protocols. Thus, it would be very useful if naturally occurring biologically active
compounds could also be used for selecting tolerant genotypes in early generations.
Detailed studies on the functions of these compounds in different genotypes under
various environmental conditions may contribute to the better understanding of the
acclimation of high economically important crops to biotic and abiotic threats.
Responses to stress factors are not linear pathways but complex integrated cir-
cuits involving multiple pathways functioning in specific cellular compartments and
tissues and the interaction of additional cofactors and/or signalling molecules, lead-
ing to highly coordinated responses to a given stimulus. Therefore, special attention
must be paid to the crosstalk between the different signalling pathways. The effi-
ciency of certain exogenously applied biologically active compounds may depend
to a great extent on the genotype. However, little is known about the physiological
and genetic background of the factors influencing the effectiveness of exogenous
materials. Consequently, an attempt must be made to discover the molecular mecha-
nisms influencing the effectiveness of these compounds. Phytohormones mediate
plant interactions with the environment as well as reflecting physiological status of
the plants. Therefore, investigations on the interactions between naturally occurring
biologically active compounds and other hormonal systems could be crucial for
understanding the mode of action of SA. Plant responses to abiotic stresses depend
on coordinated and effective crosstalk between three major processes involved in
the complex gene network: signal perception, signal transduction and the activation
of gene regulation cascades finally leading to the fine-tuning of various defence
mechanisms. If plants are to adapt well to stress situations, various elements of all
these major processes should be present and functional. If this is the case, the exter-
nal application of naturally occurring biologically active compounds may take over
the role of signals in plants and thus trigger the gene cascades involved in defence.
Various degrees of malfunctioning in the gene regulation cascade could be one rea-
son for the genotypic differences observed when naturally occurring biologically
active compounds are applied externally.

3.5 Conclusions

Both plant breeders and crop producers have an interest in finding crops capable of
tolerating environmental changes with as little damage as possible. Understanding
the mechanisms by which plants perceive environmental signals and transmit sig-
nals to the cellular machinery to activate adaptive responses is of fundamental
3  Use of Salicylic Acid and Related Compounds to Improve the Abiotic Stress… 43

Fig. 3.1  Possible work

scheme of a complex
research plan to investigate
the role and possible use of
naturally occurring
biologically active
compounds (NOBACs),
including SA

importance. The utilisation of fundamental research results in practice tends to be

slow. It is perhaps no exaggeration to state that a huge part of basic research results
lies buried in scientific papers without ever being tested in practice. Worldwide
cooperation between molecular biologists, plant physiologists and geneticists on the
one hand and plant producers and plant breeders on the other is the only way to
transfer the results of basic scientific research to practical agriculture. This could
contribute to the development of environmentally friendly methods for improving
crop productivity under various environmental conditions, in particular a technol-
ogy for the external application of naturally occurring biologically active com-
pounds to crops and vegetables to satisfy local demand and the international market
more effectively. In the long term, outputs and results will help to increase the resil-
ience of crop production in more variable environments and ultimately lead to yield
safety. They will also contribute to the optimisation of water use and the reduction
of nutrient losses in agricultural systems, thereby also reducing the environmental
impact of agricultural activities, particularly with regard to water quality.
Figure 3.1 shows a possible work scheme of a complex research plan to investi-
gate the role and possible use of naturally occurring biologically active compounds.
Further complex research related to the use of naturally occurring biologically
active compounds, such as SA or related compounds, may result in:

–– A better understanding of plant stress signalling processes

–– Increased knowledge on the effects of highly variable environmental factors at
the whole plant, cellular and molecular levels
–– Characterisation of the impacts and modes of action of biologically active
organic substances
–– Improvement of fertilisation strategies
–– Development of new techniques for the improvement of crop stress tolerance,
leading to increased biomass, yield quantity and quality
–– Development of market-ready products containing various combinations of natu-
rally occurring stress-related compounds to improve the stress tolerance of crops,
44 T. Janda et al.

leading to increased biomass, yield quantity and quality (separate products for
leaf and seed application)
–– Characterisation of the genetic materials used in breeding programmes

Acknowledgements  This work was funded by the National Research, Development and
Innovation Office (K108838).

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Emerging Trends in Physiological
and Biochemical Responses of Salicylic 4
Acid

Neha Handa, Sukhmeen Kaur Kohli, Ravdeep Kaur,

Kanika Khanna, Palak Bakshi, Ashwani Kumar Thukral,
Saroj Arora, Puja Ohri, Bilal Ahmed Mir,
and Renu Bhardwaj

Abstract
Salicylic acid (SA) is one of the important plant growth regulators that has gar-
nered focus due to its crucial role in both physiological aspects and plant defense
responses. Several studies throw light on its role in regulation of various bio-
chemical pathways that further shows its control on morphology and physiology
of plants. It is involved in conferring innate immunity to plants against both
biotic and abiotic stresses by influencing multiple processes. It interacts with
transcription factors and thus alters gene expression. It is also instrumental in
controlling the expression of PR genes through NPR1 proteins. RNA silencing
mechanisms in viral infections also become activated with SA and thus provide
immunity. Plants also gain systemic acquired resistance through SA that causes
stimulation of long-distance mobile signaling and activation of transcriptional
coactivators. The studies thus indicate that SA is involved in regulation of mech-
anisms responsible for plant morphology, physiology, and responses to stress.

Keywords
Salicylic acid • Photosynthesis • Yield • Signaling • Systemic aquired resistance

N. Handa • S.K. Kohli • R. Kaur • K. Khanna • P. Bakshi • A.K. Thukral • S. Arora

R. Bhardwaj (*)
Department of Botanical and Environmental Sciences, Guru Nanak Dev University,
Amritsar 143005, India
e-mail: [email protected]
P. Ohri
Department of Zoology, Guru Nanak Dev University, Amritsar, Punjab, India
B.A. Mir
Department of Botany, School of Life Sciences, Satellite Campus Kargil,
University of Kashmir, India

© Springer Nature Singapore Pte Ltd. 2017 47

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_4
48 N. Handa et al.

4.1 Introduction

Salicin was the first compound to be isolated from the bark of willow tree in 1828,
and this was later named as salicylic acid (SA) (Popova et al. 1997). Aspirin or ace-
tylsalicylic acid, introduced in 1898, had been a renowned compound in pharma-
ceutical formulations. SA (2-hydroxybenzoic acid) is one of the important phenolic
compounds that is present in the entire plant kingdom (Metraux 2002). SA and its
related compounds have been known for their medicinal value since ancient times.
Apart of this, various conjugates of SA have also been isolated from the different
plant species. Most commonly synthesized through phenylpropanoid pathway, SA
has acquired a status of plant hormone due to its diverse roles in influencing physi-
ological processes in plants. It affects plant growth and development through cell
elongation, cell division, and cell differentiation. It also manages various enzymatic
activities, biosynthesis of several proteins, and photosynthesis and helps in enhanc-
ing antioxidant capacity (Raskin 1992; Blokhina et al. 2003; El-Tayeb 2005). Its
role has also been established in seed germination, respiration, senescence, nodula-
tion, and yield (Vlot et  al. 2009). These effects of SA may be direct or indirect
effects due to interaction with other hormones through its influence on their biosyn-
thesis or their signaling mechanisms (Yusuf et al. 2013). Due to its phenolic nature,
SA has a potential role as a non-enzymatic antioxidant (Khan et  al. 2003; Arfan
et al. 2007).
SA has been well known for its role in disease resistance and pathogen attack.
However, recent studies have established that SA plays a wide role and protects the
plants against both biotic and abiotic stresses. First few reports of SA in relation to
disease resistance came into light with a study on Nicotiana tabacum which showed
enhanced resistance against tobacco mosaic virus with aspirin treatment (White
1979). After this, many studies indicated the significance of aspirin as well as SA in
inducing systemic acquired resistance (SAR) and accumulation of pathogenesis-­
related (PR) proteins (Kawano and Bouteau 2013). Also, studies pertaining to SA
have described its potential as an endogenous immune signal that can confer resis-
tance by activation of defense mechanisms (Fu et al. 2012; Denancé et al. 2013;
Gimenez-Ibanez and Solano 2013; Yang et al. 2013). SA has the ability to activate
both resistance in plants through inducing several signaling cascades, proteins, and
interference with gene expressions. The present chapter intends to increase our
understanding pertaining to the recent advances on physiological roles of SA as
well as the underlying signaling mechanisms leading to plant defense.

4.2 Physiological Roles of SA

4.2.1 Seed Germination

Germination is a process by which plant grows from a seed and undergoes various
morphological and physiological changes resulting in embryo formation. Process of
seed germination is completed when seed coat breaks and radical come out from it
(Hermann et al. 2007). For plant growth and yield, seed germination has significant
4  Emerging Trends in Physiological and Biochemical Responses of Salicylic Acid 49

agricultural attribute which depends on various plant hormones. The role of SA in

various developmental processes has been elucidated in different mutants of
Arabidopsis with combination of gibberellins (GAs), abscisic acid (ABA), ethylene,
and jasmonic acid (JA) (Yasuda et  al. 2008; Alonso-Ramırez et  al. 2009; Leon-­
Reyes et al. 2009). In Triticum aestivum, the pre-sowing treatments of seeds with
SA showed enhanced germination as compared to the untreated seeds (Shakirova
2007). Similarly, the use of SA also increased the germination percentage and ger-
mination rate in Anethum graveolens (Espanany and Fallah 2016). It has been
observed that seeds of wheat when treated with SA showed improvement in radicle
and plumule length (Fateh et al. 2012). SA has also been observed to have a signifi-
cant role in seed germination under stressed conditions. In salt-stressed Arabidopsis
seeds, recovery of delayed germination was observed when they were treated with
1 mM of SA (Rajjou et al. 2006). In another study on Arabidopsis, application of
SA enhanced seed germination by 40% in comparison to untreated seeds under
salinity stress (Alonso-Ramırez et  al. 2009). Similarly, Cd stressed Oryza sativa
seeds showed upregulation in germination potential in the presence of SA (He et al.
2010). SA at effective concentrations has been reported to reduce the salinity stress
by managing osmotic damage leading to enhanced seed germination (Lee et  al.
2010). Additionally, ROS biosynthesis takes place due to high concentration of SA
resulting in harmful outcome in plant growth and development (Lee et al. 2010).

4.2.2 Plant Growth

Combination of several internal and external stimuli contributes in overall growth

and development (Yusuf et al. 2013). SA plays a significant role in regulation of
plant growth, and its effect is determined by plant species, developmental stage, and
concentration of SA (Arberg 1981; Vicente and Plasencia 2011). Wheat grains
when soaked in 10−5 M concentration of SA resulted in increase in leaf number and
fresh and dry mass per plant of wheat seedlings (Hayat et al. 2005). An upregulation
in growth, pigment contents, and photosynthetic rate was reported in maize when
sprayed with 10−2  M SA (Khodary 2004). Similarly, in Tagetes erecta, it was
observed that there is increase in number of roots in response to lower concentra-
tions of SA (Sandoval-Yapiz 2004). Later, Shakirova (2007) reported an increase in
size and mass of the wheat on exogenous application of SA. Likewise, cucumber
plants, on treatment with SA, showed increased diameter of plant stem and leaf
number per plant (Sultan and Mohamed 2016). SA plays a key role in plant develop-
ment by managing stem elongation, leaf development, and differentiation of vascu-
lar system (Rubio et al. 2009). Exogenous application of SA by spraying to shoots
of soybean resulted in enhanced growth of roots and shoots (Gutierrez-Coronado
et al. 1998). Morphological parameters of okra plants showed improvement in root-­
to-­shoot ratio on spraying SA (Munir et al. 2016). In chamomile, however, the low
concentration of SA-triggered root growth in plants but higher concentration of SA
functioned in reverse order and inhibited the growth (Kovacik et al. 2009). Similar
results were also reported in cell suspension cultures of Hypericum exposed to
50 N. Handa et al.

different concentrations of SA.  At high concentrations of 250  μm, retardation in

growth and cell viability was observed, while at low concentrations, increased
growth and viability were observed (Gadzovska et al. 2012).

4.2.3 Photosynthetic Efficiency

SA is considered to be the most crucial regulator of photosynthesis, metabolic pro-

cesses, and plant-water relationship depending upon its concentration and plant
type (Yusuf et al. 2013). It affects stomatal closure (Mateo et al. 2004; Melotto et al.
2006), enzymatic activities like carbonic anhydrase, RuBisCo (Hayat et al. 2012;
Yusuf et al. 2012), chloroplast structure (Uzunova and Popova 2000), and chloro-
phyll and carotenoid contents (Fariduddin et al. 2003). Optimal levels of SA help in
proper functioning of photosynthesis. Exogenously applied SA can increase or
decrease the photosynthesis rate in plants, depending upon the species. However,
similar effects could be found in genetically different plants (such as C3 or C4 type
of photosynthesis) indicating that many other aspects like application method, dura-
tion of treatment, concentration, and environmental condition play important role
rather than species itself (Janda et al. 2014). Supplementation of exogenous SA also
provides plant protection toward different stresses. It has been reported that SA
alleviates photosynthetic disruption under heat stress by enhancing PS II efficiency
as well as RuBisCo activation (Wang et al. 2010). Furthermore, it also enhanced net
photosynthetic rate by PS II recovery under heat stress by increasing HSP21 levels
(Wang et  al. 2010). Studies were reported that pigment levels were enhanced in
wheat seedlings when 10–5 M SA was applied. Moreover, it considerably enhanced
enzymatic activities, leaf number, and fresh and dry weight (Hayat et  al. 2005).
Similar observations were reported by Ghai et al. (2002) in Brassica juncea where
an elevated hill activity and chlorophyll contents were reported to increase yield of
the crop. On the contrary, SA sometimes negatively affects stomatal functionalities,
gaseous exchange parameters, photosynthesis, chlorophyll contents, and many car-
boxylating enzymes that regulate different photosynthetic reactions and photosyn-
thetic electron transport activity (Sahu et al. 2002). It has been reported that higher
concentration of SA decreases photosynthetic as well as enzymatic activities of
RuBisCo in barley (Pancheva et al. 1996). Also, reduction in chlorophyll pigments
was observed in SA-treated black gram plants (Anandhi and Ramanujam 1997).
Similar studies were found in many other plants like Arabidopsis, cowpea, and
wheat (Moharekar et al. 2003; Chandra and Bhatt 1998). The disruption of photo-
synthetic apparatus at high SA levels occurred in barley plants due to the effects on
light-induced reactions, reaction centers, and thylakoid membranes (Uzunova and
Popova 2000). Various reports showing response of photosynthesis to SA are men-
tioned in Table 4.1.
4  Emerging Trends in Physiological and Biochemical Responses of Salicylic Acid 51

Table 4.1  Response of photosynthesis to SA

S.
no. Plant SA application Response References
1. Glycine max Exogenous (500 μM) Improved quantum efficiency Tang et al.
of PS II photochemistry(ϕ PS (2017)
II) and qP (photochemical
quenching)
Overall increase in
photosynthetic rate
2. Solanum Exogenous (0.5 mmol Upregulation of Faried et al.
tuberosum L−1) photosynthesis, water use (2017)
efficiency (WUE) and
sub-stomatal CO2
3. Phaseolus Exogenous (1 mM) Elevated chlorophyll index Chame et al.
vulgaris and net photosynthesis (2016)
4. Hordeum Exogenous Inhibited PS II and hill Maslenkova
vulgare reaction activity et al. (2009)
5. Nicotiana Exogenous Reduced PS II electron Janda et al.
tabacum transport, photosynthetic CO2 (2012)
incorporation, and stomatal
conductivity
6. Lycopersicon Exogenous (10−4 M) Decreased quantum yields of Poor and Tari
esculentum PS II photochemistry (2012)
Inhibition of guard cell
photosynthesis and stomatal
closure
7. Satsuma Exogenous Effective photosynthetic Qiu et al.
mandarin machinery (2011)
Efficient electron transport
rate and quantum yield of PS
II
Induced D1 and Deg1
protease contents
8. Triticum Exogenous (0.75 mM) Upregulation of Arfan et al.
aestivum photosynthesis and (2007)
chlorophyll a/b ratio
Reduced carotenoid contents
9. Brassica Exogenous (105M) Increased photosynthetic rate, Fariduddin
juncea chlorophyll content, and et al. (2003)
carboxylation efficiency
10. Glycine max Exogenous Enhanced photosynthesis and Khan et al.
(10−5 mol/L) stomatal conductance (2003)
Increased CO2 uptake activity
in chloroplast
52 N. Handa et al.

Table 4.1 (continued)
S.
no. Plant SA application Response References
11. Hordeum Exogenous Swelling of grana thylakoids Uzunova and
vulgare (100 μM−1 mM) and coagulation of stroma Popova
Destructed plastid structure (2000)
and reduced photosynthesis
12. Triticum Exogenous (1000 μM) Enhanced photosynthesis Sahu et al.
Aestivum under lower SA concentration (2002)
and vice versa Cytochrome
f554 dysfunction under higher
SA along with fate of
ADP-dependent stimulation
of electron transport activity

4.2.4 Crop Yield

The validity of exogenous application of plant hormone is accessed on the basis of

its biological yield. SA plays vital role in regulating different metabolic processes
occurring within plants and affected their net growth and productivity (Yusuf et al.
2013). Exogenously supplied SA positively affected early as well as total yield of
the tomato plants as reported by Yildirim and Dusan (2009). Moreover, the highest
grain yield was observed by supplementation of 0.07 mmol SA. The rise in biologi-
cal yield and productivity of the crop is probably due to elapsed senescence of
plants under SA treatment (Imran et al. 2007). This enables the plant in enhancing
photosynthesis as well as restricting loss of premature plant parts (Imran et  al.
2007). Furthermore, many phytohormones are involved in enhancing assimilates in
seeds and metabolite flow toward developing seeds so as to improve seed mass and
seed yield (Yusuf et  al. 2013). Various reports showing response of crop yield
toward SA are mentioned in Table 4.2.

4.2.5 Flowering

A successful reproductive development depends upon flowering at a particular

developmental stage and season. The transition from vegetative to reproductive
stage is controlled by day length, temperature, and light quality. However, under
environmental stress conditions, plants may show precocious flowering by surpass-
ing seasonal regulation (Martinez et al. 2004). SA is involved in flower initiation
instead of flower development. SA regulates floral transitions under stress and non-
stress conditions by involving in photoperiod and autonomous pathways (Martinez
et al. 2004). It induces flowering in members of Lemnaceae under both inductive
4  Emerging Trends in Physiological and Biochemical Responses of Salicylic Acid 53

Table 4.2  Response of crop yield to SA

S.
no. Plant SA application Response References
1. Lycopersicon Exogenous (1.5 mM) Increased number of fruits Kumar et al.
esculentum per plant and average fruit (2017)
weight
Maximum fruit yield
2. Allium sativum Exogenous (300 ppm) Elevated sodium and Shama et al.
potassium contents (2016)
Increased bulb fresh
weight, number of cloves,
and bulb diameter which
upgraded total crop yield
3. Phaseolus Exogenous (1 mM) Increased seed weight and Chame et al.
vulgaris grain yield (2016)
Enhanced grain protein
content
4. Cucumis sativus Exogenous (0.18 mM) Overall increase in total Sultan and
fruit yield Mohamed
Enhanced female flower (2016)
production, plant stem
diameter, and leaf number
per plant
5. Fragaria Exogenous (2 mM) SA-induced nutrient levels Aghaeifard
ananassa in fruit by increasing et al. (2016)
vitamin C contents,
soluble solid concentration
(SSC), and antioxidants
levels
Positive regulation of total
yield and fruit weight
6. Abelmoschus Exogenous (2 mM) Improved fruit yield and Munir et al.
esculentus seed yield (2016)
Increased number of pods
per plant and pod weight
7. Phaseolus Exogenous Enhanced biological yield Mohtashami
vulgaris L. (0.7 mmolL−1) due to rise in number of et al. (2016)
grains per pod
8. Phaseolus Exogenous (2250 μM) Upgradation of dry weight Heidari et al.
vulgaris and grain yield through (2015)
seed priming with SA
(continued)
54 N. Handa et al.

Table 4.2 (continued)
S.
no. Plant SA application Response References
9. Arachis Exogenous (300 ppm) Enhanced biological yield Mulgir et al.
hypogaea L. by increasing number of (2014)
branches per plant, pod
number, pod to peg ratio,
and kernel weight
10. Brassica juncea Exogenous (10−5 M) Increased pod number and Fariduddin
seed number et al. (2003)
Rise in seed yield due to
higher protein content
11. Vigna radiata Exogenous Higher biomass Reddy et al.
production (2002)

and noninductive photoperiod conditions. Exogenous application of SA induced

short-day flowering in Lemna gibba G3, a long-day plant (Oata 1975). Similar
results were observed by Kaihara and others (1981) in Lemna paucicostata and
Lemna minor. SA and its related compounds also induced flowering under long-day
conditions in Wolffia microspica, a short-day plant (Khurana and Maheshwari
1983). In noninductive photoperiod conditions, treatment with 10  μM SA also
induced flowering in L. paucicostata LP6, grown in Bonner-Devirian medium
(Khurana and Cleland 1992). Nitric oxide (NO) is reported to be involved in
SA-induced flowering in Lemna (Khurana et al. 2014). SA has also been reported to
induce flowering in other species. Exogenous application of SA, before reproduc-
tive stage, positively regulated growth and flowering of Calendula officinalis L.
(Pacheco et al. 2013). SA is also capable of induction of stress-induced flowering in
short-day plant Pharbitis nil (or Ipomoea nil) under nutritional stress and long-day
conditions but not under nonstress conditions. Particular behavior during stress
indicates that SA is crucial for flowering but it is not present in sufficient amount to
induce flowering (Wada et  al. 2010). Stress elevates endogenous SA levels and
activity of phenylalanine ammonialyase (PAL) to stimulate flowering (Wada et al.
2010). Low-temperature stress also induced PAL activity and stress-induced flower-
ing in P. nil and its cultivar (Wada et  al. 2014). Aminooxyacetic acid (AOA), a
known inhibitor of PAL, inhibits stress-induced flowering in P. nil which was
restored by exogenous SA treatment (Wada et al. 2010). Authors concluded that at
least partly, SA promotes stress-induced flowering in Pharbitis cultivars, and syn-
thesis of SA is stimulated by PAL enzyme. Later, Koshio and others (2015) reported
that AOA also inhibits synthesis of IAA (indole-3-acetic acid) and ACC (1-amino-
cycropropane-1-carboxylic acid) along with PAL activity. AOA suppressed stress-
induced flowering was also overcome by IAA and putrescine treatment indicating
that all these metabolic pathways work in cooperation to regulate stress-­induced
flowering. Overexpression of PnFT2 (one of the orthologs of photoperiodic path-
way flowering gene FLOWERING LOCUS T of Arabidopsis thaliana) induces flow-
ering in P. nil under stress (Wada et  al. 2010). SA induces expression of PnFT2
4  Emerging Trends in Physiological and Biochemical Responses of Salicylic Acid 55

under stressed conditions and not under nonstressed conditions which indicates that
SA regulates expression of PnFT2 to induce flowering under stress conditions
(Yamada and Takeno 2014).

4.2.6 Senescence

Senescence is a complex final stage developmental process during which plant

undergoes changes in physiology, biochemistry, and gene expression. The most vis-
ible sign of leaf senescence is yellowing of leaves which occurs due to chlorophyll
degradation and photosynthetic inhibition (Ji et  al. 2016). SA is also one of the
important regulators of leaf senescence. Endogenous level of SA is reported to
increase in leaves undergoing senescence (Lim et al. 2003). Rice mutant yellow leaf
and dwarf 1 (yld1) exhibited premature senescence and reduced plant height.
Hormone analysis of yld1 showed high accumulation of SA. Starch metabolism was
also impaired in yld1 than wild type. The results indicate correlation among SA, leaf
senescence, and starch metabolism (Deng et al. 2017). SA has crucial role in regu-
lating onset and rate of senescence. SA-3 hydrolase (S3H) enzyme, involved in SA
catabolism converts SA to 2,3-dihydroxybenzoic acid during senescence, is induced
by SA. Therefore, S3H forms important part of negative feedback control of SA
levels to prevent its over accumulation during the process (Zhang et al. 2013). SA is
also reported to regulate expression of many senescence-associated genes (SAGs)
during leaf senescence. Expression of SAGs was found to be reduced in SA-deficient
mutants of Arabidopsis. Thus measurement of endogenous SA content during
senescence and application of exogenous SA and the use of SA-deficient mutants
indicate that SA is directly or indirectly involved in controlling expression of SAGs
(Abreu and Munné-Bosch 2008). It was demonstrated by Morris et al. (2000) that
SA signaling pathway regulates the gene expression of senescence-enhanced genes
thus plays a role in developmental senescence. Authors observed that mutants and
transgenics (nahG, pad4, and npr1) defective in SA signaling pathway exhibited
altered expression of many senescence-enhanced genes. Senescence is also associ-
ated with remarkable transcriptional changes. Transcriptional factors such as
WRKY, NAC, bZIP, MYB AP2/EREBP, and C2H2-type zinc finger families are
mainly reported for their role in senescence, and their regulation results in transcrip-
tional changes in SAGs (Zhao et al. 2016; Li et al. 2016). SA regulates senescence-­
specific transcription factor WRKY53, which activates expression of
senescence-associated gene 12 (SAG12), catalase1/2/3, and oresa 9 genes (Miao
et al. 2004). Mitogen-activated protein kinase 6 (MPK6) is involved in SA-induced
leaf senescence. SA induces activation of MPK6 which in turn induces higher
expression of WRKY6 which further contributes to enhanced level of NPR1 gene
promoter, thereby inducing SA-induced leaf senescence (Chai et al. 2014). Cross
talk between SA and jasmonic acid (JA) signaling pathways is well documented in
plants, but not much is researched about their cross talk during leaf senescence. JA
and SA antagonistically regulated the expression of WRKY53 in Arabidopsis thali-
ana that reveals multiple hormone pathways control senescence (Miao and Zentgraf
56 N. Handa et al.

2007). Exogenous application of SA at low concentration (1–50 μM) delays methyl

jasmonate-induced leaf senescence by elevating nitric oxide synthase activity and
reinforcing antioxidative defense system (Ji et al. 2016).

4.3 SA Signaling in Plant Protection

4.3.1 I nduction of Transcription Factors Involved in Defense-­

Related Genes

The genes involved in plant defense get activated by SA due to its ability to induce
transcription factors that are responsible for transcription of these genes. WRKY
transcription factors are abundantly found in plants and are one of the best charac-
terized transcription factors (Chen et al. 2012). The WRKY proteins are a part of
zinc finger transcription factors which are usually present in nucleus of the plant
cells (Zheng et al. 2006). Apart from several physiological and developmental func-
tions in plants, WRKY transcription factors have imperative roles in regulating
plant responses to various stresses (Eulgem et al. 2000; Pandey and Somssich 2009;
Chen et al. 2012). SA regulates these transcription factors and further controls the
defense responses of the plants (Maleck et  al. 2000; Wang et  al. 2006). In
Arabidopsis, out of total 74 WRKY transcription factors, 49 were observed to be
responsive to exogenous SA application (Eulgem et al. 2000; Dong et al. 2003). It
was reported by Li et al. (2004) that WRKY70 expression was induced by SA, and
it further increases the expression of SA-responsive pathogenesis-related (PR)
genes. WRKY53 is another SA-induced transcription factor which, when interacts
with a protein induced by jasmonic acid (JA), aids in improving bacterial and fungal
resistance in Arabidopsis (Miao and Zentgraf 2007). SA application also results in
the activation of SA-inducible protein kinase (SIPK) which phosphorylates
WRKY1, and these together are involved in defense-related cell death (Menke et al.
2005). In Oryza sativa, it was reported that OsWRKY13 was responsible for activa-
tion of both SA-responsive genes and the genes for SA biosynthesis (Qiu et  al.
2007). StWRKY and VvWRKY1 transcription factors found in Solanum tuberosum
and Vitis vinifera, respectively, are also reported to be induced by SA (Dellagi et al.
2000; Marchive et al. 2007). SA is involved in activating another transcription fac-
tor, NtWRKT12 which binds at specific sites at PR1a promoter (van Verk et  al.
2008). Some WRKY transcription factors induced by SA, however, have negative
effects on gene expression of defense-related genes. The overexpression of
SA-induced WRKY7 in Arabidopsis, WRKY33, and WRKY25 showed reduction
in the expression of defense-related genes as well as PR1 gene (Kim et al. 2006;
Zheng et al. 2006, 2007). Also, WRKY48 not only reduced PR1 expression but also
negatively affected the resistance of Arabidopsis to Pseudomonas syringae (Xing
et al. 2008). Ethylene response factors (ERF) are transcription factors, which bind
to a target sequence GCC-box which are usually present in the promoters of PR
genes (Grennan 2008). TaERF3 in Triticum aestivum was found to be activated by
SA (Zhang et al. 2007).
4  Emerging Trends in Physiological and Biochemical Responses of Salicylic Acid 57

4.3.2 Role of NPR1 in SA Signaling

Non-expresser of PR gene 1 (NPR1) has an imperative role in regulation of PR1 and

most SA-dependent defense genes. SA aids in facilitating the interaction between
NPR1 and TGA2 and TGA3 (belonging to TGA transcription factor family) and
helps in binding with TGA-box leading to the formation of trans-activating com-
plex for RNA polymerase II (Lebel et al. 1998; Kesarwani et al. 2007; Pape et al.
2010; Herrera-Vásquez et al. 2015). Also, TGA1 and TGA4 were reported to inter-
act with NPR1 in the presence of SA, and this interaction with SA was thought to
be due to the presence of Cys residues on these transcription factors (Després et al.
2003). Under normal conditions, the NPR1 protein is present in the cytoplasm in an
oligomeric form. Pathogen attack causes SA to accumulate, and this SA causes the
disulfide bonds to reduce, and monomers of NPR1 are released which are then regu-
lated to the nucleus (Durrant and Dong 2004; Tada et  al. 2008; Spoel and Dong
2012). Inside nucleus, the NPR1 activates expression of PR genes (Kinkema et al.
2000; Mou et al. 2003).
In the absence of biotic or abiotic stress, NPR1 in the nucleus is removed with
the help of proteasome so as to check any unnecessary defense action (Spoel et al.
2009). Proteasomes are present both in nucleus and the cytoplasm and are com-
posed of large protein complexes which help in maintaining the content of specific
proteins in the cellular environment (Peters et al. 1994). Ubiquitin ligase (E3) is one
of the important ubiquitin-proteasome systems which aids in transferring ubiquitin
to a specific protein (Dreher and Callis 2007). For NPR1 degradation, cullin 3
(CUL3) E3 ligase has been reported to play an important role (Fu et al. 2012). NPR3
and NPR4 are paralogues of NPR1, and they also act as adaptor proteins for the
functioning of CUL3 E3 ligase (Fu et al. 2012). Therefore, these actively participate
in NPR1 degradation along with CUL3 E3 ligase in response to SA concentrations.
NPR3 and NPR4, both have the ability to bind with SA, but the affinity of NPR4 for
SA is higher due to the presence of many SA-binding sites (Fu et  al. 2012). In
healthy plants, the concentration of SA is low, and this causes proteasome and adap-
tor proteins to degrade NPR1 and hence prevent the activation of defense genes (Fu
et  al. 2012; Moreau et  al. 2012). But at high SA concentrations during stressful
conditions, NPR4 readily binds with SA thereby resulting in interrupting the func-
tion of CUL3 E3 ligase and adaptor proteins. This leads to the proper functioning of
NPR1 which can activate defense-related genes (Moreau et al. 2012).

4.3.3 SA and Ubiquitination

Ubiquitin consists of 76 amino acids conjugated to Lys residues in the substrate

protein (Smalle and Vierstra 2004). Ubiquitination is an important mechanism
involved in degradation of ubiquitin or proteasome-mediated proteins. It is a signifi-
cant tool to enhance plant growth, development, and defense against pathogen inva-
sion (Dreher and Callis 2007; Lee et  al. 2011a). The ubiquitin-mediated protein
58 N. Handa et al.

degradation also influenced protein modification and production (Dreher and Callis
2007). Ubiquitination network is involved in plant defense (Zeng et al. 2006).
Ubiquitin is linked to the protein in a stepwise conjugation network, involving
(1) activation of ubiquitin by ubiquitin-activating enzymes (E1) in an ATP-dependent
manner, (2) activated ubiquitin is then relocated to an ubiquitin-conjugating enzyme
(E2) induced by E1, and (3) in final step the E2 enzyme interacts with E3 enzyme,
i.e., E3 ubiquitin ligase (E3) resulting in ubiquitination of the substrate protein
(Ciechanover 1998). E3 ubiquitin ligase is categorized into two, on the basis of
presence of homology to E6-AP C terminus (HECT) or really interesting new genes
(RING) which have different constituent subunits and action mechanism (Pickart
2001). RING subunit consists of consensus sequence having Cys and His residues
which act as the binding site for ubiquitin-E2 intermediate. This intermediate has a
zinc-binding site consisting of Cys and His residues. The RING domains of the
RING protein are categorized into two types, C3-H2-C3 and C3-H-C4, based on the
presence of Cys and His residues on the fifth position (Borden and Freemont 1996).
Endogenous signal induced by pathogen attack is modulated by ubiquitination.
Plants contain several RING finger proteins; only few of them have been found to
be induced by biotic stress (Hong et al. 2007). Figure 4.1 represents the ubiquitin
conjugation network.
The RING finger proteins are specifically synthesized in plants in response to
pathogen attack and play a significant role in plant defense (Zeng et al. 2006). In a

Ubiquitin
(76 amino acid compound conjugated to Lys residue in protein
substrate)

ATP Ubiquitin activating

dependent enzyme (E1)

Activation of Ubiquitin

Ubiquitin activating
enzyme (E1)

Relocation of activated ubiquitin to ubiquitin conjugation enzymes

(E2)

E2 interacts with E3 enzyme i.e. E3 ubiquitin ligase (E3)

Ubiquitination of substrate protein

Fig. 4.1  Ubiquitin conjugation network

4  Emerging Trends in Physiological and Biochemical Responses of Salicylic Acid 59

study by Lee et al. (2011a), a gene CaRING 1 was identified which encodes a new
class of E3 ubiquitin ligase RING 1 protein in pepper plant. The expression of
CaRING 1 is induced by Xanthomonas campestris pv. vesicatoria (Xcv) an avirulent
pathogen. This RING-type protein is imperative for hypersensitive response and
increased resistance to infection. The CaRING 1 shows in vitro E3 ubiquitin ligase
activity, and it is localized in the plasma membrane. The CaRING 1-silenced plants
were found to be highly susceptible to avirulent Xcv and showed delayed reactive
oxygen species (ROS) generation, hypersensitive response, PR expression, and
sequestration of free salicylic acid in response to infection (Lee et  al. 2011a).
Another report suggests elevated resistance of tobacco mosaic virus and P. syringae
pv. tabaci infection in tobacco plants in response to overexpression of E3 ubiquitin
ligase, i.e., Os BIRF 1 (Liu et al. 2008).
SA-stimulated changes in expression of genes are found to be directly linked to
chromatin remodeling and replacement. Certain chromatin-modifying complexes
are employed at SA-responsive loci resulting in regulation of basal expression as
well as SA-triggered expression (March-Diaz et al. 2008; van den Burg and Takken
2009; Jaskiewicz et al. 2011). The basal repression of these loci requires involve-
ment of SUMO (Small Ubiquitin-Like Modifiers). These are produced posttran-
scriptionally, and their conjugation controls activity and modification of
chromatin-­modifying complexes into transcriptional complexes. SUMO conjuga-
tion also determines employment and activity of various chromatin-modifying
enzymes and hence indirectly influences SA-stimulated gene expression (van den
Burg and Takken 2009).

4.3.4 SA and RNA Silencing

RNA silencing via small RNA forms an imperative surveillance approach aiming
the nucleic acids of invading pathogens (Qu and Morris 2005; Ding and Voinnet
2007; Jaubert et al. 2011). The initial point of RNA silencing cascade is triggering
of double-stranded RNA (dsRNA) which is induced in response of replication of
plant viruses (Willmann et al. 2011). When single-stranded RNA (ssRNA) viruses
are present, the viral RNA-dependent enzyme, i.e., RNA polymerase (RdRP) in
combination with the sense ssRNA, results in generation of a dsRNA (Qi et  al.
2009; Wang et al. 2009). Moreover, if other RNA viruses are present, the two strands
don’t anneal but form a complex coiled structure that have specific region of dsRNA
(Alvarado and Scholthof 2009). Geminiviruses have RNA that is transcribed into
circular genomes which act as sources of dsRNA (Chellappan et al. 2005). Hairpin
loop of viroids also contains regions of dsRNA (Papaefthimiou et  al. 2001).
Figure 4.2 shows the formation of dsRNA for RNA silencing.
The nucleic acid sequence of pathogen virus has pathogen-associated molecular
patterns (PAMPs), and viral dsRNA (vdsRNA) is a viral PAMP (Ding 2010). The
defense surveillance cascade perceives the presence of dsRNA as abnormal RNA
molecule (Wypijewski et  al. 2009) and generates small RNA from it. This small
RNA molecule triggers the antiviral mechanisms to cleave and damage the genome
60 N. Handa et al.

Fig. 4.2  Formation of dsRNA for RNA silencing

of invading viruses (Alvarado and Scholthof 2009). The RNA silencing pathway
involves various steps like (1) perfectly paired dsRNA are formed by the action of
RdRP, (2) conversion of paired dsRNA (acts as a precursor) into small interfering
RNAs (siRNAs) (Katiyar-Agarwal and Jin 2010), and (3) from these small RNAs,
which serve as primers for generation of additional dsRNA which targets the DCL
(endoribonuclease) enzyme which amplifies the RNA silencing cascade. This reac-
tion is catalyzed by enzyme host RNA-dependent RNA polymerase (RdRP) (Voinnet
2008; Garcia-Ruiz et al. 2010). The enhanced expression of RdRP gene, i.e., RdRP
1, increased the resistance to the viral pathogen based on RNA silencing (Leibman
et al. 2011). Figure 4.3 is schematic representation of RNA silencing technique for
induction of plant tolerance to viral pathogen.
Several researchers have suggested imperative role of SA in inducing synthesis
of RdRP (Jovel et al. 2011; Lee et al. 2011b). The SA-treated plants of tobacco were
observed to have an enhanced activity of RdRP. In comparison to biologically active
SA analogs, the biologically inactive analogs weren’t able to activate plant defense
system and RdRP activity. An important tobacco RdRP gene, i.e., Nt RDRP I, was
isolated from tobacco plants, and it was observed by the treatment of biologically
active analogs (Xie et al. 2003). Another, SA-dependent activation of RdRP, i.e.,
AtRdRP 1, was reported by Yu et  al. (2003) and was isolated from Arabidopsis.
Transgenic plants were reported to express Arabidopsis NPR 1 gene (AtNPR 1).
These transgenic plants were reported to be highly susceptible to rice yellow mottle
virus (RYMV) in response to misregulation of RYMV expression by SA-mediated
RNA-dependent RdRP, i.e., RDR 1gene (Quilis et al. 2008). Resistance to cucumber
mosaic virus in Arabidopsis was acquired in response to SA application regulated
expression of RDR 1-dependent viral siRNAs (Diaz-Pendon et al. 2007).
4  Emerging Trends in Physiological and Biochemical Responses of Salicylic Acid 61

Fig. 4.3  Schematic representation of RNA silencing technique for induction of plant tolerance to
viral pathogen

Another report suggests SA and 5-chlorosalicylic acid (5-CSA) enhances the

expression of RdRP gene of cotton plants, i.e., GLRdRP (Gao et al. 2009). SA sig-
naling cascade (Lee et al. 2011b) and RNA silencing (Leibman et al. 2011) have
been found to be involved in plant defense surveillance networks against viral dis-
eases (Jovel et al. 2011). Many herbaceous plants are infected by tomato ring spot
virus (ToRSV). In these plants viral RNA is sequestered in leaves and young leaves
which emerge at later infection stages. Tissues which contain viral RNA also consist
of siRNA (ToRSV derived) and PR1a transcripts (SA signaling derived), suggesting
SA-induced defense responses and induction of RNA silencing (Jovel et al. 2011).
Another report by Rakshandehroo et  al. (2009) suggested a positive interplay
between SA-induced AOX redox signaling and SA-induced small RNA-directed
RNA silencing.
62 N. Handa et al.

4.3.5 SA and Systemic Acquired Resistance

Systemic acquired resistance (SAR) is an augmented state of plant defense in

response to wide spectrum of pathogens which is activated by local invasion in
plants. The resistance in response to SAR is detected in the systemic tissue (unin-
oculated) of the plant that is under pathogen infection (Liu et al. 2011a, b). Any kind
of localized infection by a necrotizing pathogen initiates SAR to subsequent infec-
tions caused by a broad range of pathogens virulent in nature (Jung et  al. 2009;
Maldonado et al. 2002). Accumulation of SA plays a significant role in stimulation
of local defense at initial infection site as well as in the distant tissues that are infec-
tion free for induction of SAR (Navarova et al. 2012; Shah and Zeier 2013). SA
signaling cascades have been found to be a prime requisite for regulation of SAR
(Nandi et al. 2004; Du et al. 2012). The precursor of SA biosynthesis is isochoris-
mate synthase 1 activity (Garcion et al. 2008). Isochorismate synthase 1 activity-­
deficient mutant of Arabidopsis, i.e., Arabidopsis ics1 mutant has been found to be
SAR deficient (Jung et al. 2009; Chaturvedi et al. 2012). Another important gene for
induction of sequestration of SA in the distal parts of induction of SAR is FMO1
(Flavin-Dependent Monooxygenase 1 gene) and is also required for induction of
SAR (Chaturvedi et al. 2012; Shah and Zeier 2013). Manosalva et al. (2010) sug-
gested that methyl esterase 1 (StMES1) is required for the induction of SAR in
potato and also for accumulation of free SA in distal tissues which are disease-free
(Park et al. 2007).
Initiation of SAR requires displacement of signals from the site of pathogen
invasion to the distal organs, consequently resulting in activation of SA-dependent
defense pathway (Chaturvedi et al. 2008). Earlier, Yalpani et al. (1991) suggested
that SA itself may act as a mobile signal due to the fact that it accumulates in the
phloem and is prime requirement to activate SAR. Later, several other mobile sig-
nals were identified including (1) methyl salicylate (MeSA) (Vlot et al. 2008a, b;
Manosalva et al. 2010), (2) azelaic acid is another important lipid signal involved in
SAR induction (Jung et al. 2009), (3) abeitane diterpenoid or dehydroabietinal (DA)
is another potent inducer of SAR (Chaturvedi et al. 2012), and (4) pipecolic acid, a
Lys catabolite, non-proteinaceous in nature is identified as an imperative systemic
signaling molecules (Shah and Zeier 2013). Figure 4.4 shows the classification of
Mobile Signals which Induce SAR.
Methyl salicylate (MeSA) serves as a long-distance SAR signal which occurs via
phloem in tobacco and Arabidopsis (Manosalva et al. 2010). Reaction between SA
carboxyl methyltransferase (SAMT) and methyl group of S-adenosyl-L-methionine
(SAM) molecules catalyzed by SAM results in synthesis of MeSA (Kwon et  al.
2009). The gene expression of SAMT 1 is enhanced in response to pathogen inva-
sion and SA treatment (Koo et al. 2007). Initiation and development of SAR seems
to require SAMT 1 in the primary inoculated tissues and MeSA (Park et al. 2007).
The upregulation of expression of SAMT 1genes results in conversion of SA to
MeSA completely (Kwon et al. 2009). MeSA translocates via phloem systemically
and might convert to active SA from infected parts to noninfected parts (Park et al.
2009). SA-binding protein (SABP2) has been identified and characterized in
4  Emerging Trends in Physiological and Biochemical Responses of Salicylic Acid 63

Fig. 4.4  Classification of mobile signals which induce SAR

tobacco plants (Du and Klessig 1997; Kumar and Klessig 2003; Tripathi et  al.
2010). SABP2 belongs to α/β hydrolase superfamily and is a lipase. It is frequently
occurring and binds particularly to SA (Kumar and Klessig 2003). When SABP2 is
present with MeSA as a substrate, it acts as salicylate esterase and catalysis conver-
sion of inactive MeSA to active SA (Vlot et al. 2008a, b; Manosalva et al. 2010).
Moreover, Liu et al. (2011b) suggested that the duration of light exposure which
plant perceives after primary pathogen invasion determines the quantity of MeSA
required for SAR induction.
Another important mobile signaling molecule is azelaic acid. It is a 9-carbon
dicarboxylic acid and is found to be sequestered in vascular sap of Arabidopsis
plants which is infected by Pseudomonas syringae (Jung et al. 2009). Also Zahn
(2009) reported an increase in levels of azelaic acid in plants exposed to pathogen
invasion and stimulates SAR.  Azelaic acid triggers the expression of AZI 1, i.e.,
azelaic acid-induced 1 gene. Any kind of mutation in AZI 1 results in loss of SAR
which can be triggered by azelaic acid. These responses suggest that secretion of
AZI 1 is significant for production of vascular sap that confers disease resistance
(Jung et al. 2009).
Dehydroabietinal (DA) has been shown to induce SAR (Chaturvedi et al. 2012).
It is in its most active form when applied in picomolar solution and is applied to dif-
ferent plants including Arabidopsis, tobacco, and tomato. DA when supplemented to
the leaves of Arabidopsis was quickly translocated out of the leaves and recovered
from DA untreated leaves (Chaturvedi et al. 2012). Supplementation of DA results in
enhanced accumulation of SA and expression of PR 1 gene in DA untreated leaves.
DA was found to function upstream of SA signaling and accumulation.
A non-proteinaceous amino acid pipecolic acid (Pip) is also identified as a mobile
signal in long-distance SAR cascade. It has been reported to strongly accumulate in
64 N. Handa et al.

Arabidopsis leaves which are inoculated with P. syringae pv. maculicola (Psm) as
well as in leaves treated with bacterial PAMPs flg22 and LPS (Navarova et al. 2012).
A significant elevation in systemic levels of Pip but not of SA accumulation was
found at 36 h after Psm inoculation by (Navarova et al. 2012). This suggested that
Pip accumulation occurs before SA accumulation in inoculated leaves after onset of
SAR. Song et al. (2004) suggested that ALD 1 (an aminotransferase) to be involved
in Pip biosynthesis. Pip-deficient plants with active adl1 fail to accumulate SA in
the distal tissues after pathogen inoculation and have a weakened SAR (Jing et al.
2011; Navarova et al. 2012). When Pip is exogenously supplemented to whole plant
prior to pathogen inoculation, it was observed to regain its ability to accumulate SA
and activation of SAR (Navarova et al. 2012).
Mediators are multiprotein complexes which function as transcriptional coacti-
vators and have been found to be involved in stimulation of SA-mediated SAR
induction (Kidd et  al. 2011; An and Mou 2013). Mediators refine the pathogen-­
specific and gene-specific reprogramming at transcriptional levels. This is accom-
plished by interaction of mediators with specific activator or repressor and TFs
(transcriptional factors) at the promoting sites (Conaway and Conaway 2011; An
and Mou 2013). Arabidopsis-specific mediator subunit 16 (MED16) has been
reported to positively regulate SAR (Zhang et al. 2012). Any kind of mutation in
MED16 results in reduction in levels of NPR1 protein (Zhang et al. 2012). MED16
might modulate late SA response by regulation of NPR 1 accumulation. After inoc-
ulation of Arabidopsis mutant plants with P. syringae, the plants were observed to
locally as well as systemically accumulate SA similar to that of the wild type.
Another important strategy by which SA triggers SAR is priming which is induc-
tion of unique physiological state of plant application or accumulation of SA or
treatment of pathogen-infected plants with certain synthetic compound (Slaughter
et al. 2012). The treatment results in elevated alertness with very low or no gene
stimulation (Conrath et al. 2006). Pathogen-induced genes, when primed with syn-
thetic compounds, allow the infected plants to respond more effectively to a particu-
lar invader (Conrath et al. 2006). It is considered as one of the most low-cost strategy
to increase plant defense against wide spectrum of attackers (Van der Ent et  al.
2008). SA and its functional analogs BTH [benzo(1,2,3) thiadiazole-7-carbothioic
acid S-methyl ester) and β-aminobutyric acid have been reported to induce priming
and increase resistance to fungal invasion (Hukkanen et al. 2007), oomycetes inva-
sion (Hamiduzzaman et al. 2005), bacterial invasion (Flors et al. 2008), and viral
invasion (Mandal et al. 2008).
Moreover, Beckers et al. (2009) suggested that some dormant mitogen-activated
protein kinases (MAPKs) may have an imperative role in priming in Arabidopsis.
Deposition of MAPKs prior to pathogen inoculation might be a possible mechanism
of priming during SAR. The full priming in Arabidopsis is accomplished by MPK3
and MPK6 activities. In response to resistance-stimulating avirulent strains of P.
syringae pv. tomato DC3000 and P. syringae pv. phaseolicola, enhancement in
expression of MPK3 and accumulation of SA was observed. SA and SA functional
analogs such as BTH and 4-chloro-SA activate the expression of MPK3 gene and
eventually result in priming and SAR (Beckers et al. 2009).
4  Emerging Trends in Physiological and Biochemical Responses of Salicylic Acid 65

SA arbitrates modification in the expression patterns of about 1000–2000 genes.

For such an extensive effect on genes transcriptional behavior, it might be associ-
ated with certain chromatin-remodeling complexes (March-Diaz et al. 2008). The
process of chromatin remodeling might require histone replacement, i.e., substitu-
tion of canonical histones in an octamer by some histone variants (Kamakka and
Biggins 2005). H2A-Z is one such variant which is linked to transcriptional activa-
tion as well as repression. By the action of multi-subunit complex such as SWR1
isolated from yeast, the histone H2A-Z is incorporated into the chromatin.
Arabidopsis PIE 1 (PHOTOPERIOD-INDEPENDENT EARLY FLOWERING), a
homologous SWR1 protein which interacts with histone variant H2A-Z at the N
terminus of PIE 1, suggests their role in priming process of SAR (March-Diaz et al.
2008). They demonstrated that pie1 mutant had misregulation of genes and also the
wild type treated with SA analogs such as BTH had misregulation of genes which
induced SAR.  The results suggested that chromatin remodeling might be instru-
mental for priming of several SA-sensitive loci to enhance SAR responses (van den
Burg and Takken 2009).

4.4 Conclusion

The studies have indicated that SA as endogenous plant growth regulator has imper-
ative roles in regulation of important biochemical pathways involved in physiologi-
cal processes. It has also been established that exogenous applications of SA to
healthy as well as stressed plants have promotive effects on several metabolic path-
ways. A wide array of studies pertaining to SA as immune signaling molecule has
also improved our understanding regarding its interplay with several other tran-
scriptional factors, proteins, and genes in conferring resistance against pathogens
and abiotic stresses. It has the potential to reprogram transcription through WRKY
transcription factors and activating the defense genes. The studies have established
NPR1 as a master regulator of defense genes through SA. The conversion of NPR1
to monomers, translocation, and prevention of degradation under enhanced concen-
trations of SA has proven its crucial interactions leading to activation of PR genes.
SA has a significant role in activation of certain genes which aid in providing
defense against viral infection through RNA silencing. The function of SA in SAR
is one of the most important mechanisms in plant defense as SA not only acts as
mobile signal itself but also interacts with other mobile signals as well as mediators
to induce resistance. SA is also involved in activation of SA-responsive loci through
chromatin remodeling in response to pathogen attack. Therefore, SA has been
proven to be a multifaceted growth regulator aiding in overall fortification of mor-
phological, physiological, and protection mechanisms.
66 N. Handa et al.

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Wheat Germ Agglutinin and Dehydrins
as ABA-Regulated Components 5
of SA-Induced Cadmium Resistance
in Wheat Plants

F.M. Shakirova, M.V. Bezrukova, Ch.R. Allagulova,

D.R. Maslennikova, and A.R. Lubyanova

Abstract
With the use of inhibitor of ABA synthesis fluridone, we have obtained experi-
mental evidences in favor of the key role of rapid reversible accumulation of
ABA during treatment with salicylic acid (SA) and maintenance of elevated level
of ABA in SA-pretreated seedlings subjected to abiotic stress factors in the
implementation of preadaptive and protective action of SA on wheat plants,
respectively, which was evident from their growth indicators under normal and
stressful conditions. An important contribution to the resistance of wheat to
dehydration contributes RAB (responsive to ABA) proteins, wheat germ aggluti-
nin (WGA), and dehydrins, a massive synthesis of which is observed during the
formation of kernels of wheat. However, these proteins are typical for vegetative
plants, and under the influence of unfavorable factors causing dehydration, there
is a significant increase in their levels, preceded by stress-induced accumulation
of ABA. To date, a lot of information is accumulated, indicating the effectiveness
of application of SA for reduction of the toxic effects of cadmium on different
crops. We obtained novel data supporting an important role of endogenous ABA
in the regulation of SA-induced resistance of wheat plants to cadmium stress and
involvement of WGA and dehydrins in this process.

Keywords
Salicylic acid • Abscisic acid • Wheat germ agglutinin • Dehydrins • Cadmium
stress • Triticum aestivum

F.M. Shakirova (*) • M.V. Bezrukova • C.R. Allagulova • D.R. Maslennikova

A.R. Lubyanova
Institute of Biochemistry and Genetics, Ufa Scientific Centre, Russian Academy of Sciences,
pr. Octyabrya, 71, 450054 Ufa, Russia
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2017 77

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_5
78 F.M. Shakirova et al.

5.1 Introduction

Due to the progressive technogenic contamination of soils with salts of toxic heavy
metal (HM), causing not only a decrease in yield but also a deterioration of its qual-
ity (Gallego et al. 2012; Chmielowska-Bąk et al. 2014; Liu et al. 2016; Rizwan et al.
2016), the investigation of the molecular mechanisms of plant resistance to HM
becomes a serious problem. Cadmium can be attributed to the most toxic of HM,
because it is a nonessential chemical element for the normal functioning of plants
and does not perform any physiological functions in plants (DalCorso et al. 2008;
Chmielowska-Bąk et al. 2014; Liu et al. 2016). Plant response to cadmium is char-
acterized by accumulation of ABA, stomatal closure, reduced water uptake and
flow, inhibition of chlorophyll synthesis and photosynthesis, shifts in the state of
pro-antioxidant cell systems, and disturbance of membrane structure integrity
resulting from generation of reactive oxygen species (ROS), which in total is
reflected in growth and development retardation and decline in plant productivity
(Hsu and Kao 2003; Gallego et al. 2012; Shakirova et al. 2013a, b; Chmielowska-­
Bąk et  al. 2014; Khan et  al. 2015; Vishwakarma et  al. 2017). Since exposure to
cadmium causes a disturbance of water relations, it is not surprising that under these
conditions, there is an activation of the transcription of genes sensitive to ABA
(Fusco et al. 2005; Vishwakarma et al. 2017), playing, as far as is known, a key role
in the regulation of plant defense responses during dehydration. ABA is of pivotal
importance for production of many ABA-responsive components of plant protection
(Kosová et al. 2014; Fernando and Schroeder 2016).
By now, a broad range of proteins is detected involved in plant responses to cad-
mium including those linked with antioxidant defense, toxic ions chelating and their
detoxification (Roth et al. 2006; Ahsan et al. 2009; Wang et al. 2011), as well as
proteins implicated in ABA and salicylic acid (SA) signaling (Аhsan et al. 2009;
Kang et al. 2012), indicating involvement of these stress hormones in the control of
plant resistance to cadmium. In fact, cadmium-induced accumulation of ABA (Hsu
and Kao 2003) and SA (Metwally et al. 2003) has been shown to play important role
in protection of plants against its toxic effect, while plant treatment with ABA
(Meng et al. 2009) and SA (Hayat et al. 2010; Shakirova et al. 2013a, b; Khan et al.
2015; Liu et al. 2016) was effective in diminishing the level of growth-inhibiting
action of cadmium on plants.
We have shown previously that treatment of wheat seedling with SA under nor-
mal conditions results in a fast 1.5-fold reversible increase in ABA, which, however,
does not prevent growth promotion by SA, likely due to simultaneous reversible
accumulation of indole-3-acetic acid (IAA) without visible changes in cytokinin
concentration, while SA pretreatment prevented salt-stress-induced drop of auxins
and cytokinins and maintained elevated level of ABA in seedlings thereby maintain-
ing their growth at the level close to the control (Shakirova et al. 2003a). These data
indicate a less damaging effect of stress on the SA-pretreated seedlings and the
important role of endogenous ABA in the regulation of preadaptive and protective
action of SA on wheat plants under stress (Shakirova 2007).
5  Wheat Germ Agglutinin and Dehydrins as ABA-Regulated Components… 79

The genes coding for wheat germ agglutinin (WGA) belongs to ABA-responsive
genes (Cammue et al. 1989; Skriver Mundy 1990; Shakirova and Bezrukova 2007;
Shakirova et al. 2013a, b). WGA, being a typical representative of cereal lectins and
having an affinity for N-acetyl-D-glucosamine and chitin oligomers, is a constitutive
protein, which content in plant tissue changes significantly during ontogenesis
(Raikhel et al. 1986; Raikhel and Lee 1993; Shakirova and Bezrukova 2007; De Hoff
et al. 2009; Jiang et al. 2010). Thus, significant reversible increase in WGA content
preceded by an increase in АBA level was observed in roots of wheat plants in
response to different abiotic adverse factors including cadmium stress (Cammue
et al. 1989; Singh et al. 2000; Shakirova et al. 1996, 2003b, 2013a, b; Bezrukova
et al. 2008, 2011, 2016), which is accompanied by increased excretion of protein in
the root cap and external medium (Bezrukova et al. 2011; Shakirova et al. 2013b). As
a result this lectin can participate not only in protecting the stress-weakened plants
from soilborne infection but also in maintaining the functional activity of root apical
meristem under abiotic stress conditions (Bezrukova et al. 2008, 2011, 2012, 2016).
ABA is known to be involved in the control of synthesis of water soluble proteins
dehydrins intrinsic to the plant kingdom and joined into group 2 or D 11 of LEA
(late embryogenesis abundant) proteins (Close 1996; Hanin et al. 2011; Allagulova
et al. 2003, 2007, 2015). Dehydrins are present in almost every cell compartment,
while peculiarity of their physicochemical properties, including in dispensable pres-
ence of lysine-rich 15-amino acid motif known as K-segment capable of forming
secondary structure as amphipathic-helix, enables fulfillment by these proteins of
the principally important functions of biopolymers protection from denaturation,
membrane stabilization, and preservation of cell structure integrity under stress con-
ditions (Close 1996; Allagulova et al. 2003; Hara 2010, 2013; Hanin et al. 2011;
Kosová et  al. 2014). Among the dehydration-induced plant proteins, dehydrins
characterized by high degree of thermostability and wide range of molecular mass
(from 9 to 200 kDa) are most abundant (Close 1996; Kosová et al. 2014; Zolotarov
Strömvik 2015). ABA sensitivity of dehydrin coding genes is determined by the
presence in their promoters of ABRE (ABA-responsive)-cis-elements containing
conservative ACGT sequence capable of binding ABA-activated transcription fac-
tors (TFs) from bZIP family (basic leucine zipper) AREB/ABFs (ABA-responsive
element-binding/ABA-binding factors) (Allagulova et al. 2003; Hanin et al. 2011;
Kosová et al. 2014; Zolotarov and Strömvik 2015). In promoter regions of the genes
for dehydrins, there were also other cis-regulated elements, such as, e.g., DRE/CRT
(dehydration responsive elements/C repeats), determining their sensitivity to
drought, strong salinity, and cold but not to ABA and being activated by DREB1/
CBF and DREB2 TFs (Shakirova et al. 2009; Hanin et al. 2011; Kosová et al. 2014;
Zhu et al. 2014; Wang et al. 2014; Zolotarov and Strömvik 2015).
There is evidence that the SK3-type wheat dehydrin wzy1-2 can be induced not
only by osmotic stress, cold, and ABA but also by SA, methyl jasmonate (MeJA),
and gibberellic acid (GA), due to the presence in the promoter of this gene of cis-­
acting regulatory elements, including ABA-, dehydration-, low temperature-, SA-,
MeJA-, GA-responsive elements (Zhu et al. 2014). Furthermore, 24-epibrassinolide
has been capable of activating the TADHN gene transcription independently of
80 F.M. Shakirova et al.

ABA, since seedling treatment with certain concentration of this hormone did not
change the content of endogenous ABA (Allagulova et al. 2007). Thus expression
of the genes for dehydrins may be regulated by ABA-dependent and ABA-­
independent signaling pathways (Allagulova et  al. 2003, 2007, 2015; Shakirova
et al. 2009; Hanin et al. 2011; Zhu et al. 2014). There are literature data showing
activation of synthesis and accumulation of dehydrins in plants treated with HM and
participation of these proteins in binding and neutralization of ROS, generated in
HM-treated plants, as well as in detoxification of toxic ions (Zhang et al. 2006; Xu
et al. 2008; Hara 2010; Hara et al. 2013), which serves as an evidence in favor of
important role of dehydrins in development of plant resistance to HM. It has been
shown that expression of PvSR3 gene encoding SKn-type dehydrin was strongly
stimulated by HM in leaves of bean seedlings (Zhang et al. 2006). Cadmium has
been shown to upregulate expression of dehydrin genes DHN1 and DHN6 in root
tips of barley (Tamás et al. 2010), as well as TADHN gene of dehydrin in wheat
seedlings (Shakirova et al. 2013a, b).
Based on the data obtained by us earlier showing the important role of SA-induced
reversible accumulation of ABA in the development of SA preadaptive effect on
plants and the importance of maintaining increased content of ABA in SA-pretreated
plants in development of resistance to abiotic stresses, one would expect participa-
tion of WGA and dehydrins in the implementation of protective effect of SA on
wheat plants exposed to cadmium ions (Shakirova et al. 2013a, b; 2016). The pur-
pose of this study was to identify the role of WGA and dehydrins in the manifesta-
tion of SA-induced resistance of wheat seedlings to cadmium and significance of
endogenous ABA in the regulation of the quantitative level of WGA and dehydrins
in SA-pretreated plants under stress conditions.

5.2  ignificance of Endogenous ABA in SA-Induced

S
Resistance of Wheat Plants to Cadmium Stress

Growth inhibition is one of characteristic plant responses to cadmium stress (DalCorso

et al. 2008; Gallego et al. 2012; Liu et al. 2016; Bezrukova et al. 2016). By now a lot
of evidences have accumulated showing effectiveness of application of salicylic acid
for amelioration of cadmium toxicity in different plant species (Metwally et al. 2003;
Meng et al. 2009; Hayat et al. 2010; Zhang et al. 2015; Belkadhi et al. 2016; Liu et al.
2016). Our data demonstrate significant growth inhibition of wheat seedlings best
revealed by the analysis of fresh and dry weight 8 h after exposure to cadmium, and
even 1 day after withdrawal of cadmium from the incubation medium, the growth of
these plants remained on the same low level (Fig. 5.1).
Data in Fig. 5.1 demonstrate that treatment of plants with SA during 24 h resulted
in evident growth-promoting effect on wheat seedlings, and although pretreatment
with SA did not prevent negative effects of stress on wheat seedlings, it helped to
maintain the intensity of the growth processes in these plants, at least at the level of
control plants. Furthermore, pretreatment with SA contributed to accelerated growth
restoration in seedlings during post-stress period (Fig. 5.1).
5  Wheat Germ Agglutinin and Dehydrins as ABA-Regulated Components… 81

100
(A)
Control (SA) Cd (SA)+Cd
80
Seedling fresh weight (mg)

60

40

20

0
72 80 88 96 104 112 120
10
(B)

8
Seedling dry weight (mg)

0
0 72 80 88 2496 32
104 112 48
120
Time of treatment, h

Fig. 5.1  Dynamics of fresh (a) and dry (b) mass of 3–5-day-old wheat seedlings pretreated and
untreated with 50 μM SA 8 h after exposure to 1 mM cadmium acetate and during the post-stress
recovery. 3-day-old seedlings were incubated for 24 h on the medium containing 2% sucrose in
either presence or absence of SA, and then they were placed for 8 h on the mixture of 2% sucrose
and cadmium acetate followed by transfer to 2% sucrose. Means and their standard errors are
presented (n = 3)

Plant roots are the main organs that absorb metal ions from the environment and
are also the first to show direct symptoms of heavy metal toxicity (Amirthalingam
et al. 2013; Shi et al. 2014; Bezrukova et al. 2016). Table 5.1 shows that exposure to
1 mM cadmium acetate has a very pronounced inhibitory effect on mitotic activity
of cells of root tips. Pretreatment with SA does not prevent but significantly reduces
the extent of the negative effects of stress on mitotic activity of cells of root tips and
helps to maintain this indicator in plants, at least at the level of control plants
untreated with SA (Table 5.1).
These data allow to suggest that revealed protective effect of SA pretreatment on
the investigated growth parameters is likely to be due to decreased uptake of cad-
mium by plants, which was detected in SA-pretreated different plant species (Zhang
82 F.M. Shakirova et al.

Table 5.1  Effect of pretreatment with 50 μM salicylic acid for 24 h on the mitotic index (%) of
root meristem cells of 4-day-old wheat plants before influence of 1 mM cadmium acetate, under
1 mM cadmium acetate for 8 h and 24 h after withdrawal of cadmium acetate
Variant Before influence of Cd 1 mM Cd 24 h after withdrawal of 1 mM Cd
Control 5.20 ± 0.10 3.60 ± 0.07 3.54 ± 0.06
(SA) 6.88 ± 0.09 5.33 ± 0.11 5.76 ± 0.08
Means and their standard errors are presented (n = 3)

Table 5.2  Cadmium concentration in the roots and shoots of 5-day-old wheat seedlings untreated
and pretreated during 1 day with 50 μM SA and exposed to 1 mM cadmium acetate for 24 h
Variant Roots Shoots
Seedlings untreated with SA 171.5 ± 8.5 21.2 ± 1.5
Seedlings pretreated with SA 105.3 ± 10.0   8.2 ± 1.0
Means and their standard errors are presented (n = 3)

et al. 2015; Belkadhi et al. 2016; Liu et al. 2016; Gondor et al. 2016). The obtained
data are in accordance with the results of analysis of cadmium concentration in
SA-treated and untreated plants.
Comparative analysis of cadmium content in wheat seedlings untreated and pre-
treated with SA for 24  h showed that maximal accumulation of toxic ions was
detected in roots, while in shoots the content of cadmium was about an order of
magnitude lower (Table  5.2), which is in accordance with the data showing that
roots are the site of preferential accumulation of cadmium (Bezrukova et al. 2011;
Gondor et al. 2016). However, roots and especially shoots of SA-pretreated plants
were characterized by a lower level of cadmium accumulation (Table 5.2), which, in
general, was reflected in maintenance of growth characteristics on the level close to
the control (Fig. 5.1).
Increased synthesis and accumulation of ABA having frequently a transitory pat-
tern may be characterized as the universal plant response to stressful impacts lead-
ing to disturbance of water relations (Xiong and Zhu 2003). ABA is known to play
a key role in regulation of stomata closure, resulting in a decline in transpiration and
reduction of transpiration losses (Dodd and Ryan 2016). It is of interest that in the
series of components of plant protection controlled by exogenous and endogenous
ABA, there are those involved in the range of protective action of SA (Rajjou et al.
2006; Shakirova 2007; Hayat et al. 2010; Khan et al. 2015). These data indicate in
favor of possible implication of endogenous ABA as a hormonal intermediate in the
regulation of realization of preadaptive and protective action of SA on plants
(Shakirova et al. 2013a).
Results obtained for rice cultivars contrasting in their Cd-tolerance demonstrated
important role of Cd-induced accumulation of ABA in the control of plant resis-
tance to this stress (Hsu and Kao 2003). Thus, accumulation of ABA and the decline
in cadmium concentration in roots and shoots was detected only in resistant to cad-
mium rice cultivar, while the treatment with the inhibitor of ABA synthesis fluri-
done resulted in the loss of resistance. On the contrary, plants of the cultivar,
5  Wheat Germ Agglutinin and Dehydrins as ABA-Regulated Components… 83

sensitive to cadmium, acquired resistance due to ABA treatment manifested in the

decline in cadmium accumulation in the plants. These results suggested protective
action of endogenous ABA, associated with its effect on stomatal closure and con-
sequent inhibition of cadmium flow into plants with transpiration stream (Hsu and
Kao 2003). However, our data show that wheat seedlings pretreated with SA were
characterized with significantly lower level of Cd-induced accumulation of ABA as
compared to plants untreated with SA (Fig.  5.2), while level of cadmium in
SA-pretreated plants was significantly lower than in plants untreated with this hor-
mone (Table 5.2).
We have previously suggested an important role of endogenous ABA in the man-
ifestation of the protective action of SA on wheat plants (Shakirova et al. 2003a;
Shakirova 2007; Shakirova et al. 2013a, b). In order to assess this role of endoge-
nous ABA, we carried out experiments with the pretreatment of plants with ABA
biosynthesis inhibitor fluridone (Fl), which is effective in preventing stress-induced
accumulation of ABA (Cammue et al. 1989; Hsu and Kao 2003; Bezrukova et al.
2011; Shakirova et al. 2013a, b; Stroiński et al. 2013). As can be seen from Fig. 5.2a,
pretreatment with Fl prevented SA-induced accumulation of ABA in roots of wheat
seedlings. Consequently, ABA accumulated in SA-treated seedlings was newborn
indicating participation of SA in the control of de novo ABA synthesis in wheat
plants. In connection with this, it was of interest to estimate the importance of
SA-induced reversible accumulation of ABA in the regulation of protective reac-
tions of wheat seedlings developing in plants in response to SA treatment.
Deposition of lignin in the root cell walls resulting in strengthening of their bar-
rier functions is known to contribute significantly to the development of plant resis-
tance to the HM (Kováčik et al. 2009; Bezrukova et al. 2011). We have established
earlier that SA treatment of seedlings for 24 h contributes to acceleration of lignin
deposition in the cell walls of root xylem vessels as compared to the control, which
was in this case observed on the background of manifestation of growth-stimulating
effect of SA, while under the influence of cadmium stress, additional lignin deposi-
tion was observed in the cell walls of roots of SA-pretreated wheat seedlings
(Shakirova et al. 2013a). This suggests that significant decline in the level of cad-
mium accumulation in SA-pretreated seedlings (Table 5.2) may be due to increased
barrier characteristics of root cell walls in the course of 24 h pretreatment of plants
prior to the stress.
Since SA is capable of activating phenylalanine ammonia-lyase (PAL) (Chen
et al. 2006), which is a key enzyme of lignin synthesis (Jeong et al. 2012; Dehghan
et al. 2014), it was of interest to study the effect of SA on activity of this enzyme in
roots of wheat seedlings in the course of 24 h treatment. SA was shown to cause an
almost twofold increase in PAL activity preceded by fast transient accumulation of
ABA in plants (Fig. 5.2b), which remained comparatively high even 1 day after with-
drawal of SA from the incubation medium. Since ABA also induced transcription of
PAL gene (Jeong et al. 2012), plants were pretreated with Fl to assess importance of
ABA in the control of PAL activity in roots treated with SA. Fl completely prevented
SA-induced increase in PAL activity, and activity of this enzyme was not restored
even after withdrawal of the mixture of Fl with SA from the medium (Fig. 5.2b).
84 F.M. Shakirova et al.

250
(A)

200 Control SA (Flu)+SA

*
ABA (ng g-1 FW)

150 *
*

100

50
0 2 4 6 8

20
(B)
Control SA (Fl)+SA
16
PAL activity (U g-1 FW)

12

0
12 24 48
Time of treatment, h
(C)

Control SA (Fl)+SA

Fig. 5.2  Effect of SA on ABA content (a), PAL activity (b), and the lignin deposition (c) in roots
of pretreated or untreated with fluridone 4–5-day-old wheat seedlings. 3-day-old seedlings were
incubated on the 2% sucrose solution in the presence or absence of 5 mg/L fluridone during 24 h,
then seedlings were transferred on the mixture of 2% sucrose with 50 μM SA in the presence or
absence of fluridone for 24 h. Lignin deposition was determined in the cell walls of the basal part
of the roots of 5-day-old seedlings. Values are means ± SE (n = 3)
5  Wheat Germ Agglutinin and Dehydrins as ABA-Regulated Components… 85

Fig. 5.3  Effects of 1 mM 350

cadmium acetate on the Control Cd
content of ABA in roots of 300 (SA)+Cd (Fl+SA)+Cd
4-day-old wheat seedlings
untreated or pretreated 250

ABA (ng g-1 FW)

with 50 μM SA during
24 h. Values are means ± 200
SE (n = 3)
150

100

50

0
0 1 2 3 4 5 6 7 8
Time of Cd treatment , h

The obtained data serve as evidence of important role of SA-induced accumula-

tion of ABA in the control of PAL activity in roots, and consequently, inhibition of
cadmium uptake by plants is likely to be due to the strengthening of the barrier
properties of cell walls in the course of SA pretreatment brought about by activation
of PAL and consequent acceleration of lignin deposition in root cell walls (Fig. 5.2c).
Our data show that wheat seedlings pretreated with SA were characterized with
significantly lower level of Cd-induced accumulation of ABA as compared to plants
untreated with SA (Fig. 5.3), while level of cadmium in SA-pretreated plants was
significantly lower in plants untreated with this hormone (Table 5.2).

5.3  articipation of WGA in the Protective Action of SA

P
on Wheat Plants Against Cadmium and the Role
of Endogenous ABA in the Regulation of WGA Level

Wheat germ agglutinin makes an evident contribution to the development of wheat

resistance to abiotic stresses, ABA serving as its acknowledged inductor (Raikhel
et al. 1986; Cammue et al. 1989; Raikhel and Lee 1993; Shakirova and Bezrukova
1997, 2007; Bezrukova et al. 2011; Shakirova et al. 2013a, b). In connection with
this, it was of interest to study importance of SA-induced accumulation of ABA
under cadmium stress for the regulation of the quantitative level of WGA in roots of
wheat seedlings. In order to assess the role of endogenous ABA in the regulation of
the protective action of SA on wheat plant, we carried out experiments with the
pretreatment of plants with ABA biosynthesis inhibitor fluridone, which is effective
in preventing stress-induced accumulation of ABA, when applied at concentration
of 5 mg/L (Bezrukova et al. 2011). Earlier, we found that pretreatment of seedlings
with fluridone in selected concentration completely prevents SA-induced rapid tran-
sient accumulation of ABA in the wheat plants (Shakirova 2007). It was important
86 F.M. Shakirova et al.

to compare the effects of pretreating the plants with SA and a mixture of fluridone
and SA on ABA and WGA content in wheat seedling roots under normal conditions
and under the influence of cadmium stress.
Figure 5.4a shows that a pretreatment of seedlings initially with fluridone and
then with a mixture of fluridone and SA during 24  h completely prevents the
SA-induced accumulation ABA in wheat roots. Since growing root tissues are
known to be one of the main sites of ABA-controlled synthesis of WGA in vegetat-
ing plants (Cammue et al. 1989; Shakirova and Bezrukova 2007), it was of interest
to study importance of SA-induced accumulation of ABA in the regulation of quan-
titative level of WGA in the roots of wheat seedlings. Figure 5.4b shows that pre-
treatment with SA results in reversible increase in WGA content in roots preceded
by SA-induced increase in ABA concentration; moreover, pretreatment with fluri-
done completely prevented SA-induced transitory accumulation of lectin in them.
These data point to the key role of endogenous ABA in the control of SA-induced

400
(A)

300 Control SA (FL)+SA Cd (SA)+Cd (FL+SA)+Cd

ABA (ng g-1 FW)

* *
*
200 * * *
* * *
*
* *

100

0
1 2 3 5 7
2
* (B)
*
1,6 * *
WGA (µg g-1 FW)

* * * *
*
1,2 * *
* * *

0,8

0,4

0
1 2 3 5 7
Time of treatment, h

Fig. 5.4  Effect of 1 mM cadmium acetate on the ABA (a) and WGA (b) content in roots of 4-day-­
old wheat seedlings, pretreated and untreated for 24 h with 50 μM SA in the presence or absence
of 5 mg/L fluridone
5  Wheat Germ Agglutinin and Dehydrins as ABA-Regulated Components… 87

Fig. 5.5  WGA content in 40

the incubation medium of 35 *
pretreated and untreated

WGA (ng mg-1 protein)

30
for 24 h with 50 μM SA in *
the presence or absence of 25 *
5 mg/L fluridone 4-day-old 20
wheat seedlings after 7 h
15
influence of 1 mM
cadmium acetate 10
5
0
Control (SA) Cd (SA)+Cd (FL+SA)
+Cd

accumulation of WGA and involvement of this protein in the preadaptive effect to

possible stresses exerted by SA on wheat plants.
Furthermore it was important to compare the effects of pretreating the plants
with SA and a mixture of SA and fluridone on ABA and WGA content in wheat
seedling roots exposed to cadmium stress. Figure  5.4a shows that incubation of
seedlings in cadmium led to a rapid reversible accumulation of ABA in untreated
with SA roots, which is not surprising, since this response is a typical stress reaction
(Shakirova et al. 2013a; Asgher et al. 2015). SA-pretreated seedlings were charac-
terized by visibly lower level of cadmium-induced accumulation of ABA, suggest-
ing lower extent of injurious effect of cadmium on SA-pretreated plants. At the
same time a pretreatment of seedlings initially with fluridone and then with a mix-
ture of fluridone and SA during 24  h completely prevents of cadmium-induced
accumulation ABA in roots (Fig. 5.4a).
Figure 5.4b shows that the effect of cadmium acetate leads to a transient increase
in the level of content of WGA in wheat seedling roots, which is also not surprising,
since the literature contains information on the involvement of this lectin in the
responses of plants not only to drought, salinity, and hypo- and hyperthermia
(Cammue et al. 1989; Shakirova et al. 1993, 1996; Singh et al. 2000; Bezrukova
et al. 2012) but also to HM (Bezrukova et al. 2011, 2016). At the same time, plants
pretreated with SA for 24  h are characterized by lower level of accumulation of
wheat lectin (Fig. 5.4b) indicating a less damaging effect of cadmium on them, in
contrast to SA-untreated plants. Co-pretreatment with fluridone and SA sharply
reduced the stress induced of lectin accumulation in roots (Fig. 5.4b).
The obtained results indicate in favor of the key role of endogenous ABA in the
regulation of this protective component in the plants pretreated with SA and an
important role of maintaining increased concentration of endogenous ABA in
SA-pretreated seedlings in the regulation of the accumulation of WGA, contributing
to the development of plant resistance to cadmium stress.
Since WGA is excreted into the external medium (Bezrukova et al. 2011, 2016;
Shakirova et al. 2013b), it was expected that the stress-induced reversible accumula-
tion of lectin will be accompanied by increasing its release into the medium of
seedling incubation. The data presented in Fig. 5.5 demonstrate that already after
7 h of cadmium incubation, WGA content in the medium exceeded that of control
88 F.M. Shakirova et al.

Table 5.3  The effect of 1 mM cadmium acetate in the presence or absence of 1 mg/l WGA or
1 mg/l gliadin for 7 h on mitotic index in the root meristem cells of 4-day-old wheat seedlings
pretreated with 50 μM SA or 1 mg/l WGA in the presence and absence of fluridone
(SA) (Fl+SA) (Fl+SA) + (Fl+SA) + (Fl+WGA)
Variant Control + Cd + Cd Cd+WGA Cd+gliadin (WGA)+Cd + Cd
MI 5.2 ± 5.3 ± 3.7 ± 5.1 ± 0.11a 3.6 ± 0.09b 5.2 ± 0.12a 3.6 ± 0.11b
After 7 h 0.13a 0.10a 0.07b
exposure
to 1 mM
cadmium
acetate
Mean data of three independent replicates and their SEs are presented
Means followed by different letters are significantly different at P < 0.05 (ANOVA, LSD test)

by twofold. In SA-pretreated plants, the level of the WGA content was already sig-
nificantly lower, reflected in about 50 % decline in lectin content in the medium
(Fig. 5.5). In the variant of SA pretreatment in a mixture with Fl, the WGA content
remained on the level close to that of the control due to prevention by fluridone of
cadmium-induced accumulation of ABA (Fig. 5.5).
Similar pattern was observed when lectin level in incubation media of seedlings
was analyzed. Consequently, activation of reversible accumulation of lectin in the
roots of SA-pretreated seedlings exposed to cadmium, as well as excretion of WGA
into the external media, was caused by maintenance of increased concentration of
ABA in these plants.
It was discovered that WGA participates in stimulation of division in apical root
meristem under normal conditions (Shakirova et al. 2004), in prevention of stress-­
induced inhibition of cell division in the root tips as well as in acceleration of recov-
ery of root cell growth by division during the post-stress period (Bezrukova et al.
2008; Shakirova et al. 2013a). To estimate the extent of involvement of WGA in
protective action of SA on plant growth processes under cadmium stress, we carried
our analysis of MI in roots of SA-pretreated and untreated seedlings in the presence
or absence of WGA exposed to cadmium action.
The data presented in Table  5.3 show pronounced growth-inhibiting effect of
cadmium acetate on cell division in root apical meristem already 7 h after the start
of the treatment, while SA-pretreated plants were characterized by considerably
smaller level of detrimental effect of cadmium on cell growth and maintenance of
root MI on the control level under these conditions. The defensive effect of SA on
this indicator was not detected in the presence of Fl (Table  5.3), pointing to the
important role of endogenous ABA in protective action of SA on wheat plants under
cadmium stress.
Our earlier studies revealed that plant treatment with WGA contributes to the
decline in detrimental effect of NaCl salinity on cell division in root apical meristem
(Bezrukova et al. 2008). This suggested that prevention of protective action of SA
on cell growth in the root tips exposed to cadmium stress under combined pretreat-
ment with Fl is due to inhibition of SA-induced enhancement of the accumulation
of WGA in roots and its appearance in the external medium (Fig. 5.5).
5  Wheat Germ Agglutinin and Dehydrins as ABA-Regulated Components… 89

To test this hypothesis, we performed combined addition of cadmium acetate

with WGA into incubation medium of plants pretreated with a mixture of SA and
Fl. Table 5.3 shows that joint treatment of these seedlings with cadmium and WGA
contributed to prevention of growth-inhibiting action of cadmium and to mainte-
nance of root MI at the level close to that of the control or SA-pretreated plants
exposed to cadmium. The observed effect is a result of the treatment with exactly
WGA, since a wheat storage protein gliadin had no protective effect on division of
root cells in plants pretreated with a mixture of SA and Fl either under stress
(Table 5.3).
As seen in Fig. 5.6, the exposure to 1 mM cadmium acetate for 8 h resulted in
significant increase in MDA accumulation in the roots of 4-day-old wheat seed-
lings. At the same time, under stress SA-pretreated wheat seedlings were character-
ized by reduced levels of this indicator in comparison with SA-untreated seedlings.
Pretreatment with Fl completely prevented SA-induced protective effect on wheat
plants, and level of lipid peroxidation was consistent with those exposed to cad-
mium and untreated with SA. It is necessary to note that, by itself, SA pretreatment
resulted in slight increase in lipid peroxidation of plants in comparison with the
control (Fig.  5.6), which, however, did not prevent manifestation of the growth-­
stimulating action of SA.
In total, the data obtained point to the key role of endogenous ABA in the control
of cadmium-induced activation of reversible accumulation of WGA in roots of
seedlings pretreated with SA and its excretion into external medium and important
contribution of WGA in protective action of SA in wheat seedlings under cadmium
stress.

125

100 * * *
MDA (nmol g-1 FW)

* *
75

50

25

0
Control (SA) (WGA) Cd (SA)+Cd (WGA)+Cd (Fl+SA) (Fl+WGA)
+Cd +Cd

Fig. 5.6  Malondialdehyde content in the roots of 4-day-old wheat seedlings untreated and pre-
treated for a day with 50 μM SA or 1 mg/l WGA in either presence or absence of 5 mg/l fluridone
8 h after exposure to 1 mM cadmium acetate
90 F.M. Shakirova et al.

5.4  he Role of Endogenous ABA in SA-Induced

T
Accumulation of Dehydrins in Wheat Plants in Response
to Cadmium Stress

The key role in protecting plants against stress factors causing disturbance of water
relations is ascribed to proteins dehydrins (Close 1996; Allagulova et  al. 2003;
Hanin et al. 2011; Kosová et al. 2014; Graether and Boddington 2014). By now a lot
of evidences have been obtained in support of involvement of dehydrins in plant
response to HM (Hara 2010; Hara et al. 2013; Wang et al. 2014; Allagulova et al.
2015). The data indicate important role of dehydrins in plant defense against nega-
tive effect of HM. This was convincingly demonstrated on transgenic tobacco plants
over-expressing BjDHN2/BjDHN3 dehydrin genes of hyper-accumulator Brassica
juncea (Xu et al. 2008). These plants were characterized by increased resistance to
ions of cadmium and zinc as shown by decreased level of electrolyte leakage and
production of MDA on the background of accumulation of metals in roots and
leaves as compared with the control. Furthermore, BjDHN2/BjDHN3 transgenic
plants were characterized by increased capacity to restore growth during post-stress
period (Xu et al. 2008).
It has been shown that expression of PvSR3 gene encoding SKn-type dehydrin
was strongly stimulated by HM in leaves of bean seedlings (Zhang et al. 2006). It
was revealed that dehydrins implicate in neutralization of ROS generated by HM, in
binding of HM and detoxification of toxic ions (Hara 2010; Hara et  al. 2013).
Cadmium has been shown to upregulate expression of dehydrin genes DHN1 and
DHN6 in root tips of barley (Tamas et al. 2010), as well as TADHN gene of dehydrin
in wheat seedlings (Shakirova et al. 2013a).
A large number of dehydrin genes are identified in wheat, which encode seven
types of dehydrins (KS, SK3, YSK2,Y2SK2, Kn, Y2SK3, YSK3) and separate in 32
homologous clusters (Wang et al. 2014). The gene amplification differed among the
dehydrin types, and members of the YSK2- and Kn-type DHNs are more numerous
in wheat than in other cereals. The relative expression of all of these DHN clusters
was analyzed using an in silico method in seven tissue types (i.e., normal growing
shoots, roots, and reproductive tissues; developing and germinating seeds; drought-
and cold-stressed shoots) as well as semiquantitative reverse transcription poly-
merase chain reaction in seedling leaves and roots treated by dehydration, cold, and
salt, respectively. Transcripts of certain types of DHNs accumulated specifically
according to tissue type and treatment, which suggests their differentiated roles in
wheat abiotic stress tolerance (Wang et al. 2014).
We discovered that 24  h exposure of wheat seedlings to cadmium resulted in
significant changes in the content of dehydrins differing in responsiveness to this
stress (Fig. 5.7). Thus, cadmium-induced sharp accumulation of 22 kDa dehydrin
and especially 28 kDa in plants and less than twofold increases in the level of dehy-
drins with molecular mass of 55 и 69 kDa. Furthermore, plants pretreated with SA
were characterized by significantly lower level of cadmium-induced accumulation
of all of the four dehydrins (Fig. 5.7), which is quite expected taking into account
that these plants accumulated less cadmium (Table 5.2).
5  Wheat Germ Agglutinin and Dehydrins as ABA-Regulated Components… 91

((A))
[kDa] [kDa]

120

85
69

55
50

35
28

25
22

1 2 3 4 5
Treatment

Fig. 5.7 Immunoblot analysis of dehydrin expression in 5-day-old wheat seedlings. (a)

Representative Western blot. (b) Densitometric analysis of 22 kDa, 28 kDa, 55 kDa, and 69 kDa
dehydrin bands expressed as a percentage of values for corresponding control. 1 control, 2 50 μM
SA (24 h), 3 1 mМ cadmium acetate (24 h), 4 pretreatment with SA for 1 day and then exposition
on cadmium acetate (24 h), 5 pretreatment with the mixture of 5 mg/L fluridone and SA for 1 day,
and then exposure on cadmium acetate (24 h). Bars represent means ± SE values (n = 3) (Shakirova
et al. 2016)
92 F.M. Shakirova et al.

There are data in the literature showing SA-induced transient accumulation of

transcripts of TADHN dehydrin gene in wheat seedlings (Shakirova et al. 2013a)
and SA-induced enhancement of synthesis of dehydrin proteins in Arabidopsis
seeds during first hours of germination (Rajjou et al. 2006).
Furthermore, Fig.  5.7 shows that 24  h pretreatment of wheat plants with SA
caused 1.5-fold increase in the content of dehydrins with molecular mass of 22 and
28  kDa, showing the greatest sensitivity to cadmium. These results serve as evi-
dence in favor of involvement of dehydrins in defense reactions of plants grown
under normal conditions and as a result are likely to contribute to development of
their resistance to subsequent action of stress factors.
By now, a lot of data are accumulated indicating implication of both ABA-­
dependent and ABA-independent signaling pathways in the control of expression of
dehydrins in response to different stress factors, inducing disturbance of water
regime, which is caused by the presence in the promoters of dehydrin genes of dif-
ferent interacting combinations of cis-regulatory elements capable of binding to
variety of trans-factors (Shakirova et  al. 2009; Hanin et  al. 2011; Graether and
Boddington 2014; Kosova et al. 2014; Wang et al. 2014; Zolotarov Strömvik 2015).
PvSR3 gene of dehydrin from bean is an example of ABA-independent regulation of
transcription, which expression was sharply upregulated by different HM, Cd being
among them, and, furthermore, was upregulated by exogenous SA but was not
responsive to ABA (Zhang et al. 2006). In connection with this, it is of special inter-
est to study importance of increased level of ABA in SA-pretreated wheat seedlings
exposed to the action of cadmium in the control of dehydrin content in them and
contribution of these proteins to realization of protective action of SA.
Data of Fig. 5.7 illustrate complete prevention of Cd-induced accumulation of all
of the four dehydrins in plants pretreated with the mixture of SA and fluridone.
Obtained data convincingly demonstrate the key role of endogenous ABA in the
control of SA-induced changes in the level of dehydrin proteins under stress and in
realization of protective action of SA in general on wheat plants.
This is supported by the data showing significant decline in the extent of detri-
mental effect cadmium on mitotic activity of cells in the root apical meristem of
SA-pretreated seedlings (Table 5.3) and the decline in the level of electrolyte leak-
age and MDA accumulation as compared to plants untreated with SA (Fig. 5.8),
whereas the presence of fluridone completely prevented protective action of SA
pretreatment on stressed wheat plants.
The obtained data indicate involvement of WGA and dehydrins in SA-induced
preadaptation of wheat plants to possible stresses due to SA-induced transient accu-
mulation of ABA in the course of treatment under normal conditions and in realiza-
tion of protective action of SA pretreatment associated with maintenance in them of
increased level of ABA. In total, this is reflected in preventing the negative effect of
cadmium on the growth of SA-pretreated wheat plants and in acceleration of their
growth recovery during post-stress period.
5  Wheat Germ Agglutinin and Dehydrins as ABA-Regulated Components… 93

120
(A)
MDA, nM g-1 FW 100

80

60

40

20

80 Control SA Cd (SA)+Cd (Flu+SA)

+Cd(B)
Electrolyte leakage, µS g-1 FW

60

40

20

0
Control SA Cd (SA) + Cd (FL+SA) +
Cd

Fig. 5.8  Malondialdehyde content (a) and electrolyte leakage (b) in the roots of 4-day-old wheat
seedlings untreated and pretreated for a day with 50  μM SA in either presence or absence of
5 mg/L fluridone 8 h after exposure to 1 mM cadmium acetate

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Salicylic Acid-Mediated Defence
Signalling in Respect to Its Perception, 6
Alteration and Transduction

Kusum Verma and S.B. Agrawal

Abstract
Salicylic acid (SA) is a small phenolic molecule, well known for its remedial
properties in the case of human health. In plants, as a phytohormone, it regulates
many physiological aspects like adventitious root initiation, germination of
seeds, stomatal closure, flowering, senescence and thermogenesis. As a signal-
ling molecule, defence responses mediated via SA against a variety of biotic and
abiotic stresses are already well reported. It can be synthesized from cinnamic
acid via phenylalanine ammonia-lyase into the cytosol while in the chloroplasts
from isochorismic acid by isochorismate synthase; however, differences occur
on the basis of growth conditions, developmental stage and plant species. Since
long research is in progress to study its signal transduction pathways triggered
via plant stress responses, and a major work has been done in exploring SA sig-
nalling with special attention to its receptors identification. The use of several
biochemical methods and high-throughput screen development makes the way
easy to hunt for SA receptors and to explore SA signal perception by identifying
various SA-binding proteins. However, genetic evidences are less that confirms
that these binding proteins are the true receptors of SA. Huge expression data
confirms the connection and coordination between SA and other signalling net-
works, and the components of this convoluted network are universal under biotic
and abiotic stresses. Fascinatingly, NPR1 (non-expressor of pathogenesis-related
genes 1) was anticipated to be a SA receptor in this signalling pathway. Both
NPR1-independent and NPR1-dependent signalling pathways have been
reported, but very less is known about the intermediates of this NPR1-independent
signalling pathway. Lots of work is going on to explore SA-overexpressing
mutants for studying the downstream targets and regulation of SA biosynthesis.

K. Verma • S.B. Agrawal (*)

Laboratory of Air Pollution and Global Climate Change, Center for Advanced Studies,
Department of Botany, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2017 97

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_6
98 K. Verma and S.B. Agrawal

Here we focused on the SA level regulation mechanism with special attention on

the perception and transduction of a signal from SA-induced defence response.
Studying SA-mediated plant defence will facilitate the advancement of disease-­
resistant crops involving genetic manipulation of the SA signalling pathway
components in a positive direction.

Keywords
Salicylic acid • Biotic stress • Abiotic stress • Signalling • Perception • NPR1

6.1 Introduction

In plants, salicylic acid (SA; o-hydroxybenzoic acid) is a very important phytohor-

mone of phenolic nature that plays a role as a key signalling molecule during plant
defence (Vlot et al. 2009; Robert-Seilaniantz et al. 2011; Pieterse et al. 2012; Janda
and Ruelland 2014). It imparts an essential function in the regulation of biochemical
as well as physiological processes like cell growth, respiration, stomatal aperture,
senescence, seed germination, seedling development and thermotolerance over
whole life period of the plant (Boatwright and Pajerowska-Mukhtar 2013; Vlot
et al. 2009). Biotic and abiotic stresses in plants also involve the role of this signal-
ling molecule. The first mention of SA having its role in plant defence was in 1979
(White 1979). An extensive study has been made to explore the participation of SA
as a signal iota in systemic acquired resistance (SAR) and in local defences (Shah
and Klessig 1999; Dempsey et al. 1999). As a result of local infection caused due to
a wide range of pathogens, a sharp state of defence mediated via salicylic acid
occurred throughout a plant known as SAR.  Methyl salicylate esterase, methyl
salicylate (MeSA), a lipid-derived molecule (glycerol-3-phosphate)-dependent fac-
tor, a lipid transfer protein (DIR1), dehydroabietinal, azelaic acid and pipecolic acid
are the molecules involved in SAR as systemic mobile signals. Several multiprotein
complexes serve as mediators known to prime SA-mediated SAR functioning as
transcriptional coactivators. Quiescent mitogen-activated protein kinases (MAPKs)
are the important candidates required for priming; however, pipecolic acid is known
to be an endogenous mediator of this defence mechanism. SA accumulation in
plants in response to microbial attack induces expression of pathogenesis-related
(PR) proteins, ultimately allowing the plant to respond in a resistant manner
(Malamy et al. 1990; Métraux et al. 1990). Furthermore, level of SA accumulation
in plants is directly correlated with the pathogen resistance where less SA accumu-
lation is responsible for susceptibility and high accumulation makes a plant resis-
tant. Gaffney et  al. (1993) and Delaney et  al. (1994) reported that plants with
reduced SA levels due to ectopic expression of a bacterial SA-hydroxylase gene
(NahG) were highly sensitive to pathogen attack as they were inefficient to trigger
defence responses.
Moreover, in case of abiotic stresses such as heat, chilling, heavy metals, osmotic
stress, drought and salinity, the SA response has also been studied (Horvath et al.
6  Salicylic Acid-Mediated Defence Signalling in Respect to Its Perception, Alteration… 99

2007; Khan et al. 2010; Vicente and Plasencia 2011). During these abiotic stresses,
to reveal the SA function, either the SA-deficient transgenic plants expressing NahG
that degrades SA to catechol or mutant plants defective in perception of SA or pro-
duction of SA were used by several authors. Some of these mutants, for example,
cep (constitutive expression of PR-1 gene), express PR genes and accumulate ele-
vated levels of SA. As these mutants were high in SA levels, the related genes most
likely encode components that play role upstream to SA or may encode for a target
of a different pathway that later invade the SA pathway (Klessig et al. 2000). By
contrast, the mutant eds5 (enhanced disease susceptibility 5) has a mutation
upstream to SA and, similar to the sid2 (SA induction deficient 2) mutant, accumu-
lates low SA and thus shows enhanced susceptibility to pathogen attack (Rogers and
Ausubel 1997). In this line, the most studied npr1 mutant (non-expressor of PR
genes), which lose its function to express PR genes, does not increase the resistance
even after treatment with SA (Cao et al. 1994).
There are two distinct pathways for the synthesis of SA that utilize different
precursors. In the cytoplasm, phenylpropanoid pathway takes place through phe-
nylalanine, and in the chloroplast, SA is synthesized via the isochorismate path-
way (Fig. 6.1). Further, SA synthesized in plants undergoes glycosylation and/or
methylation, where cytosolic SA glucosyltransferases catalyze the glucose conju-
gation reactions (Lee and Raskin 1999; Song 2006) and SA carboxyl methyltrans-
ferase convert SA to MeSA, and this MeSA is a volatile derivate which functions
as long-­distance signalling molecule in Arabidopsis and tobacco SAR as men-
tioned above (Shulaev et al. 1997; Chen et al. 2003; Park et al. 2007; Vlot et al.
2008). A study conducted by Chen et al. (1993) reports that SA specifically inhib-
its the activity of catalase in vitro and enhanced H2O2 concentration in vivo that
leads to induced expression of defence-related genes associated with SAR. Thus,
the high levels of H2O2 acting as a signalling molecule mediate the action of SA
in SAR (Chen et al. 1993). Since constitutive accumulation of SA is often linked
with reduced plant growth and fitness (Chandran et al. 2014; Pajerowska-Mukhtar
et al. 2012), biosynthesis of SA and signalling mediated via SA are strictly con-
trolled. However, SA basal levels differ widely among species in a range up to
100-fold (Raskin et al. 1990).
Major study on this hormone is related to exploring its function in the systemic
and local responses against microbial pathogens and on defining the transduction
pathways leading to SA-induced gene expression. Jumali et al. (2011) showed that
most genes responding to acute SA treatment are related to stress and signalling
pathways which eventually led to cell death. Moreover, other phytohormone signal-
ling pathways mediated via ethylene, abscisic acid and jasmonic acid are well inter-
connected with SA signalling pathway (Pieterse et al. 2012; Derksen et al. 2013).
Finding SA targets and the understanding of its molecular function in plant pro-
cesses will help in exploring complex SA signalling, ensuring its importance in
plant defence mechanisms. In this chapter, we summarize the recent advancements
made in the SA research and focused mainly on SA perception and signalling mech-
anisms during stress.
100 K. Verma and S.B. Agrawal

Fig. 6.1  Mechanisms, SA biosynthesis, metabolism, homeostasis, transport and signalling during
biotic and abiotic stresses. Enzymes involved in SA production and metabolism are shown in red
fonts. BA2H benzoic acid-2-hydroxylase, ICS1 isochorismate synthase 1, IPL isochorismate
pyruvate-­lyase, PAL phenylalanine ammonia-lyase, SABP2 SA-binding protein 2, SAGT SA gly-
cosyltransferase, SAMT1 SA carboxyl methyltransferase. Concentrations shown are Km values of
SAMT1 (15µM) and SAGT (20µM). Dashed lines with arrows indicate transport across the mem-
branes. Blue text denotes mode of transport. Question marks denote unidentified mechanisms. ABC
denotes ATP-binding cassette transport protein. SA surrounded by pink colour indicates free
SA. SA conjugated forms are MeSA methyl salicylate, MeSAG methyl SA O-ß-glucose, SAG SA
O-ß-glucoside, SGE salicyloyl glucose ester. SA-binding receptors are depicted in boxes. NPR1/2/3
= non-expressor of PR (pathogenesis-related) proteins 1/2/3. ‘Oligomeric’ means the oxidized
form of NPR1, whereas ‘monomeric’ denotes reduced form of NPR1. Dotted line connecting
NPR1  in the nucleus and ICS1 depicts the inhibition of ICS1 following activation of defence
response SA biosynthesis and metabolism. A simplified scheme showing the two SA biosynthetic
pathways. Question marks highlight steps where direct evidence is still missing. The three dashed
lines leading from cinnamic acid to SA indicate that three proposed pathways exist. ICS isochoris-
mate synthase, IPL isochorismate pyruvate-lyase, PAL phenylalanine ammonia-lyase, MeSA
methyl salicylate, SAG salicylic acid 2-O-β-d-glucoside, SGE salicyloyl glucose ester, AA amino
acid (Source: Modified from Dempsey et al. 2011)

6.2  lant Defence Responses Involving SA and Link

P
Between Biotic and Abiotic Stresses

Plant defence is comprised of several overlapping layers that include PAMP-­triggered

immunity (PTI), effector-triggered susceptibility (ETS) and effector-triggered
6  Salicylic Acid-Mediated Defence Signalling in Respect to Its Perception, Alteration… 101

immunity (ETI; Jones and Dangl 2006). Receptors for cognate pattern recognition
identify the microbial epitopes or PAMPs (pathogen-associated molecular patterns)
such as chitin or flagellin for the initiation of ground-level defences like PTI. However,
these responses trigger the accumulation SA (Meng and Zhang 2013). It is reported
that Arabidopsis sid2 mutants synthesize small quantities of SA and are faulty in
PTI/basal responses (Nawrath and Métraux 1999; Wildermuth et al. 2001). Normally
when phytotoxins and effector proteins inside the cell target some host proteins and
interfere with PTI, it results in increased pathogenicity and host susceptibility.
However, there are different mechanisms by which phytotoxins and effectors sup-
press plant defences involving PR gene expression and SA accumulation (Xin and
He 2013). Plants use ETI to overcome the repression of plant defence by phytotoxins
and effector proteins. During ETI, a specific resistance (R) receptor protein first rec-
ognizes the effector protein either indirectly or directly. After an effector protein
recognition by its R receptor, a signalling cascade starts that results in PR gene
expression and SA accumulation and undergoes programmed cell death, a kind of
hypersensitive response (Jones and Dangl 2006). This form of resistance is highly
specific and affords a high level of protection to plants. Both PTI and ETI also initiate
a significant defence response in which long-distance signalling gets induced as an
effect of an initial local infection to rescue the distant uninfected plant parts against
future pathogen infection, i.e. known as SAR (Champigny and Cameron 2009; Shah
and Zeier 2013). Similar to PTI and ETI, those plants which are faulty in SA accu-
mulation show low levels of SAR. However, SA themselves is not a signalling com-
ponent for SAR (Vernooij et al. 1994); the conjugate of SA, MeSA, participates in
SAR (Liu et al. 2011).
Besides defence against pathogen attack, abiotic stresses like ozone exposure
and ultraviolet light, both trigger accumulation of SA, expression of PR proteins
and resistance to viruses in tobacco (Yalpani et al. 1994). Sharma et al. (1996) dem-
onstrated the defensive role of SA in Arabidopsis thaliana under ozone stress,
because the transgenic line of SA-deficient NahG proved to be much susceptible to
ozone stress. The mRNA expression under ozone stress requires SA accumulation.
Related studies demonstrated that Arabidopsis thaliana under ozone stress became
more resistant to virulent phytopathogenic strains of Pseudomonas syringae. Above
inferences denote that the pathways of pathogen- and ozone-induced resistance
depend on SA and also interconnected.
Under ozone stress, induction of antioxidant system was highly SA dependent as
it was found to be defective in NahG plants (Rao and Davis 1999). While Arabidopsis
thaliana (genotype Cvi-0), highly sensitive to ozone show HR-like cell death due to
high levels of SA accumulation that leads to higher levels of ROS generation(Rao
et al. 2000). In Arabidopsis, simulated acid rain caused necrosis of leaves and up-­
regulated the expression of gene related to pathogen resistance pathway mediated
via SA (Lee et al. 2006). NahG plants inefficient to accumulate SA showed severe
necrosis in comparison to wild type against simulated acid rain. Thus, the
SA-mediated signalling is not only related to the SAR induction but is also seems to
be crucial for the regulation of abiotic stress responses.
102 K. Verma and S.B. Agrawal

The defensive effect of SA under an abiotic stress has been recently reported by
many workers. Strobel and Kuc (1995) demonstrated that cucumber and tobacco
showed SA-induced tolerance against Cu stress, and the similar was later reported
in sunflower plants (El-Tayeb et al. 2006). Exogenous application of SA on two rice
plants grown in the presence of Hg2+ or Pb2+ accelerates germination of seeds and
growth of seedlings (Mishra and Choudhuri 1997). Later Metwally et  al. (2003)
reported alleviation of Cd-caused accumulation of MDA content and increased
fresh weight of root and shoot after SA pretreatment in seedlings of barley. The
reason behind the consequences was SA-induced suppression of Cd toxicity effects
that also reduced antioxidant capacity. Amelioration of Cd-induced growth inhibi-
tion, desiccation and decrease in chlorophyll content by SA treatment was also
demonstrated by Drazic and Mihailovic (2005) in soybean grown under Cd stress.
In these plants, SA changed the distribution of Cd in different organs instead of
decreasing the Cd uptake. Cd-induced toxicity effects on photosynthetic efficiency
get reduced when maize seeds were presoaked with SA (Krantev et al. 2008). Al
was shown to be responsible for the increased SA levels in the roots of Cassia tora
L. plants that resulted in tolerance to Al via enhancing the efflux of citrate and thus
hindering uptake of Al (Yang et al. 2003). Intracellular free SA content was raised
in maize after Cd treatment (Pál et al. 2005).

6.3 SA Induced Biotic and Abiotic Stress-Specific Genes

SA has not only been involved in stress responses related to pathogens (Vlot et al.
2009) but has also been demonstrated to function in defence signalling against vari-
ous abiotic stress factors. Among the signalling molecules, SA has received a par-
ticular attention because it regulates various plant processes against environmental
stresses through signalling and crosstalk between other existing growth factors or
substances (Horvath et  al. 2007). Numerous studies have shown that exogenous
application of SA can reduce salinity-induced toxicity symptoms in different plant
species (Hayat et al. 2010; Horvath et al. 2007; Ashraf et al. 2010). In plants, biotic
and abiotic stresses initiate a range of signal transduction cascade. SA is known to
induce a large number of genes under environmental stresses. In tobacco cell cul-
tures, PR genes and MAPK pathway both were induced by SA (Zhang and Klessig
1997). Under broad spectrum of environmental stresses, plants involve MAPK path-
ways for their signal transduction cascade (Zhang and Klessig 2001). Van Loon
et al. (2006) advocated that pathological or related situations can induce PR pro-
teins. However, the biotic and abiotic stress tolerance in plants induced by SA may
have various causes.
SA influences several other genes that may relate to certain kinds of environmen-
tal stress. Recently it was reported that SA can induce both, a chloroplast elongation
factor having its significant role in plant adaptation against environmental stresses
and also topoisomerase genes (Singh et al. 2004; Hettiarachchi et al. 2005). In vivo
expression of TLC1, a member of the long terminal repeat retrotransposon families,
can also be induced by SA. Transcription and expression of retroelements of this
6  Salicylic Acid-Mediated Defence Signalling in Respect to Its Perception, Alteration… 103

family was found to be induced in response to many stresses like salinity and
wounding (Tapia et al. 2005). In plants, there are many reports revealing the impor-
tant role of heat shock proteins (HSPs) in thermotolerance (Clarke et al. 2004). The
exogenously applied SA (0.1 mM) was able to induce the synthesis of class I cyto-
solic family Hsps such as Hsp17.6 and Hsp70 with protein-refolding activity and
known to increase the heat tolerance in pea plants (Pan et  al. 2006). Cronje and
Bornman (1999) reported SA-mediated change in the expression of Hsp70/Hsc70 in
the cell suspension cultures of tomato. In Arabidopsis plants, Hsp17.6 was known
to be induced by SA in a concentration-dependent way, but was not able to induce
Hsp101, belonging to ATP-dependent Hsp100/ClpB family of chaperones and func-
tions in disaggregation of protein complexes induced under heat stress (Clarke et al.
2004). Since it was found that both npr1 mutants and NahG transgenic plants were
capable of inducing Hsp17.6 and Hsp101, it was assumed that the presence of SA is
crucial for basal but not for acquired thermotolerance (Clarke et al. 2004). A recently
isolated gene, Oshsp18.0-CII, can be induced by SA. However, this induction was
much weaker than its induction by heat shock. Chang et al. (2007) assumed that this
heat shock gene might be post-transcriptionally regulated. Ubiquitous proteins and
cyclophilins (Cyp) possess intrinsic peptidyl-prolyl cis-trans isomerase activity for
the catalyzation of X-Pro peptide bonds rotation and assist the folding of proteins
in vivo. Treatment of SA on bean leaves leads to accumulation of Cyp mRNA. The
result demonstrated that this gene might be involved in SA-mediated signal trans-
duction pathway and might be a stress-regulated protein (Marivet et  al. 1995).
Abundant proteins related to late embryogenesis, dehydrins, have known to be accu-
mulated in plants facing not only certain biotic and abiotic stresses but also respond
to SA exposures (Shen et al. 2004). SA is also known to increase the expression of
an osmotin gene which plays a role in stress-related signal transduction (Kim et al.
2002). According to various reports, Arabidopsis mutants in which SA accumula-
tion and synthesis have been altered showed alteration in tolerance to salt (Cao et al.
2009; Hao et al. 2012; Miura et al. 2011). Moreover, the signalling pathway behind
and the downstream targets of SA signalling network involved in plant protection
during abiotic stress remain obscure. Thus, here in this review we further discussed
SA biosynthesis, signal perception, receptors, signal transduction and related
aspects in plants facing environmental stresses under following sections.

6.4 Biosynthesis of SA in Plants, Its Control and Regulation

6.4.1 Biosynthetic Pathways

In plants, there are two major SA biosynthetic pathways identified: the phenylala-
nine ammonia-lyase (PAL) and the isochorismate (IC) (Mustafa et al. 2009; Kawano
et al. 2004). In the shikimate pathway, chorismate is the end product which is com-
monly utilized in both the pathways for the production of SA (Dempsey et al. 2011).
PAL and IC synthase (ICS) are the critical enzymes for these pathways, respectively
(Wildermuth et al. 2001; Fig. 6.1). However, plants utilize the phenylalanine route
104 K. Verma and S.B. Agrawal

most commonly for the synthesis of SA. In this pathway, benzoic acid 2-hydroxy-
lase finally catalyzes benzoic acid hydroxylation at C-2 position, and SA gets syn-
thesized after a sequence of reactions. Synthesis of benzoic acid takes place in
various steps starting from trans-cinnamic acid either via a non-oxidative pathway
or a β-oxidation of fatty acids (Mustafa et al. 2009; Verberne et al. 1999). Cinnamic
acid is formed in a reaction catalyzed by PAL enzyme using phenylalanine as a
substrate. The PAL is a key enzyme of phenylpropanoid pathway, which involves in
the generation of different types of phenolics, and the enzyme can also be induced
by various biotic and abiotic stresses (Yalpani et  al. 1993). In the isochorismate
pathway, chorismate is converted to isochorismate as a result of ICS activity, which
is subsequently catalyzed by isochorismate pyruvate-lyase (IPL) to give rise to SA
(Mustafa et al. 2009). According to Wildermuth et al. (2001) in Arabidopsis during
the plant defence responses, SA biosynthesis was found to be governed by ICS.
Gene homologs of PAL and ICS are ubiquitous and found all over plant kingdom
(Yuan et al. 2009; Sadeghi et al. 2013; Kim and Hwang 2014) which suggests that
the biosynthetic pathways of SA are very important to survive during the course of
evolution. The ICS1 mutants of Arabidopsis were unable to accumulate SA against
pathogen attack (Wildermuth et  al. 2001). Infection to any pathogen, however,
decreased accumulation of SA up to 50% in the Arabidopsis mutants showing 10%
reduction in the activity of PAL as compared to the wild type (Huang et al. 2010).
Thus, during immunity in plants, the IC biosynthetic pathway plays a major role for
SA synthesis. In this route, chorismate is converted into IC with the help of ICS in
the chloroplasts (Garcion et  al. 2008), and IC is ultimately transformed to SA
(Dempsey et  al. 2011). This SA has exported from chloroplast with the help of
EDS5, a MATE transporter (Serrano et al. 2013). The lack of SA accumulation in
eds5 mutants showed the importance of this export mechanism in the distribution
and accumulation of SA inside the cell (Nawrath et al. 2002; Ishihara et al. 2008).

6.5  egulation and Control of SA Biosynthesis

R
During Stress

The SA biosynthesis is controlled at the time of mRNA synthesis and later at post-­
transcriptional levels. At transcriptional level, MYBs (myeloblast; MYB96 and
MYB30), WRKYs (WRKY28 and WRKY46) and WIPK (wound-induced mitogen-­
activated protein kinase)-activated transcription factors positively regulate ICS path-
way, thereby leading to increased production of SA in plants (Vidhyasekaran 2015).
At post-transcriptional level, the RNA-binding proteins (RBP) have their role in mul-
tiple post-transcriptional processes. For example, an RBP of A. thaliana, AtRBP-
defence related 1 (AtRBP-DR1), has found to control SA biosynthesis mediated via
ICS1, as a loss-of-function mutants of AtRBP-DR1 accumulated lesser SA and lines
overexpressing it accumulated higher SA content than wild type (Qi et al. 2010).
The key components of SA signalling, including genes and proteins, regulate SA
level in turn to pathogen attack. A large number of those genes encode proteins that
participate either upstream of SA synthesis or in signal transduction of
6  Salicylic Acid-Mediated Defence Signalling in Respect to Its Perception, Alteration… 105

SA. SA-induced genes, such as PR1, PR2 and PR5 including the transcription factor
WRKY38, have been recognized as the main components of the SA signalling net-
work (Eulgem and Somssich 2007). According to Hruz et al. (2008), this type of
signalling is closely connected with plant-pathogen response which represents a
biotic stress. In spite of biotic stresses, SA signalling has also been activated by
abiotic stresses like ozone, UV and also gamma irradiation that are well known to
trigger SA accumulation.
A tight regulation of SA biosynthesis is a preliminary requirement, as constitu-
tive accumulation of SA has negative impacts on proper growth of plants (Chandran
et al. 2014). Accumulated data show that transcriptional control of ICS1 by calcium
signalling is a major way to start SA biosynthesis. In the cytosol calcium ions (Ca2+),
concentration transiently increases upon immune receptor activation through Ca2+
channels. Elevation of intracellular Ca2+, called Ca2+ signature, is decoded by Ca2+
sensor proteins, such as calmodulin (CaM) and Ca2+-dependent protein kinases
(CDPKs; Dodd et al. 2010; Poovaiah et al. 2013; Boudsocq and Sheen 2013; Schulz
et al. 2013). After binding to CaM, it regulates intended protein activities, thereby
relaying Ca2+-dependent downstream responses. Immunological responses in
Arabidopsis involve the CBP60g (calmodulin-binding protein 60 g), a CaM-binding
transcription factor, and SARD1 (systemic acquired resistance deficient 1), a homo-
log of CBP60g; both are known to control ICS1 transcription (Wang et al. 2011;
Zhang et al. 2010; Wan et al. 2012). However, SARD1 and CBP60g have found to
be partially redundant for the expression of ICS1 and accumulation of SA during
defence responses. Truman et al. (2013) reported some other related homolog of
CBP60g, CBP60a which regulates ICS1 expression negatively upon binding to
CaM. Possibly, after pathogen infection, SARD1 and CBP60g bind to the promoter
of ICS1 and initiate its expression, where it partly removes the CBP60a, a negative
regulator from the ICS1 promoter. Recently, the CDPKs (CPK4, CPK5, CPK6 and
CPK11) were known to interact with the WRKY transcription factors (WRKY8,
WRKY28 and WRKY48) via phosphorylating them during ETI. Gao et al. (2013)
reported such types of ETI responses mediated by the RPM1 (resistance to P. syrin-
gae pv. maculicola 1) or RPS2 (resistance to P. syringae 2); both are immune recep-
tors associated with plasma membrane. Mutants of WRKY8 or WRKY48 have been
known to compromise in pathogen-induced ICS1 expression (vanVerk et al. 2011).
Overall, these findings suggest that during immune responses like ETI, ICS1 tran-
scription occurs through transcription factors like WRKY upon activation of CDPK-­
dependent transmission of Ca2+ signatures. Calcium signalling along with regulation
of ICS1 also influences the continuance of SA accumulation via the EDS1 (enhanced
disease susceptibility 1) transcription that encodes an essential regulator for the
positive feedback loop (Du et  al. 2009; Feys et  al. 2001). A calmodulin-binding
transcription activator 3/signal responsive gene 1 (CAMTA3/SR1) binds to the
EDS1 promoter to repress its transcription. CAMTA3/SR1 mutants were reported to
be high in SA levels and increased immunity towards P. syringae and Botrytis cine-
rea. Recently Zhang et al. (2014) published that an interacting protein of C­ AMTA3/
SR1 links it to ubiquitin-mediated degradation and enhances the expression of
EDS1 and thereby immunity for P. syringae.
106 K. Verma and S.B. Agrawal

6.6 SA Receptors: A Hopeful Journey for Its Search

As an immune signal, it must bind to some target or receptor for the activation of
downstream signalling. This idea led to great efforts in the last 15 years to find out
the SA receptors. Klessig and his colleagues found potential SA receptors by isolat-
ing SA-binding proteins (SABPs) that are mainly of four types. The SABPs that
have been identified include a chloroplastic carbonic anhydrase, a catalase, a cyto-
plasmic ascorbate peroxidase and a methyl salicylate esterase (Du and Klessig
1997; Kumar and Klessig 2003; Park et al. 2007; Vlot et al. 2008, 2009). Subsequent
studies reported that the activity of SA-binding protein 2 (SAPB2) and SA methyl-
transferase 1 (SAMT1) was crucial in distal tissues for the perception of SAR signal
(Park et al. 2009). Although SABP2 is needed for SAR signalling, it does not act as
a SA receptor. As an alternative to the traditional biochemical approach using
tobacco plants, Klessig’s group recently isolated additional SABPs in Arabidopsis
using a combined technology that involves photo affinity labelling and surface plas-
mon resonance (Tian et al. 2012). These SABPs were the glutathione-S-­transferases,
GSTF2, GSTF8, GSTF10 and GSTF11, and the subunit E2 of the α-ketoglutarate
dehydrogenase. However, the role and importance of these SABPs as SA receptors
remain to be determined. So far, genetic analyses reveal that none of them are the
bona fide SA receptor, because these molecules do not have functional roles in plant
immune signalling.

6.6.1 NPR Family as Possible SA Receptors

Recently on the basis of ligand–receptor binding method, it is reported that non-­

expressor of pathogenesis-related (NPR) proteins, NPR1, NPR3 and NPR4, are the
possible SA receptors in Arabidopsis thaliana (Wu et  al. 2012; Fu et  al. 2012).
These three proteins contain a conserved domain at N-terminal, BTB/POZ (broad
complex, tramtrack, and bric-à-brac/poxvirus and zinc finger) and an ankyrin repeat
in the middle of the protein (Liu et al. 2005). Further, Wu et al. (2012) demonstrated
that SA binds to NPR1 when both are present in equilibrium, by an equilibrium
dialysis ligand-binding technique. A strong binding takes place between NPR1
C-terminal transactivation (TA) domain and Cys 521 and Cys 529 via the transition
metal copper (Rochon et al. 2006; Wu et al. 2012). When SA is lacking, the TA
domain of NPR1 is repressed by the BTB domain and therefore fails to start the
expression of SA response genes. On the other hand, upon pathogen infection, SA
concentration increased, which aid the binding of Cys 521 and Cys 529 to SA
through copper (Fig. 6.2). The BTB domain of NPR1 indicates that, like other pro-
teins containing BTB domain, NPR1 may also interact with Cullin 3 (CUL3) ubiq-
uitin E3 ligase and mediate substrate degradation. However, the NPR1 substrate has
not yet been identified. NPR1 itself undergoes degradation by the proteasome both
before and after SAR induction (Spoel et al. 2009). Fu et al. (2012) reported that
NPR3 and NPR4, both paralogs of NPR1, act as adaptor proteins for the CUL3
ubiquitin E3 ligase that particularly prepare NPR1 for degradation in a manner
6  Salicylic Acid-Mediated Defence Signalling in Respect to Its Perception, Alteration… 107

Fig. 6.2  Models for SA perception. (a) Direct SA binding to NPR1 modulates its activity. In
unstressed conditions, the C-terminal transactivation domain of NPR1 is repressed by the
N-terminal BTB/POZ domain, keeping NPR1  in an inactive state (green). NPR1 perceives SA
through Cys 521/529 via the transition metal copper, which triggers a conformation change of
NPR1, resulting in derepression of the transactivation domain and activation of NPR1 (yellow). (b)
NPR1 accumulation is regulated by SA through the SA receptors NPR3 and NPR4. Pathogen
infection triggers SA accumulation. In the case of low SA, the SA receptor NPR4 triggers NPR1
degradation through the 26S proteasome. When SA levels are intermediate, NPR1 protein accumu-
lates. High SA concentrations trigger the SA receptor NPR3-mediated NPR1 degradation. Thus,
only intermediate levels of SA achieve NPR1 accumulation, thereby activating SA-mediated tran-
scriptional reprogramming

dependent on SA concentration. NPR1 and NPR4 interaction occurs when there is

no SA in the medium; however, in the presence of SA, the interaction disrupts which
is responsible for the NPR1 and NPR3 binding. Fu et  al. (2012) found that the
NPR1 has no considerable activity for SA binding under various conditions, but
NPR3 and NPR4 are able to bind SA with different affinity. Since the affinity of
NPR4 for SA is high and low in the case of NPR3 for SA, low levels of SA should
108 K. Verma and S.B. Agrawal

inhibit the degradation of NPR1, whereas increased levels of SA may enhance the
process. According to the proposed model, when there is no infection, NPR1 has
been constantly removed by NPR4 via CUL3–NPR4-mediated degradation; how-
ever, the basal levels of SA also disrupt a few of the NPR1–NPR4 interactions. This
allows some NPR1 to escape degradation, which is essential for keeping basal
immunity (PTI). Subsequent pathogen infection leads to the induction of higher SA
levels in the surrounding infected tissues, which enhance the interaction between
NPR1 and NPR3, responsible for the degradation of NPR1 mediated via CUL3–
NPR3. NPR1 degradation leads to programmed cell death (PCD) at the site of infec-
tion. The reason behind is NPR1 acts as a negative regulator of PCD during
ETI. Besides this in systemic tissues, a medium level of SA is not sufficient to bring
about NPR1–NPR3 interaction but known to be enough to disrupt NPR1–NPR4
interaction and, consequently, enables NPR1 to accumulation, leading to the activa-
tion of SAR. Therefore, as receptors of SA, NPR3 and NPR4 seem to regulate the
NPR1 homeostasis and modulate the function of NPR1  in SAR, ETI and basal
immunity. However, contradicting results for SA receptor identification are due to
various experimental approaches undertaken to test the direct binding of NPR1 to
SA. Crystallography of NPR1, NPR3 and NPR4 might be helpful in unravelling the
SA-binding sites of these receptors. Moreover, in plants, salinity tolerance is medi-
ated via both NPR1-dependent and NPR1-independent pathways (Jayakannan et al.
2015).
Two high-throughput techniques were adopted by Tian et al. (2012) and Moreau
et al. (2013) for the identification of possible candidates for SABPs; the first involves
photo affinity cross-linking to 4-azido SA, followed by mass spectroscopy and
immuno-selection-based identification, while the second utilizes 4-azido SA cross-­
linking and immuno-detection of SABPs on a protein microarray. Manohar et al.
(2015) reported the identification of nine new SABPs, based on at least two inde-
pendent assays, and provided a list of more than 100 candidate SABPs identified by
the above two high-throughput screens. Overall, these researches would be expected
to have a long-lasting impact on future research in plant immunity.

6.6.2 SA Receptors Other Than NPR1 Family

A large number of evidences indicate that there are SA-dependent but NPR1-­
independent pathways to regulate defence gene expression (An and Mou 2011). For
example, in a genetic screen for the suppressors of npr1, SNI1 (suppressor of npr1-­
1, inducible) and SNC1 (suppressor of npr1-1, constitutive) were identified as nega-
tive regulators of defence responses (Li et  al. 1999, 2001). In the sni1/npr1 and
snc1/npr1 double mutants, SA-mediated defence gene expression was restored in
previous one and activated constitutively in later one. Recently, SNI1 was discov-
ered to be a subunit of the SMC5/6 complex involved in responses related to DNA
damage (Yan et al. 2013). Further studies demonstrated that SA could activate the
responses related to DNA damage to potentiate expression of defence genes in an
NPR1-independent manner. Hence, there is likely existence of other SA receptors to
6  Salicylic Acid-Mediated Defence Signalling in Respect to Its Perception, Alteration… 109

arbitrate NPR1-independent pathways. Other than regulation of defence gene

expression, SA is well known to have several physiological roles in plants. In sus-
pension cultures of tobacco, SA treatment was able to provoke the superoxide gen-
eration that further leads to increased cytosolic free calcium levels (Kawano et al.
1998). Du et  al. (2013) recently reported that protein trafficking via clathrin-­
mediated endocytosis involves SA interference. Compared to gene regulation, these
responses are very quick, indicating the existence of other receptors for SA in these
early SA responses. According to recent studies, salicylate is able to bind and acti-
vate AMPK (AMP-activated protein kinase), an energy sensor molecule which is
highly conserved among eukaryotes (Hawley et al. 2012). In Arabidopsis, two of
the AMPK homologs known as Snf1-related kinases (SnRKs) function as the main
regulators of transcriptional networks during energy and stress signalling (Hrabak
et al. 2003; Baena-Gonzalez et al. 2007). Since the SA-activated defence responses
are also energy-demanding processes, it is worthwhile to investigate whether SnRKs
could be possible SA receptors.

6.7 SA Perception

Several researches on SA receptor identification explored the SA perception and

execution of downstream signals. In this regard, initially biochemical methods iden-
tified several SA-interacting proteins whose activities got affected upon SA binding
(Chen and Klessig 1991; Du and Klessig 1997; Tripathi et al. 2010; Moreau et al.
2013; Murli Manohar et  al. 2015). However, these SA-binding proteins failed to
explain many SA-mediated responses including transcriptional reprogramming,
and thus how SA is perceived still holds some mystery.
Recently in Arabidopsis, the three NPR family members (NPR1, NPR3 and
NPR4) were suggested as SA receptors (Wu et al. 2012; Seyfferth and Tsuda 2014).
Among them, NPR1 is a key regulator of SA-mediated reprogramming of transcrip-
tional events and immunity and functions as a coactivator (Pajerowska-Mukhtar
et al. 2013). Structurally NPR1 consists of a BTB/POZ domain, an ankyrin repeat
and a sequence for its localization to nucleus (Fig. 6.2). Mutations in npr1 lead to
complete loss of SA-mediated transcriptional reprogramming and high susceptibil-
ity to pathogens (Volko et al. 1998; Dong 2004). Therefore, NPR1 was thought to
be a SA receptor (Wu et al. 2012). Biochemical studies indicate that SA binding to
NPR1 triggers a conformational modification (Fig.  6.2). Further protein deletion
studies put forward that, in NPR1, the C-terminal transactivation domain is intramo-
lecularly inhibited by the N-terminal BTB/POZ domain. Due to this suppression,
later transactivation domain gets released upon SA binding. Thus, the study sug-
gested NPR1 as a SA receptor and as a signal transducer in SA signalling. However,
in the absence of SA, NPR1 undergoes degradation via the pathway of 26S protea-
some (Spoel et al. 2009). Upon pathogen infection, when SA increases, NPR1 accu-
mulates; however, full induction of SA-responsive genes requires NPR1 turnover.
Thus, for SA response, regulation of NPR1 level is critical.
110 K. Verma and S.B. Agrawal

According to published reports, SA also binds to NPR3 and NPR4 which are
other known NPR1 paralogs. Compared to NPR4, the binding affinity of SA to
NPR3 is very low. NPR1 undergoes proteasomal degradation after binding to NPR3
or NPR4 by their Cullin 3 (CUL3) E3 ubiquitin ligase activity. This NPR1 degrada-
tion, along with the differences in SA-binding affinity towards its paralogs, answers
many aspects related to NPR1 homeostasis at the infection site in the distal tissues
for providing the basal resistance. Upon infection, the SA concentration increases at
the site of infection. Due to increased SA concentration, the interaction between
SA-bound NPR3 and NPR1 leads to the proteasomal degradation of NPR1 and thus
promoting local cell death. However, at the distal site of infection, a moderately
high concentration of SA occurs which is higher than the basal concentration but
not as high as at the infection site. This reduces NPR1–NPR3 interaction and allows
NPR1 to penetrate the nucleus leading to PR-1 transcription (Fig. 6.3). In case of no
infection, very low basal SA concentration favours the interaction of NPR1 with
unliganded NPR4, while some NPR1 escape degradation and become responsible
for the NPR1-dependent expression of defence-related genes to provide basal resis-
tance to plants (Fu et al. 2012; Kaltdorf and Naseem 2013). Interestingly, SA favours
NPR1–NPR3 interaction as compared to NPR1–NPR4 interaction. The above
model in which when SA is absent NPR4-mediated NPR1 degradation averts NPR1
accumulation; however, high SA concentrations also prevent NPR3-mediated NPR1
accumulation. Thus, NPR1-mediated signalling occurs only at intermediate levels
of SA (Fig.  6.3). This model is reliable with the observation that NPR1 protein
accumulates in high concentrations at the site of infection in a leaf. Although this
model is attractive, further validation is required to confirm the mechanism of SA
perception in plants to address fundamental questions.

6.7.1 Perception in Plants with High Basal Level of SA

It is reported that SA plays a significant role in protecting rice from oxidative dam-
age during pathogen infections rather than functioning as signalling molecule to
induce defence gene expression (Yang et al. 2004). As compared to Arabidopsis, the
main function of SA is different in rice; however, rice has all the homologs of
Arabidopsis NPR1, NPR3 and NPR4 (Yuan et  al. 2007). Similar to Arabidopsis
NPR1, rice NPR1 (designated OsNPR1 or NH1) is also a positive regulator of
defence responses (Yuan et al. 2007). The rice homolog NPR3 (NH3) shares the
highest homology with NPR3 and NPR4 of Arabidopsis. Interestingly, NPR3 and
NPR4 regulate immune responses negatively in Arabidopsis (Zhang et al. 2006) as
compared to rice NH3 which plays a positive role for the same (Bai et al. 2011). In
rice, addition to NPRs, WRKY45 plays a key role in SA signalling (Shimono et al.
2007). It will be of significance to test whether rice perceives SA signal through
NPR homologs or through other components such as WRKY45.
6  Salicylic Acid-Mediated Defence Signalling in Respect to Its Perception, Alteration… 111

Fig. 6.3  Model of the biosynthesis and action of salicylic acid on the induction of biotic and
abiotic stress tolerance

6.8 Transcriptional Reprogramming Mediated Via SA

SAR involves extensive reprogramming of transcription. SA modulates the expres-

sion of about 2000 genes. Chromatin structure is very important for the gene expres-
sion regulation, and chromatin states could control cellular memory. NPR1 induces
high levels of chromatin modification on transcription factor gene promoters
(Vidhyasekaran 2015) and controls the expression of various genes (Wang et  al.
2006). NPR1 function regulation is mediated by SA binding directly or by changes
in redox status triggered by SA (Mou et al. 2003). When SA is absent, NPR1 exists
as an oligomer; however, SA-mediated changes in the redox potential of a cell
reduce cysteine residues present in NPR1 via thioredoxins (TRXh3 and TRXh5)
that result in the NPR1 monomerization (Tada et  al. 2008). Thus, SA-triggered
monomerization of NPR1 and nuclear accumulation are important steps for NPR1-­
mediated transcription. However, just NPR1 nuclear localization is not sufficient for
reprogramming of transcriptional events, as the occurrence of SA as well is required
for complete PR1 induction (Spoel et  al. 2003). Additional NPR1 regulation
involves its phosphorylation (Spoel et al. 2009) triggered by SA in the nucleus at the
N-terminus (Ser 11/15) via kinase(s) yet to be determined. Phosphorylation of
NPR1 contributes to its recruitment to a ubiquitin ligase, resulting in proteasome-­
mediated degradation of NPR1. This step is required for the proper control of
112 K. Verma and S.B. Agrawal

transcription by NPR1, which further allows the fresh NPR1 to reinitiate the next
cycle of transcription. NPR1 interacts with specific transcription factors to regulate
the transcription of various SA-responsive genes. Major transcription factors identi-
fied belong to a subclass of a family of basic leucine zipper transcription factor
(TGA; Gatz 2013). There are ten TGA transcription factors which are encoded by
Arabidopsis genome, and all are known to bind the consensus DNA sequence, i.e.
TGACG. Interaction analysis of TGA transcription factors and NPR1 by yeast two-­
hybrid showed specificity for TGA2/TGA5/TGA6 (clade II) and TGA3 (clade III;
Zhou et al. 2000; Hepworth et al. 2005). Genetic analysis studies by Seyfferth and
Tsuda (2014) revealed that TGA2, TGA5 and TGA6 are involved in repression of
PR1 transcription when SA is absent.

6.9 SA Signal Transduction

Accumulation of SA initiates a multifaceted signalling cascade that includes PR

gene expression. According to current knowledge of SA signalling, NPR1 plays a
central role in the regulation of SA-induced signal transduction (Vlot et al. 2009; An
and Mou 2011; Yan and Dong 2014). SA accumulation leads to a change in redox
status of a cell that facilitates the monomerization of a cytosolic oligomer pool of
NPR1, which interact with TGA transcription factors in the nucleus to regulate gene
expression (Mou et al. 2003). Although NPR1 plays a central role in signalling, it is
not able to bind SA in a usual ligand-binding assay, which implies that it is not the
SA receptor. Further investigation in this line demonstrated that NPR1 homologs
NPR3 and NPR4 bind SA and control protein stability of NPR1 to intervene SA
signalling (Fu et al. 2012). According to the above findings, levels of SA affect the
NPR3 or NPR4 ability to prepare NPR1 for ubiquitinylation and degradation by the
proteasome. At the minimum and maximum levels of SA, the NPR3 and NPR4
direct degradation of NPR1, thus preventing SA signalling. At the intermediate lev-
els, NPR1 does not undergo degradation and can play a part in SA-induced signal
transduction (Yan and Dong 2014). This ensures that SA provokes defence gene
expression only when it is needed and prevents constitutive immune signalling that
is usually detrimental for the plant growth and development (Rivas-San Vicente and
Plasencia 2011).

6.9.1 SA-Mediated Signal Transduction Pathways and Networks

6.9.1.1 Protein Phosphorylation

Phosphorylation of proteins is a common post-translational modification used in
plant signalling. Conrath et al. (1997) reported that okadaic acid, a protein phospha-
tase inhibitor suppresses PR-1 expression in the presence of SA.  In a study of
tobacco suspension culture cells, SA induces a transient and rapid activation of a
protein kinase (48 kDa) called SIPK (SA-induced protein kinase) (Vlot et al. 2009;
6  Salicylic Acid-Mediated Defence Signalling in Respect to Its Perception, Alteration… 113

Zhang and Klessig 1997). Further study on transgenic tobacco (BY2 suspension
cells) revealed that SIPK:GFP was both cytosolic and nuclear. Later on, Menke
et al. (2005) reported that SIPK phosphorylates a WRKY1 transcription factor in
the tobacco. The induction of a CK2-like (casein kinase II-like) activity via SA
phosphorylates the NPR1-interacting TGA2 transcription factor. In vitro SA treat-
ment facilitates this interaction (Kang and Klessig 2005). In the nucleus, NPR1 is
phosphorylated (Ser11 and Ser15) in response to SA treatment in vivo, which facili-
tates its interaction with the CUL3-based ubiquitin ligase and stimulates turnover
(Spoel et al. 2009).

6.9.1.2 Cell Redox Status and Reactive Oxygen Species (ROS)

SA signalling is linked to cell redox status as NPR1 is monomerized by the reduc-
tion of disulphide bonds. The cell redox status is controlled by the levels of glutathi-
one and the balance between their reduced (GSH) and oxidized (GSSG) forms. A
GSH/GSSG ratio of 15:1 leads to monomerization of NRP1, whereas a ratio lower
than 7.5:1 prompt oxidation of the monomeric form and consequently production of
the oligomeric form (Mou et al. 2003). In plants, direct application of SA caused the
increase of GSH+GSSG and GSH/GSSG after 8  h (Spoel and Loake 2011).
Interestingly SA binds and inhibits the glutathione-S-transferase activities of three
enzymes (GSTF8, GSTF10 and GSTF11). Binding of SA to GSTF can modulate
glutathione homeostasis and thus cell redox status. However, it is largely controlled
via glutathione levels and the ratios between oxidized and reduced forms (Tian et al.
2012). There is an assumption that under stress conditions, SA and ROS have their
role in self-amplifying feedback loops as a high SA concentration brings about a
high H2O2 content (Fauth et al. 1996; Rao et al. 1997). Besides this, H2O2 treatment
causes the accumulation of SA (León et al. 1995). It will be very interesting to find
out whether SA is upstream or downstream of H2O2.

6.9.1.3 SA and Phospholipid Signalling

Phospholipase D (PLD, EC 3.1.4.4) is known to be induced rapidly upon SA accu-
mulation (Krinke et al. 2009). The PLD enzyme activity leads to creation of impor-
tant signalling molecule phosphatidic acid (PA). Exogenous treatment with SA
augments the level of PA or the PLD activity in Arabidopsis, Brassica napus and
Glycine max (Kalachova et al. 2012; Profotova et al. 2006; Rainteau et al. 2012).
Krinke et al. (2009) demonstrated the significance of PLD activity in the regulation
of SA-induced changes in transcriptome in the presence of quencher of PA. PR-1 is
also among those of PLD-dependent genes. In Arabidopsis suspension culture cells,
SA also induces a phosphatidylinositol-4-kinase (type-III PI4K, EC 2.7.1.67) lead-
ing to the generation of phosphatidylinositol-4-phosphate (PI4P) and
phosphatidylinositol-­4,5-bisphosphate PI-4,5-P2 (Krinke et al. 2007). This depicts
a noticeable overlap between PLD- and PI4K-controlled genes. PR-1 is controlled
by both the pathways under defence regulation. In Arabidopsis, the expression of
PI-4,5-P2-dependent PLDs, PLD-1, PLD-2 and PLD-3, is induced by SA (Janda
et al. 2013; Zhao et al. 2013). These observations might indicate that they have their
role in SA-induced signal transduction.
114 K. Verma and S.B. Agrawal

6.9.1.4 Nitric Oxide (NO)

SA signalling involves NO that is another signalling molecule. NO donors induce
SA accumulation, while pathogen defence mediated via NO requires SA (Grun
et al. 2006; Wendehenne et al. 2004). For example, in the NahG mutant, NO-induced
local and systemic resistance against TMV infection and require SA (Song and
Goodman 2001). Thus, NO might be a component of SA signalling pathway. The
mode of action of NO is still unclear, but a possible target could be NPR1 through
S-nitrosylation, which leads to the stabilization of oligomeric NPR1 forms (Tada
et al. 2008).

6.10 Conclusion and Future Perspectives

Phytohormone SA regulates various physiological phenomenons by providing plant

resistance against abiotic and biotic stresses (Fig. 6.3). Stress-induced SA is synthe-
sized in the chloroplasts via the isochorismate pathway in the presence of ICS1
enzyme. A number of researches have shed light into our understanding of
SA-mediated signalling, through the discoveries of calcium signalling as the major
switch for SA biosynthesis, NPR family as SA receptors and the molecular studies
on their functional mechanisms at transcription level. Recently many researchers
made their focus on searching SA receptors, and consequently NPR1, NPR3 and
NPR4 came to describe SA signalling. However, intriguing situation arises in con-
nection with NPR1 which is both a SA receptor and a major SA main effector. But
it is likely to be also true for other SA-binding proteins that have enzymatic activi-
ties and which, therefore, are also SA effectors. SA induces huge transcriptional
reprogramming in which NPR1 acts as the central coactivator of TGA transcription
factors. Recently, there are two alternatives, but not exclusive models for SA per-
ception mechanisms are available. According to first model, NPR1 itself perceives
SA, triggering an NPR1 conformational change, thereby activating SA-mediated
transcription. The second model states that NPR3 and NPR4 perceive SA, thereby
regulating NPR1 protein accumulation. NPR1 does not bind SA directly; however,
this regulation is made by SA-induced phosphorylation and redox changes. The
data regarding signalling pathways related to SA accumulation and control in
response to biotic stress factors are abundant; however, study of these aspects under
abiotic stresses needs further consideration. Ozone, UV exposure and H2O2 treat-
ments increase SA accumulation (León et al. 1995; Neuenschwander et al. 1995;
Sharma et al. 1996; Surplus et al. 1998; Yalpani et al. 1994). The signalling path-
ways controlling SA levels in response to these abiotic conditions have been less
studied compared to pathogen attack. NPR1 is a keystone of SA signal transduction.
NPR1-independent pathways also exist, but reports concerning the identification of
components and intermediates of these pathways are limited. Although SA plays a
key role in SAR, the full understanding of how the signal spreads to other plant tis-
sues and organs remains elusive. An improved understanding of SA signal transduc-
tion pathways will surely help to build a relationship between regulatory networks
of phytohormones that integrate the crosstalk between SA and ethylene, jasmonic
6  Salicylic Acid-Mediated Defence Signalling in Respect to Its Perception, Alteration… 115

acid, abscisic acid, cytokinins, auxin, brassinosteroids and gibberellic acid. Redox
status can alter SA level; however, SA controls gene expression via lipid signalling.
In fine words, SA signalling is complex, and over the coming years, further advances
will be required to better understand this interesting story. Again, perhaps
SA-overexpressing lines will be good tools to answer such questions.

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Role of Salicylic Acid in Heavy Metal
Stress Tolerance: Insight into Underlying 7
Mechanism

Sukhmeen Kaur Kohli, Neha Handa, Ravdeep Kaur,

Vinod Kumar, Kanika Khanna, Palak Bakshi,
Ravinder Singh, Saroj Arora, Rupinder Kaur,
and Renu Bhardwaj

Abstract
Heavy metal (HM) toxicity is one of the leading abiotic stresses, which is very
unsafe and risky for plants. With a fleeting enhancement in industrialization and
urbanization due to population explosion, heavy metals inclusion into the envi-
ronment has become a major concern for the environmentalists. These are most
deleterious pollutants and cause phytotoxicity in plants. Heavy metal accumula-
tion in plants results in generation of oxidative stress. Salicylic acid (SA) is an
imperative endogenous plant hormone. It has a crucial role in regulation of vari-
ous physiological and metabolic processes in plants. It is considered as one of the
most important signaling molecule involved in both abiotic and biotic stress tol-
erance. Application of optimal concentrations of SA enhances plants’ tolerance
to heavy metal stress by modulating levels of several metabolites including com-
ponents of antioxidative defense cascade, osmolytes, secondary metabolites, and
metal-chelating compounds.

Keywords
Antioxidants • Phytotoxicity • Phytohormones • Salicylic acid • Secondary
metabolites

S.K. Kohli • N. Handa • R. Kaur • K. Khanna • P. Bakshi • R. Singh • S. Arora

R. Bhardwaj (*)
Department of Botanical and Environmental Sciences, Guru Nanak Dev University,
Amritsar, Punjab 143005, India
e-mail: [email protected]
V. Kumar
Department of Botany, DAV University, Sarmastpur, Jalandhar 144012, India
R. Kaur
Biotechnology Department, DAV College, Amritsar, India

© Springer Nature Singapore Pte Ltd. 2017 123

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_7
124 S.K. Kohli et al.

7.1 Introduction

Heavy metal (HM) contamination has becoming a major challenge all over the
world (Nagajyoti et al. 2010). Addition of heavy metals to the environment is chiefly
attributed to natural sources (weathering of rocks), industrial sources (metal extrac-
tion plants, chemical industries, and power plants), agricultural sources (pesticides,
fertilizers, polluted manure, and sewage sludge), burning of fossil fuels, and efflu-
ents from automobiles (Mishra and Tripathi 2008; Asati 2013). Additionally, min-
ing, smelting, and agricultural operations lead to contamination of heavy metals like
Cd, Cu, and Zn in various areas of the world (Herawati et al. 2000). Heavy metals
such as cadmium (Cd), lead (Pb), chromium (Cr), copper (Cu), nickel (Ni), mercury
(Hg), and cobalt (Co) are toxic environmental pollutants. Metals like Cu and Zn are
essential for plants but higher concentrations of them prove fatal for plants (Mourato
et al. 2012). These heavy metals become toxic contaminants of soil due to various
reasons like (i) rate of addition of metal by anthropogenic activities is far more than
natural processes, (ii) they get randomly localized in the environment and there are
more chances of direct exposure, and (iii) metals are usually added to the environ-
ment in bioavailable forms (Wuana and Okieimen 2011).
Bioavailability and mobility of metal ions in soil is directly influenced by root
exudates and biological fauna (microorganisms) in its vicinity (Wenzel et al. 2001).
Availability of metals is also affected by several other factors including pH of soil,
organic matter content, and solubility of heavy metals (Palmer and Guerinot 2009;
Miller and Cramer 2004). Higher plants have developed a highly effective mecha-
nism for uptake and accumulation of metal ions as well as other inorganic molecules
from the soil. This uptake is dependent upon transportation cascade, indicating that
different heavy metals are transported through similar channels across the plasma
membrane of roots (Manara 2012). Metal pollution causes phytotoxicity in plants
which resulted in generation of oxidative stress (Das and Jayalekshmy 2015). Plant
growth, biomass, and productivity sharply get affected by bio-amplification of
heavy metals (Singh et al. 2010; Sharma 2012). Different levels of food chain get
affected with the heavy metals such as Zn, Cu, Pb, Mn, Ni, Cr, Cd, and As which are
present in the wastewater and are used for irrigation (Singh et  al. 2010). Due to
heavy metal toxicity, there is aggregation of reactive oxygen species (ROS) and
methylglyoxal (MG) in plants which results in lipid peroxidation, oxidation of pro-
teins, inactivation of enzymes, DNA damage, and other metabolic components (Das
and Jayalekshmy 2015). Photosynthesis, water relation, and mineral nutrition also
get repressed due to heavy metal stress in plants (Li et al. 2013; Yadav et al. 2014;
Ali et al. 2013). It was reported by Sangwan et al. (2014) that inhibition of activity
of nitrogenase, nitrate reductase, nitrite reductase, glutamine synthetase, and gluta-
mate dehydrogenase enzymes resulted in stunted growth of cluster beans under Cr
toxicity. Arsenic stress in seedlings of sunflower results in takeover of Mg of chlo-
rophyll with As which results in affected photosynthesis and growth (Yadav et al.
2014). Stomatal conductance, cellular CO2 concentration, transpiration rate, and net
photosynthetic rate get decreased in Cr-stressed plant due to breakdown of thyla-
koid membrane, chloroplast thickening, and rise in plastoglobulin in leaves of
7  Role of Salicylic Acid in Heavy Metal Stress Tolerance: Insight into Underlying… 125

barley plant (Ali et al. 2013). It was reported by Cui et al. (2014) that disruption in
–SH group of protein due to Hg results in irregular conversion of protein structure.
Wide arrays of reports suggest potential role of plant growth regulators (PGRs)
including auxin, gibberellins, cytokinins, abscisic acid, brassinosteroids, ethylene,
and salicylic acid in improving crop yield and tolerance to heavy metal stress (Bajguz
2010; Divi et al. 2010). Salicylic acid (SA) is an important phenolic plant growth
regulator (PGR), which regulates several physiological functions in plants, including
stomatal closure, flowering, and thermogenesis (Mohsenzadeh et al. 2011; Miura and
Tada 2014). It is also reported to maintain the postharvest quality of fruits, e.g., fruit
ripening is delayed as a result of inhibition of ethylene synthesis in response to salic-
ylate supplementation in plants (Srivastava and Dwivedi 2000). SA also regulates
photosynthesis, osmolyte metabolism, antioxidative defense cascade, and water rela-
tions of plants under abiotic stresses such as low temperature, salinity, etc. (Wang
et al. 2012; Miura and Tada 2014). Besides these, it is also involved in mediating
acclimatization to phytotoxicity due to heavy metals (Zhou et al. 2009; Zengin 2015).
Pretreatment of plants with SA provides protection from heavy metals, e.g., in rice
under Pb and Hg stress (Mishra and Choudhuri 1999), barley under Cd stress
(Metwally et al. 2003), alfalfa under Hg stress (Zhou et al. 2009), Indian mustard
plants under Pb stress (Jazi et  al. 2011), Thlaspi under Cd stress (Llugany et  al.
2013), pea under Pb stress (Ghani et al. 2015), and maize under Cd stress (Gondor
et al. 2016). Alleviation of Hg toxicity was reported by Zhou et al. (2009) in Medicago
sativa plants in response to exogenous application of SA. A similar study was done
by Ahmad et al. (2011), suggesting reduction in Cd stress by exogenous application
of SA in mustard plants. Moreover, Popova et al. (2009) observed that SA has an
ability to counter heavy metal stress by sequestration of reactive oxygen species. It
has also been found to have a prominent role in regulating photosynthetic machinery
by affecting various parameters such as chloroplast structure, stomatal closure, and
activities of photosynthetic enzymes such as carbonic anhydrase and ribulose-
1,5-bisphosphate carboxylase/oxygenase (RuBisCo) (Slaymaker et al. 2002).
In the present chapter, an attempt has been made to understand involvement of
SA in heavy metal tolerance in plants. It covers studies on heavy metal contamina-
tion in soil, metal phytotoxicity, and biosynthesis of SA and its role in acclimatiza-
tion of plants to metal stress.

7.2 SA Biosynthesis in Plants

Biosynthesis of SA is suggested to occur via two alternate pathways, viz., phenylala-

nine pathway (Ogawa et al. 2006; Garcion and Métraux 2006; Sawada et al. 2006) and
isochorismate pathway (Wildermuth et al. 2001; Garcion et al. 2008). Phenylalanine
pathway in tobacco plants is reported to be active in tobacco (Ogawa et al. 2006),
while in Arabidopsis, both isochorismate and phenylalanine pathways were reported
(Mauch-Mani and Slusarenko 1996; Wildermuth et al. 2001; Ferrari et al. 2003).
In phenylalanine pathway, phenylalanine ammonia lyase (PAL), which is a chief
regulator of phenylpropanoid pathway, also plays a key role in SA biosynthesis.
126 S.K. Kohli et al.

Phenylalanine acts as a substrate of PAL and gets converted to trans-cinnamic acid.

The decarboxylation of side chain of trans-cinnamic acid leads to the formation of
benzoic acid through two probable routes (Fig. 7.1). In the first route of β-oxidation
pathway, benzoic acid is formed from trans-cinnamic acid through cinnamoyl-CoA
and benzoyl-CoA intermediates. The second route for the formation of benzoic acid
involves non-β-oxidation pathway in which para-hydroxybenzaldehyde is the inter-
mediate (Ribnicky et al. 1998; Chong et al. 2001). Benzoic acid so formed from
either of the two mechanisms is converted to SA in the presence of the enzyme
benzoic acid 2-hydroxylase (BA2H) (León et al. 1993, 1995). In the same phenyl-
alanine pathway, the formation of SA can also take place from trans-cinnamic acid
by its 2-hydroxylation to form ortho-coumaric acid, followed by decarboxylation
mechanism by β-oxidation to produce SA (Lee et al. 1995; Fig. 7.1).
The second pathway of SA biosynthesis (Fig. 7.2) starts with chorismate from
shikimic acid pathway, and it is converted to isochorismate, and the reaction is cata-
lyzed by the enzyme isochorismate synthase (ICS) (Gaille et al. 2002, 2003). The
formation of SA from isochorismate is thought to be catalyzed by isochorismate
pyruvate lyase (IPL) (Wildermuth et al. 2001; Strawn et al. 2007). This enzyme or
gene coding for similar proteins as bacterial IPL, however, has not been detected in
plants (Chen et al. 2009). But in Arabidopsis, two genes, viz., PBS3 coding for acyl-
adenylate-/thioester-forming enzyme family and EPS coding for BAHD acyltrans-
ferase superfamily, have been found to be instrumental in SA biosynthesis (Chen
et al. 2009; van Verk et al. 2011).
In order to overcome impact of heavy metal stress in plants, they develop differ-
ent strategies, one of them being involvement of phytohormones. Modulation of a
large number of antioxidative enzymes like superoxide dismutase (SOD),

Fig. 7.1  Schematic outline of phenylalanine pathway in SA biosynthesis (PAL phenylalanine

ammonia lyase, BA2H benzoic acid 2-hydroxylase)
7  Role of Salicylic Acid in Heavy Metal Stress Tolerance: Insight into Underlying… 127

Fig. 7.2 Schematic
outline of isochorismate
pathway in SA
biosynthesis (Adapted
from Wildermuth et al.
2001; Gaille et al. 2003;
Chen et al. 2009) (ICS
isochorismate synthase)

peroxidase (POD), catalase (CAT), and glutathione-S-transferase (GST) and nonen-

zymatic antioxidants consisting proline, ascorbic acid, and glutathione also detoxi-
fies ROS (Yadav et al. 2014; Singh and Prasad 2014; Xu et al. 2009). Quenching
action of ROS depends upon their localization in cell compartments. In stress condi-
tions, proline acts as osmolyte which enhances antioxidant enzyme (Parida and Das
2005; Ashraf and Foolad 2007).

7.2.1 SA and Growth and Development

Reduction in plant growth due to metal stress is the primary visible symptom that
indicates the extent of toxicity induced. Numbers of reports on several heavy metal-
treated plants have established the growth-promoting effects of SA, thereby sug-
gesting its active involvement in stress protection (Table 7.1).

7.2.2 SA and Photosynthetic Efficiency

Numerous studies have demonstrated that heavy metals affect photosynthetic

machinery by distorting chloroplast ultrastructure (Najeeb et al. 2011), diminishing
photosynthetic pigment biosynthesis (Parmar et al. 2013), inducing stomatal clo-
sure (Leal-Alvarado et al. 2016), and hampering the activity of photosystems I and
II and enzymes of the Calvin cycle (Popova et al. 2012). Salicylic acid (SA) is one
of the main regulators of photosynthesis. It affects chlorophyll and carotenoid con-
tents, leaf and chloroplast ultrastructure, activities of enzymes of photosynthetic
carbon reduction pathway, and stomatal closure (Rivas-San Vicente and Plasencia
2011). SA is a crucial signaling molecule that modulates abiotic and biotic stress
128 S.K. Kohli et al.

Table 7.1  Role of SA in growth promotion of heavy metal-stressed plants

Heavy SA
S. no. metal concentration Plant Response References
1. Pb 0.1 mM/L Oryza sativa Root and shoot Chen et al.
lengths enhanced (2007)
significantly
2. Cd 500 μM Pisum sativum Root and shoot fresh Popova et al.
weights enhanced (2008)
with SA
3. Cd 500 μM Zea mays Root and shoot Krantev et al.
growth, shoot fresh (2008)
weight and root dry
weight enhanced with
SA
4. Cd 0.1 mM Oryza sativa Vigor index, He et al. (2010)
germination, and root
length improved with
SA
5. Pb 5 and 10 μM Brassica napus Root and shoot Jazi et al. (2011)
lengths, leaf area, and
root and shoot dry
weights, root and
shoot fresh weights,
specific leaf area and
leaf weight ratio
improved due to SA
application and
reduced toxic effects
of Pb
6. Cd 500 μM Zea mays Fresh weights of roots Mohsenzadeh
and shoots enhanced et al. (2011)
with SA
7. Cd 250 and Helianthus Fresh weight and Moradkhani
500 μM annuus length of stems and et al. (2012)
roots, fresh weight
and number of leaves
enhanced in
Cd-stressed plants
8. Cd 2.0 mM Pisum sativum Root and shoot fresh Gaballah and
weights enhanced Rady (2012)
with SA
9. Pb 10−4 M Pisum sativum Plant height, number Ratushnyak et al.
of tendrils, number of (2012)
leaves, and length and
width of leaves
enhanced with SA
10. Cd 300 mg/L Raphanus Root and shoot Raza and Shafiq
sativus lengths increased (2013)
with SA
(continued)
7  Role of Salicylic Acid in Heavy Metal Stress Tolerance: Insight into Underlying… 129

Table 7.1 (continued)
Heavy SA
S. no. metal concentration Plant Response References
11. Ni 0.5 mM Triticum Plant height, fresh Siddiqui et al.
aestivum weight, dry weight, (2013)
and leaf area
improved with SA
12. Pb 5 and 10 μM Brassica napus Root and shoot Jazi and Oregani
lengths, root and (2014)
shoot dry weights,
and leaf area
increased with SA
13. Cd 10−5 M Cicer arietinum Root and shoot Alyemeni et al.
lengths and fresh and (2014)
dry mass enhanced
significantly
14. Cd 250 and Linum Shoot/root ratio Belkadhi et al.
1000 μM usitatissimum increased with SA (2014)
15. Cd 0.1 mM Oryza sativa Improvement in root Fatima et al.
and shoot lengths and (2014)
fresh and dry weights
of both roots and
shoots with SA
16. Cu 1 and 2 mM Tagetes erecta Plant height, root Afrousheh et al.
length, leaves (2015)
number, leaf area, and
fresh and dry weights
of shoot and root
increased with SA
17. Cd 100 mg/L Anethum Improved germination Espanany and
graveolens rate, germination Fallah (2016)
percentage, radicle
elongation, plumule
elongation, radicle
dry weight, plumule
dry weight, and vigor
index with SA

responses in plants. It attenuates heavy metal (HM) toxicity on photosynthetic appa-

ratus and improves photosynthetic efficiency under HM stress (Shi et  al. 2009).
Effects of exogenous application of SA on photosynthetic parameters are discussed
in Table 7.2.

7.2.3 SA and Antioxidative Defense Response

The prime response of plants to heavy metals exposure is overproduction of reactive

oxygen species. Some metals generate ROS directly, by participating in Haber-
Weiss reactions or indirectly by disturbing antioxidative defense system, electron
130 S.K. Kohli et al.

Table 7.2  Effect of exogenous application of SA on photosynthesis in response to heavy metal

stress
S. Heavy SA Effect on photosynthetic
no. metal concentration Plant parameters References
1. Cu 500 μM SA Helianthus Stress-elevated Chl a/Chl b ratio El-Tayeb
annuus L. was lowered et al. (2006)
2. Pb 100 μM SA Oryza sativa Restored chlorophyll content Chen et al.
(2007)
3. Cd 500 μM SA Pisum sativum Improved chlorophyll content, Popova
protected photochemical activity et al. (2008)
of chloroplast membranes and
photosynthetic carboxylation
reactions
4. Cd 500 μM SA Zea mays L. Enhanced chlorophyll content, Krantev
CO2 fixation rate, ribulose et al. (2008)
1,5-bisphosphate carboxylase
(RuBPC), and
phosphoenolpyruvate
carboxylase (PEPC)
5. Ni 1000 μM SA Zea mays L. Ameliorated negative effects of Wang et al.
Ni on chlorophyll and β-carotene (2009)
contents, and net photosynthetic
rate
6. Cd 500 μM Triticum Enhanced chlorophyll content, Moussa and
aestivum L. 14 CO2 fixation, and activities of El-Gamal
PEPC and RuBPC and abscisic (2010)
acid. Also diminished damage to
chloroplast ultrastructure
7. Ni 10 μM SA Brassica Improved contents of pigments, Yusuf et al.
juncea leaf gaseous exchange (2012)
parameters, and carbonic
anhydrase activity
8. Cd 200 μM SA Lolium perenne Increased chlorophyll content, Wang et al.
L. net photosynthetic rate, (2013)
transpiration rate
9. Cd 500 μM SA Poa pratensis Increased chlorophyll and K, Ca, Guo et al.
L. Mg, and Fe content (2013)
10. Mn 10 μM SA Brassica Improved water relations and Parashar
juncea photosynthetic attributes along et al. (2014)
with carbonic anhydrase activity
11. Cd 100 μM SA Arachis Improved chlorophyll content Xu et al.
hypogaea L. and net photosynthetic rate 2015
12. Cd 1 μM SA Iris hexagona Increased the chlorophyll content Han et al.
and net photosynthetic rate via (2015)
improving the ultrastructure of
chloroplasts
(continued)
7  Role of Salicylic Acid in Heavy Metal Stress Tolerance: Insight into Underlying… 131

Table 7.2 (continued)
S. Heavy SA Effect on photosynthetic
no. metal concentration Plant parameters References
13. Cd 100 μM SA Cucumis melo Elevated chlorophyll content, Zhang et al.
L. photosynthetic capacity, and (2015)
PSII photochemistry efficiency
14. Cd 50 μM SA Brassica napus Increased chlorophyll and Ali et al.
carotenoid content and leaf (2015)
gaseous exchange parameters
except internal CO2, and reduced
damage to chloroplast
15. Cd 100, 200, Lolium perenne Improved chlorophyll content Bai et al.
300 μM SA L. (2014)

transport chain, or metabolism of essential elements (Yadav 2010). Overproduction

of ROS, namely, superoxide radical, hydrogen peroxide, hydroxyl radical, and sin-
glet oxygen, is highly toxic and causes oxidative damage to nucleic acids, proteins,
carbohydrates, and lipid peroxidation (Gill and Tuteja 2010). To combat uncon-
trolled oxidation, plants possess antioxidative defense system that includes enzy-
matic (superoxide dismutase, SOD; guaiacol peroxidase, POD; catalase, CAT;
dehydroascorbate reductase, DHAR; monodehydroascorbate reductase, MDHAR;
glutathione reductase, GR; ascorbate peroxidase, APOX; glutathione peroxidase,
GPOX; and glutathione-S-transferase, GST) and nonenzymatic antioxidants (gluta-
thione, GSH; ascorbic acid, ASA; phenolic compounds, α-tocopherols, etc.) for
scavenging and detoxification of ROS (Gill and Tuteja 2010; Sharma et al. 2012).
SA is widely reported to provide protection against heavy metal-induced oxida-
tive injuries, thereby lowering extent of lipid peroxidation by enhancing antioxida-
tive capacity of plants. Table 7.3 discusses effects of exogenous application of SA
on antioxidative defense system in plants in response to heavy metal stress. While
most of the studies reflect that SA induces activities of antioxidative enzymes under
metal stress (Chen et al. 2007; Guo et al. 2007, 2009; Wang et al. 2009; Parashar
et al. 2014; Zhang et al. 2015), some have reported decline in CAT activity under
metal stress (Krantev et al. 2008; Guo et al. 2013; Pandey et al. 2013). In some cases
SA-mediated stress responses are related to an increase in H2O2 generation by inhib-
iting CAT activity and promoting peroxidases activity (Krantev et  al. 2008).
Exogenous application of SA slightly induced NADH oxidase activity (which stim-
ulates H2O2 generation) in mercury-exposed roots of alfalfa. H2O2 acts as secondary
messenger and triggers signaling cascades for activating defense mechanisms under
stress when produced at moderate levels (Zhou et al. 2009). Few studies indicate
that SA being an iron-chelating molecule acts as an antioxidant as it can directly
scavenge hydroxyl radicals which may result in lowering of metal-induced increase
in antioxidative enzyme activity (Ahmad et al. 2011).
132 S.K. Kohli et al.

Table 7.3  Effect of exogenous application of SA on antioxidative defense system in plants in

response to heavy metal stress
S. Heavy SA Effect on antioxidative defense
no. metal concentration Plant system References
1. Pb 100 μM SA Lowered H2O2 content by
Oryza sativa Chen et al.
enhancing activities of SOD, (2007)
CAT, and APOX
2. Cd 10,000 μM SA Oryza sativa Enhanced activities of Guo et al.
antioxidative enzymes (SOD, (2007,
POD, and CAT) and contents of 2009)
nonenzymatic antioxidants (GSH
and NPT). Lowered hydrogen
peroxide and MDA content
3. Cd 500 μM SA Zea mays L. Enhanced APOX and SOD Krantev
activities while lowered MDA, et al. (2008)
electrolytic leakage and CAT
activity
4. Cd 500 μM SA Pisum sativum Lowered oxidative injuries Popova
et al. (2008)
5. Ni 1000 μM SA Zea mays L. Enhanced level of enzymatic Wang et al.
antioxidant (SOD, APOX, (2009)
MDHAR, DHAR, and GR) and
nonenzymatic antioxidants (GSH
and ASA) in chloroplast. Lowered
superoxide anion generation rate,
H2O2 and MDA contents, and
lipoxygenase activity
6. Cd 500 μM SA Cannabis Enhanced SOD and POD Shi et al.
sativa L. activities (2009)
7. Hg 200 μM SA Medicago Increased activity of NADH Zhou et al.
sativa oxidase, APOX, POD, and (2009)
content of ascorbate and
glutathione in the roots. Slightly
decline SOD activity
8. Cd 500 μM SA Poa pratensis Enhanced SOD, POD, and APOX Guo et al.
L. activity. Reduced MDA, hydrogen (2013)
peroxide, and CAT activity
9. Al 60 μM SA Oryza sativa Decreased superoxide anion and Pandey
L. hydrogen peroxide content and et al. (2013)
lipid peroxidation. Elevated
activities of SOD, GPOX, and
APOX were suppressed, whereas
Al-induced decline in CAT
activity was enhanced.
(continued)
7  Role of Salicylic Acid in Heavy Metal Stress Tolerance: Insight into Underlying… 133

Table 7.3 (continued)
S. Heavy SA Effect on antioxidative defense
no. metal concentration Plant system References
10. Mn 10 μM SA Brassica Enhanced antioxidative enzymes Parashar
juncea and lowered level of electrolyte et al. (2014)
leakage, lipid peroxidation, and
H2O2accumulation
11. Cd 1 μM SA Iris hexagona Increased activities of SOD, Han et al.
POD, and CAT, while decreased (2015)
TBARS content and NADH
oxidase activity
12. Cd 100 μM SA Cucumis melo Reduced superoxide anion Zhang et al.
L. generation and lipid peroxidation, (2015)
and increased activities of
antioxidant enzyme (SOD,
GPOX, CAT, and APOX)
13. Cd 50 μM SA Brassica Enhanced activities of SOD, Ali et al.
napus POD, APOX, GPOX, and GR and (2015)
lowered activity of CAT and
MDA content
14. Cd 100, 200, Lolium Enhanced activity of antioxidative Bai et al.
300 μM SA perenne L. enzymes and lowered H2O2 and (2014)
MDA accumulation

7.2.4 SA and Metal Nutrition

Mineral nutrients are essential for the growth and development of plants, and sur-
vival under various environmental stress conditions. Various workers reported that
mineral nutrients play an important function in alleviation of abiotic stress (Iqbal
et al. 2011; Nazar et al. 2011, 2015). Salicylic acid regulates the uptake and metabo-
lism of many mineral elements and thus improve the growth and development of
plants under abiotic stress (Chen et al. 2011; Khokon et al. 2011; Wang et al. 2011;
Tufail et al. 2013; Nazar et al. 2015). Salicylic acid also plays a protective function
in membrane integrity and ions regulation including mineral nutrient absorption
(Alpaslan and Gunes 2001; Gunes et al. 2007). Salicylic acid is involved in regula-
tion of the absorption of various elements, i.e., Cu, Ca, Zn, P, Mn, and Fe, and thus
reduces oxidative stress under Pb stress conditions (Wang et  al. 2011). Salicylic
acid-induced variations in photosynthesis may be due to the mineral contents of Ca,
N, K, and P in Brassica juncea varieties diverging in salt tolerance (Syeed et al.
2011). Aghaeifard et al. (2016) studied the effect of salicylic acid and humic acid on
yield, leaf mineral elements of strawberry. Application of salicylic acid significantly
increased the phosphorus and calcium contents in the leaves of the plants.
134 S.K. Kohli et al.

7.2.5 SA and Osmolytes

To prevent the toxic effects of abiotic stress-induced reactive oxygen species (ROS)
generation, plants have developed mechanisms which enhance their alteration to
ionic and osmotic stresses. However, in order to maintain osmotic balance, plants
have appropriate protective mechanisms known as osmoregulation which are medi-
ated by osmolytes, i.e., amines, soluble sugars, glycinebetaine, prolines, etc. These
compounds helped in maintenance of turgor under stressed plants (Misra and
Saxena 2009). Glycinebetaine is an effective compatible solute for protection
against osmotic, salt, and metal stresses and also helps in osmotic adjustment (Wang
et al. 2010; Bharwana et al. 2014; Khan et al. 2014). Glycinebetaine accumulation
in stressed plants modifies the osmotic balance of cells, protects activity of RuBisCo,
provides stability to membrane integrity, and also detoxifies the toxic ions (Ashraf
and Foolad 2007). Salicylic acid-mediated enhancement in content of glycinebeta-
ine can increase overall growth of plants (Misra and Misra 2012). Enhancement in
level of glycinebetaine led to rise in biomass of Rauwolfia serpentina. Proline accu-
mulation is one of the effective mechanisms used by plants for survival under heavy
metal stress. Proline detoxifies the excess level of ROS, modifies osmotic balance,
provides stability to enzymes and proteins, and protects the biological membranes
(Iqbal et al. 2014). Moreover, Iqbal et al. (2016) studied that phytohormones and
osmolytes are involved in reducing the toxic effects of Cd. Proline is an important
osmolyte which maintains cellular homeostasis through osmotic regulation. Various
workers reported that salicylic acid is involved in enhancing the proline metabolism
under abiotic stresses (Misra and Saxena 2009; Khan et  al. 2013). Salicylic acid
induced activity of enzymes involved in proline biosynthesis, i.e., γ-glutamyl kinase
and pyrroline-5-carboxylate reductase under salinity stress along with enhanced
level of proline. Upregulation of proline biosynthetic enzymes, i.e., γ-glutamyl
kinase and pyrroline-5-carboxylate reductase, and downregulation of proline oxi-
dase activity were involved in enhancing the proline content (Misra and Misra
2012). Sugar alcohol, i.e., mannitol, and soluble sugars accumulation have been
also studied to contribute in plant stress tolerance as osmoprotectant (Cheng et al.
2009). Increased levels of polysaccharides and soluble sugars respectively with
100 m μmol/L, and 0.5 and 1.0 mM of salicylic acid improved the growth of the
plant (Yuan et al. 2014; Luo et al. 2014).
Under the combined application of salicylic acid and calcium, the aluminum
toxicity is reduced in Glycine max (Lan et al. 2016). It was analyzed by Keshavarz
et al. (2016) that the content of proline in canola plant leaves during 0, 24, and 48 h
after foliar application of salicylic acid, and it was observed that maximum proline
content was found for 24 h after treatment of salicylic acid. Salicylic acid enhanced
the growth and stimulated the accumulation of proline in wheat plants (Shakirova
et al. 2003). Treatment of tomato plants with 0.01 mM salicylic acid via root drench-
ing enhanced the growth of plant and stimulated the accumulation of soluble sugar
(Wasti et al. 2012). Various abiotic stresses, i.e., salinity, heavy metal, low tempera-
ture, and drought stress, induce osmotic stress leading to loss of turgor. Several
studies reported that the treatment of salicylic acid affects osmotic stress responses.
7  Role of Salicylic Acid in Heavy Metal Stress Tolerance: Insight into Underlying… 135

The addition of 0.05 mM salicylic acid to hydroponic solution containing media and
polyethylene glycol reduced the toxic effects of osmotic stress in wheat seedlings
(Marcinska et al. 2013).

7.2.6 SA and Secondary Metabolites

Secondary metabolites are compounds that have no direct participation in life pro-
cesses of plants, but they have an imperative role in increasing plants tolerance and
acclimatization to stressful environment (Seigler 1998). They are a distinguished
class of biological entities, which are primary source of food additives, flavors,
pharmaceuticals, and industrial biochemicals. Sequestration of such metabolites
aids plant to adapt to the changing environment and to combat stress conditions
(Ramakrishna and Ravishankar 2011). These compounds are accumulated in plants
in relatively lesser quantities when compared to primary metabolites (Croteau et al.
2000; Dewick 2002). The contents of heavy metals such as silver, lanthanum, mer-
cury, and cadmium were reported to be significantly influenced by synthesis of sec-
ondary metabolites (Marschner 1995). Heavy metals (Cu and Cd) have been
reported to enhance the content of secondary metabolites including shikonins
(Mizukami et al. 1977) and digitalins (Ohlsson and Berglund 1989). Another report
by Trejo-Tapia et al. 2001 suggests Cu-induced elevation in contents of betalains in
Beta vulgaris plants.
Many researchers are employed in enhancing the content of phenolic compounds
such as anthocyanins through application of phytohormones in Fragaria ananassa
(Nakamura et al. 1999) and Daucus carota (Narayan et al. 2005). Elevation in con-
tents of herniarin and its precursor (Z)- and (E)-2-β-d-glucopyranosyloxy-4-
methoxycinnamic acid and umbelliferone was recorded in leaves of Matricaria
chamomilla in response to treatment with salicylic acid (Dučaiová et  al. 2013).
Another report by Dong et al. (2010) suggested that there was an elevation in accumu-
lations of phenolic compounds such as salvianolic acid B, caffeic acid, and the phe-
nylalanine lyase (PAL) activity as a result of exogenous application of salicylic acid.
Ni- and Cd-stressed Matricaria chamomilla plants when treated with 50 μM of
SA resulted in enhancement in activity of PAL as well as elevation in contents of
total soluble phenols (Kovacik et al. 2009). Moreover, Kovacik et al. (2010) reported
that Scenedesmus quadricauda plants under Cu stress treated with 25 μM of SA
showed enhancement in contents of secondary metabolites including protocatechuic
acid and aldehyde, indicating their imperative role in Cu tolerance. Also there was
elevation in content of p-OH benzoic acid and aldehyde, vallinic acid, caffeic acid,
and p-coumaric. Moreover, Lopez-Orenes et al. (2014) also observed an enhance-
ment in the contents of β-caroboline alkaloids in Zygophyllum fabago due to supple-
mentation with 0.5 mM of SA.
136 S.K. Kohli et al.

7.2.7 SA and Metal-Chelating Compounds

One of the most significant mechanisms by which plants counter heavy metal toxic-
ity is activation of sulfur assimilation cascades, which eventually enhance the content
glutathione. In response to increase in levels of glutathione, the de novo synthesis of
metal-ligating compounds such as phytochelatins (PCs) is also enhanced (Ederli
et al. 2004). The sulfur-rich biomolecules are widely present in the cells and regulate
different functions in plant system independently (Mishra et al. 2009). The increase
in sulfur reduction cascade activity is due to modulation of sulfur uptake and trans-
port and also activation of enzymes triggering the pathway (Rausch and Wachter
2005; Herbette et al. 2006). Chelation of heavy metal ions with high-affinity ligands
is primary mechanism for heavy metal detoxification and acclimatization in plants
under metal stress. In response to heavy metal stress, plants synthesize two types of
metal-chelating compounds, i.e., phytochelatins (PCs) and metallothioneins (MTs)
(Yang et al. 2005). PCs are considered as biomarkers for early detection of heavy
metals in plants (Hossain et al. 2006). The PCs are classified in the category of non-
protein-bound thiols along with glutathione and other –SH-rich compounds (Kafel
et al. 2010). The contents of total nonprotein thiol contents have been reported to be
enhanced in several plant species such as Ceratophyllum demersum under Cd stress
(Mishra et al. 2009); Silene vulgaris under Cd stress and combined stress of Zn, Cd,
and Pb (Nadgorska-Socha et al. 2011); and Vaccinium myrtillus under Cd, Pb, and
Zn stress (Kandziora-Ciupa et al. 2013).
PCs are synthesized in the cytosol and are actively transported in the form of
M-PC complexes, i.e., metal-phytochelatin complexes (Manara 2012; Song et al.
2014). PCs have an ability to bind with the toxic metals such as Cu, Cd, Ag, Ni, etc.
Its synthesis is induced in response to several heavy metals including Hg, Ag, Cd,
Au, Cu, Ni, Zn, As, and Pb; however Cd is considered as the strongest inducer (Grill
et al. 1987, 1989; Vatamaniuk et al. 2001). The other important class of metal che-
lating compounds are MTs. Cu-treated plant species have been reported to induced
synthesis of MTs like proteins. MTs are ubiquitously present in the plant system,
have a low molecular weight, proteinaceous in nature, and have the ability to bind
to metal ions (Rauser 1999). There are number of reports which suggests imperative
role of SA in modulating contents of metal-chelating compounds, indicating its
active participation in heavy metal toxicity amelioration and tolerance. Similar
observation of enhancement in content of sulfur and total nonprotein thiol contents
by tenfold was recorded in Hordeum vulgare plants under Cd stress in response to
supplementation with SA (500 μM) (Metwally et al. 2003). Elevation in contents of
glutathione was observed in Medicago sativa plants under Hg stress by application
of 200 μM of SA (Zhou et al. 2009). Moreover, Gondor et al. (2016) reported that
there was a substantial enhancement in contents of PCs and GSH in response to
exogenous application of SA (0.5 mM) to Zea mays plants under Cd stress.
7  Role of Salicylic Acid in Heavy Metal Stress Tolerance: Insight into Underlying… 137

7.3 Conclusion and Future Prospects

Plant under the effect of heavy metals generates huge amount of ROS which eventu-
ally results in oxidative stress. Oxidative stress causes lipid peroxidation, oxidation
of protein and nucleic acids at cellular level. The acclimatization of plants to heavy
metal toxicity is dependent upon uptake, accumulation, degradation, sequestration,
and tolerance mechanisms. Application of PGRs is one such tolerance mechanism.
SA plays an imperative part in modulation of heavy metal stress responses. It
increases tolerance of plant to heavy metal stress by regulating levels of various
metabolites antioxidative enzymes, antioxidants, metal nutrients, osmolytes, sec-
ondary metabolites, and metal-chelating compounds. Owing to the significance of
SA in heavy metal stress management, further investigations are directed toward
identification of molecular and signaling regulators of heavy metal stress tolerance
to better understand the underlying mechanisms and development of optimal strate-
gies for crop improvement under heavy metal stress conditions.

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Heat Stress Tolerance in Plants: Action
of Salicylic Acid 8
Rahat Nazar, Noushina Iqbal, and Shahid Umar

Abstract
Heat stress is a major abiotic stress of global concern, affecting plant growth and
production of plants, particularly crops worldwide. It occurs due to disturbance
in plant metabolism as a consequence of excess generation of reactive oxygen
species (ROS), which leads to oxidative stress. Plants adopt different strategies
to overcome the adverse effects of heat stress. The molecular mechanism of the
heat stress responses and breeding of heat-tolerant plants is essential to protect
the food production. Recently, the role of salicylic acid has received attention in
the regulation of numerous developmental processes under heat stress condition
and has emerged through cross talk between chemical signaling pathways. The
present review focuses on improving our understanding on the mechanism to
induce thermotolerance in plants by salicylic acid interaction and gives an insight
into some scientific approaches to modulate plants’ responses for high tempera-
ture tolerance.

Keywords
Heat stress • Phytohormones • Salicylic acid • Thermotolerance

8.1 Introduction

High temperature has been recognized as a serious threat to crop production world-
wide. The continuous increase in greenhouse gases causes a 1–4 °C rise in average
global temperature during the twenty-first century (Gong et al. 1997; IPCC 2007).
The high day and night temperatures will become more common in the future and

R. Nazar (*) • N. Iqbal (*) • S. Umar

Department of Botany, Jamia Hamdard University, New Delhi 110062, India
e-mail: [email protected]; [email protected]

© Springer Nature Singapore Pte Ltd. 2017 145

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_8
146 R. Nazar et al.

may represent a tremendous environmental hurdle to global food production sug-

gested by many climate modeling studies (Lobell et al. 2011; Cairns et al. 2012).
Temperature is known to strongly influence the distribution and abundance patterns
of both plants and animals, due to the physiological constraints of each species
(Thomas et al. 2004). Therefore, changes in temperature due to climate change are
expected to be one of the important drivers of change in natural and managed sys-
tems. Plants, in particular, as sessile organisms, cannot move to favorable environ-
ments; consequently, plant growth and developmental processes are substantially
affected lethally by high temperature stress (Lobell and Asner 2003; Lobell and
Field 2007).
Unconstrained emission of greenhouse gases (GHG) by various anthropogenic
activities increases the existing concentration of these gases; among them carbon
dioxide (CO2), methane (CH4), chlorofluorocarbons (CFCs), and nitrous oxide
(N2O) are the major sources of contribution to the rise in global temperature (Smith
and Olesen 2010). Keeling et al. (2009) reported that annual-average atmospheric
CO2 concentration rose from about 315 ppm (parts per million) to about 369 ppm
from 1959 to 2001. As stated by the Intergovernmental Panel on Climate Change
(2007), the concentration of CO2 will continue to rise about 500–1000 ppm by the
year 2100. These continuous changes in climate will result in loss of about 15–37%
of species of plants and animals by the year 2050 (Thomas et al. 2004). Thus, for
knowledge-based breeding and selection criteria for heat-tolerant lines, an under-
standing of thermotolerance mechanism through molecular approach has recently
received much attention.
One of the immediate consequences of temperature stress is the change in mem-
brane fluidity which might represent a potential site of perception and/or injury to
plants (Horvath et al. 1998). According to Schöffl et al. (1999), the high temperature
stress causes severe cellular injury and eventually leads to death of the cells, which
could be attributed to a cataclysmic breakdown of cellular organization. Coleman
and McConnaughay (1994) reported that at 35–45 °C, the degradation of protein
accelerates, but the synthesis of protein decreases. Heat stress induces the rapid
production and accumulation of reactive oxygen species (ROS) and can disrupt
chloroplast functions (Apel and Hirt 2004).
For the alleviation of adverse effects of heat-stressed induced factors, plants have
adopted numerous strategies responded by complex and often interconnected sig-
naling pathways (Rasmussen et al. 2013). The mechanisms that are regulated at the
molecular level expedite plants to survive under heat stress conditions. Exogenous
applications of osmoprotectants, signaling molecules, nutrients, etc. have shown
beneficial effects on plants growing under high temperature stress. Although the
physiological and biochemical aspects of thermotolerance are relatively well dis-
cussed, this review is focused on the involvement of salicylic acid signaling under
heat stress and how salicylic acid modulates the crop heat tolerance.
Phytohormone signaling pathways interact with metabolites and play an impor-
tant role in regulation of growth and development of plants under stressful condi-
tions. They also play a significant role in plant perception and response to abiotic
stress. Studies in relation to the role of phytohormones in alleviation of heat-induced
8  Heat Stress Tolerance in Plants: Action of Salicylic Acid 147

stress are few, but there is sufficient significant literature available. Auxin, abscisic
acid (ABA), gibberellic acid (GA), cytokinins (CKs), jasmonic acid (JA), salicylic
acid (SA), ethylene, brassinosteroids (BRs), nitric oxide (NO), and polyamines
(PAs) are found to play an essential role in alleviating heat stress by regulating plant
growth and development processes (Karagezov 2002; Clarke et al. 2009; Zhang and
Wang 2011; Asensi-Fabado et al. 2013; Khan et al. 2013; Song et al. 2013; Lubovská
et al. 2014).

8.2 Impact of Heat Stress on Global Warming

CO2 is a gas with greenhouse properties. The continuous increasing of the atmo-
spheric CO2 concentration leads to increases in temperature and makes food crops
vulnerable to heat stress, a phenomenon widely known as global warming.
Temperatures above the normal optimum are termed as heat stress which affects the
normal cellular homeostasis. It has been thus predicted that mean temperatures will
rise between 1.4 and 4.0 °C by the year 2100 (IPCC 2007). Plant growth and pro-
ductivity are severely affected by global warming. Due to global warming, the heat
waves will increase in frequency and magnitude over most land areas (IPCC 2011)
and increased the risk of heat stress. Rosenzweig and Parry (1994) reported that the
impact of global warming will differ regionally, and it is envisaged that developing
countries will be affected to a greater extent, thereby resulting in increased food
insecurity therein. It may exacerbate the deleterious effects of leaf overheating and
affect number of metabolic processes, including photosynthesis, respiration, meri-
stem initiation, water transport, and phenology during heat stress season (Atkin and
Tjoelker 2003; Ghannoum and Way 2011). Therefore, plant responses and adapta-
tion to elevated temperatures, and the mechanisms underlying the development of
heat tolerance, need to be better understood for important agricultural crops.

8.3  nalysis of the Impact of Heat Stress on Physiological,

A
Biochemical, and Molecular Mechanism in Plants

Heat stress may result in various physiological, biochemical, and molecular changes
in plant metabolism including protein denaturation and perturbation of membrane
integrity (Levitt 1980). Many of these changes in plants that appear during heat
stress acclimation are reversible, but if the stress is much higher than irreversible,
changes may also occur resulting in cell death. Hemantaranjan et al. (2014) reported
that heat stress alters the tertiary and quaternary structures of membrane-bound pro-
teins, resulting in increased membrane permeability, which is evident from increased
loss of electrolytes. This shows that heat stress alters the integrity and functioning
of biological membranes.
On onset of the heat stress, hypocotyls and petioles elongate to change the plant
architecture; this was similar to the morphological responses of shade avoidance
under heat stress (Hua 2009; Tian et  al. 2010). High temperature reduces plant
148 R. Nazar et al.

growth by affecting the shoot net assimilation rates and thus the total dry weight of
the plant (Wahid et al. 2007). It affects the stability of various proteins, membranes,
and RNA species and cytoskeleton structures and the efficiency of enzymatic reac-
tions in the cell and creates metabolic imbalance (Ruelland and Zachowski 2010;
Suzuki et al. 2011, 2012).
High temperature reduces cell size and growth by causing loss of cell water con-
tent (Ashraf and Hafeez 2004; Rodriguez et al. 2005). Reduction in net assimilation
rate (NAR) is also another reason for reduced relative growth rate under heat stress
as observed in maize and millet (Wahid 2007) and sugarcane (Srivastava et  al.
2012).
High temperature stress has a considerable influence on the photosynthetic
capacity of plants especially of C3 than C4 plants (Crafts-Brandner and Salvucci
2002). They also suggested that inhibition of net photosynthesis by moderate heat
stress is the result of inability of Rubisco activase to maintain Rubisco in an active
form (Salvucci and Crafts-Brandner 2004). The primary site of heat stress-induced
injury in chloroplast is the carbon metabolism of the stroma and photochemical
reactions in thylakoid lamellae (Marchand et al. 2005; Wang et al. 2009). Thylakoid
membrane is highly susceptible to heat stress, and the major alterations that take
place inside the chloroplast are the altered structural organization of thylakoids, loss
of grana stacking, and swelling of grana (Ashraf and Hafeez 2004; Rodriguez et al.
2005). It also adversely affects the photosystem II (PSII) activity (Morales et  al.
2003) and reduces the amount of photosynthetic pigments (Marchand et al. 2005).
Heat stress causes many pre- and postharvest morphological changes in plants
including scorching and sunburns of leaves and twigs, branches, and stems, leaf
senescence, abscission, shoot and root growth inhibition, fruit discoloration, and
damage (Rodríguez et al. 2005). It has also been reported that heat stress causes
damage to leaf tip and margins, rolling and drying of leaves, and necrosis, as was
observed in sugarcane (Omae et al. 2012).
Heat stress uncouples light-harvesting complex I and II of photosystems (Hemme
et al. 2014) and alters other metabolic pathways resulting in accumulation of ROS,
most commonly singlet oxygen (1O2), superoxide radical (O2·−), hydrogen peroxide
(H2O2), and hydroxyl radical (OH·) which are responsible for oxidative damage in
plants (Asada 2006). In order to tolerate heat stress, plants produce heat stress factor
(HSF) and heat shock protein (HSP). HSF serves as the terminal component of sig-
nal transduction and mediates the expression of HSP. According to Chakrabortty
and Pradhan (2011), catalase (CAT), ascorbate peroxidase (APX), and superoxide
dismutase (SOD) showed an initial increase in their activities at lower temperature
stress before declining at 50 °C, while that of peroxidase (POX) and glutathione
reductase (GR), they found decline at all temperature ranges (20–50 °C). In addi-
tion, tolerant varieties showed maximum activities of antioxidant enzymes at tem-
perature range between 35 and 40 °C, while susceptible varieties showed at 30 °C.
MBF1c, a highly conserved transcriptional coactivator, is upregulated through heat
stress. Suzuki et al. (2008) reported that MBF1c is a key regulator of basal thermo-
tolerance and provided evidence for the existence of a coordinated heat stress-­
response network involving salicylic acid, trehalose, and ethylene-signaling
8  Heat Stress Tolerance in Plants: Action of Salicylic Acid 149

pathways under the control of MBF1c. In Arabidopsis, DREB2A, which has a nega-
tive regulatory domain in its middle region, plays an important role in heat stress.
The deletion of this region transforms DREB2A into a constitutively active form
(DREB2A CA). The overexpression of DREB2A CA in transgenic Arabidopsis
enhanced tolerance to heat/drought stress (Sakuma et al. 2006). The “omics” tech-
nologies have provided novel opportunities and expectations for the identification
of transcriptional, translational, and posttranslational mechanisms and signaling
pathways that regulate the plant responses to abiotic stress including high tempera-
ture (Aprile et al. 2009; Hasanuzzaman et al. 2013). In addition, microarray tech-
nology has recently become a powerful tool for the systematic analysis of expression
(or transcriptome) profiles of large numbers of genes that are induced or repressed
by heat treatment (Liu et al. 2011; Yeh et al. 2012). Recent microarray studies in
Arabidopsis deficient with APX gene, however, have found that certain HSPs are
expressed typically under other stress conditions, although expression of HSPs
under heat stress occurs normally (Penueli et  al. 2003). In an experiment on the
combined effects of CO2 and temperature on the grain yield, Mitchell et al. (1993)
observed that a temperature of 27 °C or higher applied midway through anthesis
could result in a high number of sterile grains and considerable yield losses. Wheeler
et al. (1996) used a temperature gradient tunnel system to demonstrate that a tem-
perature of 30 °C or elevated prior to anthesis significantly reduced the grain num-
ber and, subsequently, yield. Crop yields are predicted to decrease approximately
10% for every one-degree increase in temperature.

8.4 Phytohormone Signaling in Heat-Stressed Plants

Plants have evolved a series of complex response mechanisms to receive and

respond to diverse external signals. Figure  8.1 shows various major strategies
involved in heat stress tolerance. Phytohormones are important components in mul-
tiple signaling pathways. They act synergistically or antagonistically to regulate
plant growth and development, nutrient allocation, and source-sink transitions and
induce defense mechanism by inducing gene expression. The studies on the influ-
ence of phytohormone signaling in mitigating high-temperature stress are less;
however, in recent years, a progress has been gained toward the elucidation of phy-
tohormone signaling in heat stress plants (Clarke et al. 2009; Sakata et al. 2010;
Baron et al. 2012; Piterková et al. 2013). Indoleacetic acid (IAA) and ABA induce
signal transduction pathway and also regulate Kentucky bluegrass growth and qual-
ity under heat stress (Li et al. 2014). Auxin modulates positively and negatively the
expression of certain genes through two related families of proteins, the AUX/IAA
proteins and auxin response factors (ARFs) (Ulmasov et  al. 1997; Rouse et  al.
1998). In Arabidopsis, β-aminobutyric acid (BABA, a nonprotein amino acid)
enhanced acquired high temperature tolerance, possibly through ABA pathway
(Zimmerli et al. 2008). Song et al. (2006) showed that NO act as a signal in induce-
ment of thermotolerance in plant by activating active oxygen scavenging (AOS).
150 R. Nazar et al.

Fig. 8.1  Diagrammatic representation of major tolerance strategy under heat stress

Increased NO production was also observed in tobacco leaf cells and heat-stressed
alfalfa (Gould et al. 2003). Involvement of brassinosteroids (BRs) in plant responses
to heat stress is also reported (Ogweno et al. 2008; Zhang et al. 2013). SA acts as a
stress signaling substrate and has been found to protect potato, mustard, tomato,
bean, Arabidopsis, and wheat from heat stress (Dat et  al. 1998; Larkindale and
Knight 2002; Khan et al. 2013). Ethylene (Suzuki et al. 2008; Li et al. 2011) and
CKs (Arnison et al. 1990; Sayed 1999) have been reported to transmit heat stress
signal in plants and alleviate detrimental effects of high temperature stress.
Figure 8.2 represents phytohormones signaling under heat stress.
8  Heat Stress Tolerance in Plants: Action of Salicylic Acid 151

Heat stress

Perception of stress

Signal transduction

Activation of stress responsive genes

MAP kinase cascade ROS accumulation

Phytohormones signaling
RECEPTOR
MOLECULES

Signal sensed by phytohormones receptor molecules

Up regulation of S Induction of Increases heat

and N assimilation osmoprotection shock protein

Up regulation of genes Chaperon signaling

Maintains cell
of S and N assimilation and transcription
water balance
pathways activation

Increased N and S Reestablishment of cellular

allocation to homeostasis, functional and Protection from
photosynthesis structural protection of proteins oxidative stress
and membranes

Improved photosynthesis

Induces Heat
Tolerance

Fig. 8.2  Diagrammatic representation showing the role of phytohormone-mediated tolerance in

heat-stressed plants. ROS reactive oxygen species
152 R. Nazar et al.

8.5 Heat-Mediated Modulation of Phytohormones

A surplus of studies have shown promising roles of phytohormones such as IAA,

GA, CK, ethylene, ABA, SA, NO, JA, BRs, and PAs in plant heat stress tolerance
(Table 8.1). A brief introduction of phytohormones and their modulation in heat-­
stressed plants are discussed. Optimal growth and development of plants depend
from different environmental factors. The phytohormones regulate key physiologi-
cal events under normal and stressful conditions for plant growth and development.
Thus, considerable attention has been devoted in alleviating the detrimental effects
of high temperatures in plants through the exogenous application of
phytohormones.

8.6 Salicylic Acid

Phytohormones help plant in adjusting to heat stress by modifying the plants’ abil-
ity to adapt under changing environments either through induction of osmolyte syn-
thesis, increase in antioxidants, or utilization of nutrients. Endogenous levels of SA
and its glucoside have been shown to increase in plants subjected to heat, ozone, or
UV. Khan et al. (2013) reported that SA treatment under heat stress increased pro-
line production which increased osmotic potential enabling the plants for higher
water intake resulting in positive influence on stomatal aperture and photosynthetic
machinery leading to higher efficiency of PS II and increased Rubisco activity that
cumulatively resulted in increased photosynthesis. In cucumber plants (Cucumis
sativus L.), foliar spraying with 1 mM SA induced heat tolerance, as shown by the
lower electrolyte leakage parameter, lower H2O2, higher catalase activity and lipid
peroxide levels, and higher Fv/Fm chlorophyll a fluorescence value, whereas the
hydroponic application of the same concentration had the opposite effect (Shi et al.
2006). SA is found to increase the photosynthetic rate in grape leaves under heat
stress (Wang et al. 2010) and can alleviate the heat-induced damage in plants by
upregulating the antioxidant system (Wang and Li 2006).
SA is known to stabilize the trimers of heat shock transcription factors and to aid
them in binding to the heat shock element in the promoter of HSP genes (Jurivich
et al. 1992). Wang et al. (2010) studied that before heat stress, SA did not affect
level of HSP21, but the HSP21 immune signal increased in both SA-treated and
control leaves during heat stress. Thermotolerance can be induced in potato plants,
however, by treatment with an acetyl-SA spray (Dat et al. 1998), and the induced
thermotolerance is extremely long lasting (Lopez-Delgado et al. 1998). Exogenous
application of SA or acetylsalicylate has been shown to enhance thermotolerance in
tobacco and Arabidopsis (Dat et al. 1998; Lopez-Delgado et al. 1998; Clarke et al.
2004). Wang and Li (2006) reported that spraying 0.1 mM solution of SA decreased
thiobarbituric acid reactive substances and relative electrolyte leakage in young
grape leaves and increased antioxidative enzymes, indicating that SA can induce
intrinsic heat tolerance in grapevines. Pan et al. (2006) reported that inhibition of
SA synthesis using inhibitors of SA synthesis under heat stress not only reduced the
endogenous SA content but also the level of heat tolerance.
8  Heat Stress Tolerance in Plants: Action of Salicylic Acid 153

Table 8.1  Phytohormone-mediated heat stress tolerance in plants

Phytohormones Plants Parameters influenced References
Auxin Arabidopsis Expression of MsCPK3 + Davletova et al. (2001)
gene involved in hormone
synthesis
Expression of YUCCA + Sakata et al. (2010)
auxin biosynthetic gene
Abscisic acid Arabidopsis Reduces oxidative stress − Larkindale and Knight
(2002)
Abscisic acid Cicer arietinum Osmolytes + Kumar et al. (2012)
Gibberellic Raphanus Amylase activity + Cavusoglu and Kudret
acid sativus (2007)
Hordeum
vulgare
Gibberellic Phaseolus Amylase activity + Mansoor and Naqvi
acid vulgaris (2013)
Gibberellic Arabidopsis Modulates SA biosynthesis + Alonso-Ramírez et al.
acid Seed germination and (2009)
establishment
SA signaling and HSP + Zhang and Wang
accumulation (2011)
Cytokinin Zea mays L. Modulating the activity of + Caers et al. (1985)
antioxidant enzymes
Cytokinin Nicotiana Antioxidant enzymes + Synkova et al. (2006)
tabacum
Cytokinin Arabidopsis Leaf longevity and + Xu et al. (2010)
photosynthetic capacity
Cytokinin Nicotiana Antioxidant defense, + Lubovská et al. (2014)
tabacum source-sink relation
Cytokinin Agrostis CAT and SOD activity + Liu and Huang (2002)
stolonifera Action of protease −
Upregulation of heat shock + Veerasamy et al.
proteins (2007)
Leaf senescence −
Enhance antioxidant + Xu and Huang (2009)
metabolism
Jasmonic acid Raspberry Total phenolics + Ghasemnezhad and
Javaherdashti (2008)
Jasmonic acid Arabidopsis Photosystem II + Heckatorn et al.
(1998)
Role in stomatal control + Munemasa et al.
(2011)
Salicylic acid Cucumis sativus Electrolyte leakage − Shi et al. (2006)
L. H2O2 −
Salicylic acid Vitis vinifera Antioxidants + Wang and Li (2006)
HSP21 + Wang et al. (2010)

(continued)
154 R. Nazar et al.

Table 8.1 (continued)
Phytohormones Plants Parameters influenced References
Salicylic acid Triticum Proline + Khan et al. (2013)
aestivum
Ethylene Arabidopsis TBARS − Larkindale and Knight
(2002)
HSP, APX1 + Wu et al. (1994)
Brassinosteroid Brassica juncea Synthesis of heat shock + Dhaubhadel et al.
proteins (1999)
Brassinosteroid Lycopersicon Carboxylation efficiency + Ogweno et al. (2008)
esculentum and antioxidant system
Brassinosteroid Cucumis sativus H2O2 level + Xia et al. (2009)
Brassinosteroid Oryza sativa Pollen germination + Thussagunpanit et al.
Seed setting + (2013)
Brassinosteroid Cucumis melo Upregulation of + Zhang et al. (2013)
L. photosynthetic pigment
Photochemical activity +
Brassinosteroid Solanum Stomatal conductance + Wu et al. (2014)
melongena Quantum efficiency of PSII +
Nitric oxide Oryza sativa Expression of transcripts + Uchida et al. (2002)
for stress-related gene
Small heat shock protein 26 +
Rubisco activity and + Song et al. (2013)
carotenoid
Nitric oxide Phragmites ABA-induced + Song et al. (2008)
communis Trin. thermotolerance
Active oxygen-scavenging + Song et al. (2006)
enzyme
Nitric oxide Lycopersicon Regulation of HSP70 + Piterková et al.
esculentum production (2013))
Nitric oxide Festuca Recovery of PSII + Chen et al. (2013)
arundinacea
Polyamines Phaseolus Recovery of root and + Basra et al. (1997)
vulgaris hypocotyls
Polyamines Glycine max Membrane integrity + Amooaghaie and
Moghym (2011)
Polyamines Cucumis sativus Antioxidant system + Asthir and Deep
Electrolyte leakage − (2011)
Polyamines Arabidopsis Expression of heat shock + Sagor et al. (2013)
related gene
Polyamines Trifoliate orange Antioxidant enzymes + Fu et al. (2014)
seedlings Expression of stress-related +
gene
Polyamines Oryza sativa Antioxidant system, + Mostofa et al. (2014)
glyoxalase system +
(+), (−) symbols indicate increase or decrease, respectively
8  Heat Stress Tolerance in Plants: Action of Salicylic Acid 155

Larkindale et  al. (2005) screened Arabidopsis mutants and NahG transgenic
plants for their basal and acquired thermotolerance and showed that ABA, ROS, and
SA play a role in the development of acquired thermotolerance. However, in another
experiment on Arabidopsis plants, SA was shown to be essential for basal but not
for acquired thermotolerance (Clarke et al. 2004).
Liu et al. (2008) reported that another strategy by which SA induces heat toler-
ance is through the changes in the activities of plasma membrane H+ and Ca2+-
ATPase. Liu and Huang (2005) reported that the thermotolerance induced by
exogenous SA was related to the Ca2+-CaM system in grape plants. SA increased
the activity of H+-ATPase in grape plant leaves and kept the stability of the plasma
membrane H+-ATPase following heat stress. SA induced an increase of the Ca2+
level in cells and activated the Ca2+-CaM system, which further stimulated the activ-
ity of Ca2+-ATPase. Finally, the Ca2+ level in the cytoplasm was regulated and main-
tained at a normal level by Ca2+-ATPase to protect cells and to avoid injury resulting
from heat shock.
Khan et al. (2015) reported that SA proves beneficial for plants both under opti-
mal or stress environments. It modulates the production of osmolytes and other
metabolites and plant nutrient status to protect plants under abiotic stress condi-
tions. SA induction in Cleopatra was not sufficient to protect PSII from photoinhibi-
tion, resulting in higher malondialdehyde (MDA) buildup (Zandalinas et al. 2016).
Galani et al. (2016) suggested that the exogenous application of SA mitigated the
deleterious effects of heat stress on Gossypium hirsutum seedlings by reducing
membrane damage through reduced electrolyte leakage, MDA content, and H2O2
content. It reduced dehydration losses through triggering antioxidative defense sys-
tem and increasing proline accumulation. Thus SA offers an efficient, economic,
and simple means to enhance heat tolerance in cotton.

8.7 Conclusion and Future Prospects

From the current review, it clearly emerges that heat stress is a major concern for
crop production worldwide as it affects the growth, development, and productivity
of plants. The increase in temperature reduces overall plant growth and productivity
and disturbs the physiological as well as molecular processes. Responses and adap-
tations of plants to heat stress and the mechanisms underlying the development of
heat stress tolerance need to be understood for the agricultural crops. Phytohormones
regulate a number of signaling networks involving developmental processes and
plant responses to environmental stresses including biotic and abiotic stresses. One
strategy that provides a more targeted approach involves manipulating the expres-
sion of genes through the phytohormones, which would affect a subset of stress-­
responsive genes, play a protective role in regulating antioxidant defense system,
increase the synthesis of chaperones, induce the expression of heat shock proteins,
elevate the level of osmolytes, and increase the expression of cell detoxification
enzymes for redox homeostasis in the development of heat stress tolerance mecha-
nism. In this review an attempt was made to summarize the role of phytohormones
156 R. Nazar et al.

under heat stress in regulating a number of signaling networks, modulate the expres-
sion of HSP, and induce different metabolites such as antioxidant and osmoprotec-
tants and different metabolic pathways and processes. This will give more insight in
the mechanism of heat stress tolerance in order to develop heat-tolerant genotypes.
Since the output of plant hormone action depends on specific hormone combina-
tions rather than on the independent activities of each hormone and different hor-
mones affect overlapping processes, therefore, a cross talk of phytohormones
especially salicylic acid in regulating all the abovementioned mechanisms might
also exist. Further study needs to be elucidated to understand the cross talk between
phytohormones and heat stress tolerance through the regulation of different physi-
ological and molecular mechanisms in order to develop heat-tolerant genotypes.

Acknowledgment  RN and NI are thankful to University Grant Commission, New Delhi, for
funding in the form of Dr. D.S. Kothari Postdoctoral Fellowship and to the head of the Department
of Botany, Jamia Hamdard University, New Delhi, for providing research facilities.

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Salicylic Acid: Molecular Basis of Stress
Resistance in Plants 9
Uğur Uzuner, Aykut Sağlam, and Asım Kadıoğlu

Abstract
Plants and their casual pathogens are in a continuous evolutionary battle and
combat with each other in order to overwhelm their counter mechanisms. Beyond
physical barriers and natural openings in plants, all pathogens retain a plethora
of various strategies to overcome plant defense mechanisms. Conversely, plants
employ mainly two-tiered immune system to resist pathogen attacks, on molecu-
lar basis, basal and durable immune response, both which stem from highly com-
plicated and genetically controlled pathways including the incorporation of
major gene(s) and multiple genes with minor effects, respectively. Plant immune
response and following pathogen resistance are accomplished through percep-
tion of pathogen-derived effectors, elicitors, and conserved specific pathogen
molecules. Recent advances in biotechnology, bioinformatics, next-generation
sequencing, and all omics platforms provide useful and novel insights over tre-
mendous examples of persistent plant-pathogen combat such as the discovery of
new resistance genes, miRNAs, siRNAs, transcription factors, and such. Rising
environmental and ecological disarrangements, however, further affect pathogen
populations remarkably, mostly encouraging the development of highly aggres-
sive or novel pathogens, pointing to recurrent struggling between pathogen and
plants.
In this review, we disclose insights into genetic and molecular aspects of basic
plant resistance systems to highlight recent advancements and role of salicylic
acid over various stress conditions.

U. Uzuner (*) • A. Sağlam

Molecular Biology and Genetics, Karadeniz Technical University, 61080 Trabzon, Turkey
e-mail: [email protected]
A. Kadıoğlu
Biology, Karadeniz Technical University, 61080 Trabzon, Turkey

© Springer Nature Singapore Pte Ltd. 2017 163

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_9
164 U. Uzuner et al.

Keywords
Plant • Pathogen • Basal • Durable • Resistance genes • miRNAs • siRNAs
• Transcription factors • Perception

9.1 Introduction

The increasing human population in the world brings out rising environmental and
ecological problems with that. Over 7 billion humankind need to maintain their
lives by gathering nutrients from their living lands. The global future life planning
attempts enforce us to develop new strategies in order to provide enough food to
rising world population, by combatting with adverse ecological, environmental,
pathological, and global changes (Anderson et al. 2016; Huesing et al. 2016). The
indispensable and biggest threat to human and plant life today is obviously global
warming, which adversely affects not only ecosystems but also pathogen popula-
tions (Juroszek and von Tiedemann 2011). These sorts of alterations are becoming
much more noticeable for every one of us and further speed up due to the rapid
technological and industrial developments. The rapid environmental and ecological
variations also affect pathogen populations remarkably and mostly trigger the devel-
opment of highly aggressive or novel pathogen agents, not just for plants but also
for human health. One of the most obvious signs of these alterations is augmented
pandemics and outbreaks among both human and plant populations such as viral
outbreaks, intercontinentally moving plant diseases (Hubbard et al. 2015; Leclerc
et al. 2014; Lidder and Sonnino 2012). To adapt altering world-living conditions
and develop the best strategies to heal adverse conditions or at least secure the equi-
librium to dramatic changes occurring in our environment, an immediate counteract
is also requisite. Providing new strategies in order to get the highest crop yield from
our agricultural efforts, we also need to evolve crop-growing strategies and further
increase the efforts to supply sufficient yield to meet with increasing world popula-
tion needs. Beyond conventional breeding endeavors to reach these goals, one of the
major challenges is to overcome disease developing strategies evolving consistently
and employed by pathogens on cereal crops.
Developing new strategies to alleviate plant disease occurrences and increase
crop resistance by eliminating the pathogenicity agents from environment, improv-
ing the production capacity in arable lands, saving freshwater resources, and declin-
ing fuel use for agricultural applications is imperative for efficient food production
(Bale et al. 2008; Ratnadass et al. 2012; Turner et al. 2013). Plant disease resistance
is important for food production, reducing the use of lands, saving freshwater
resources, and declining fuel use for agricultural applications. Disease resistance is
carried in both natural and cultivated plant populations; however, they haven’t
always protected themselves from pathogens (Dangl et  al. 2013). Plants possess
preformed defenses such as cuticle, cell walls, antimicrobial chemicals (glucosides,
saponins, etc.), antimicrobial proteins, enzyme inhibitors, and detoxifying enzymes
that break down pathogen-derived toxins and infection-induced responses of their
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 165

immune systems (Lutz 2012; Zhang et al. 2013). When plants exposure to biotic or
abiotic stimuli, plant’s induced resistance (IR) mechanism takes into action, if the
physical barriers are not successful for the restriction of pathogen infection. IR
mainly follows two different but advanced physiological routes: systemic acquired
resistance (SAR) and induced systemic resistance (ISR). SAR is usually initiated
right after a pathogen attack in plants; upon the accumulation of phytohormone
salicylic acid (SA), induction of pathogenesis-related (PR) gene expression, and
often the development of hypersensitive response (HR) (Harel et al. 2014).
Membrane proteins termed pattern recognition receptors (PRRs) are the first level
of microbe recognition, and they perceive molecular signatures characteristic of
pathogen-associated (or microbe-associated) molecular patterns (PAMPs) (Zhang
et  al. 2013). A chain of signaling events which is referred to general defense
responses in plants starts with PAMP recognition and results in plant SAR (Nicaise
et al. 2009).
Most soilborne microorganisms such as plant growth-promoting rhizobacteria
and fungi are able to promote induced systemic resistance (ISR) with the coloniza-
tion at plant roots. ISR is mainly triggered and regulated through the sophisticated
cross talk occurring between ethylene (ET) and jasmonic acid (JA) plant hormones
and helps plants to provide more efficient response to pathogen (bacteria, fungi,
nematodes, and oomycetes) (Harel et al. 2014). These responses include differences
in cell wall composition, accumulation of chitinases and glucanases as pathogenesis-­
related proteins, and synthesis of phytoalexins (Annapurna et al. 2013).
New defense strategies mostly employed in the light of recent molecular biol-
ogy-, bioinformatics-, plant pathology-, systems biology-, and biotechnology-based
advances, beyond plant breeding systems, are currently focusing on not only the
integration of R genes into cereal crops but also some coordinated novel approaches
including the co-expression of defense pathway-related TFs, miRNAs, and siRNAs,
RNAi-based neutralization of viral pathogenicity agents (mostly proteins), and
manipulation of defense response regulators to channel the targeted specific path-
way to confer desired and timed immune response.
In this review, molecular aspects of plant resistance will be discussed, including
the genetic basis for plant resistance, signaling pathways, perception of pathogens,
and recent trends to tackle with plant pathogens to confer durable resistance.

9.2 Major Disease Resistance Systems in Plants

9.2.1 Basal Defense

9.2.1.1 MAMP-/PAMP-/DAMP-Triggered Immunity (PTI)

Like the anatomy of human and animals, plants also harbor highly sophisticated
response systems against biotic and abiotic stress conditions and employ them with-
out no doubt when needed. This advanced similarity between plant and animal sys-
tems with respect to the mode of action of defense responses affected the naming
called as plant innate immunity or plant immune system, in particular through the
166 U. Uzuner et al.

recent findings established in the last two decades. The excessive complexity of
plant defense response network is recognized as highly intricate, and this phenom-
enon is unceasingly unraveled by means of high-throughput and proficient new
technology-based findings unrevealed through reverse genetics, transcriptomics,
bioinformatics, next-generation sequencing, and such.
Mainly, plants employ two major defense systems when encountered with all
kinds of stress conditions: basal and durable immune defense responses. Basal
immune response is triggered upon the identification of pathogen molecules by
extracellular plant cell surface receptors and/or through natural openings. These
kinds of receptors seeming to be “plant cell bodyguards” or “threat informers” are
also called as pattern recognition receptors (PRRs). PRRs are indispensable for
plant tissues for the recognition of pathogen, herbivore attacks, even for the identi-
fication of adverse and rapid environmental alterations. PRRs are a class of plasma-­
membrane-­bound extracellular receptors of plant cells and eligible to recognize the
conserved pathogen-associated molecular patterns (PAMPs), also called as
microbial-­associated molecular patterns (MAMPs) of pathogen. This PRR-initiated
defense response is called as PAMP-triggered immunity (PTI) (Boller and He 2009;
Dodds and Rathjen 2010). In addition, the new coming findings point out the pres-
ence of some other plant PR receptors functional in both animal and plant cells
which can also recognize endogenous host molecules following any cell or tissue
damage (Mogensen 2009). In other words, the host cell molecules can also trigger
host defense response, by informing the possible threats to host integrity. The mol-
ecules such as oligouronides, cellodextrins, and cutin monomers, most likely
released from damage-exposed host cells, alerting defense responses in the host
organisms are so-called as DAMPs (damage-associated molecular patterns). In
addition to PAMPs and MAMPs, it is therefore strongly accepted that endogenous
host DAMPs are also responsible for the induction of PTI response conditionally
(Postel and Kemmerling 2009).

9.2.2 Durable Defense

9.2.2.1 Effector-Triggered Immunity (ETI)

The second layer of plant defense also known as durable immune response is mainly
induced through the recognition of pathogen effectors by plant R proteins, and this
defense is called as effector-triggered immunity (ETI), leading to rapid and acute
defense responses in plants, such as HR (Muthamilarasan and Prasad 2013). ETI
was formerly called “R gene-based immunity” or “vertical immunity” through the
casual result of “gene-for-gene theory.” The exact description of gene-for-gene the-
ory was reported as “For every dominant avirulence (Avr) gene product in the
pathogen, there is a cognate resistance (R) gene protein in the host, and the direct or
indirect interaction of these proteins leads to the activation of host defense responses,
which result in the restriction of pathogen invasion” (Stergiopoulos and Wit 2009).
Because of its strength and intensity to pathogen invasion, PTI response is dif-
ferentiated from ETI. Thus, it is suggested that PTI response of plants is moderately
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 167

weaker and employed by a slower mode of action against pathogen attacks as com-
pared to ETI. On the other hand, PTI and ETI trigger similar physiological altera-
tions in plant compartments to stress conditions; both are typically eligible to
recognize abnormal conditions and pathogen attacks through PRRs, which are
mostly extracellular or transmembrane proteins. In other words, both immune
responses need PRR proteins at certain degrees in order to transfer their signals to
downstream response components.
ETI is induced in infected plants as major defense response against many biotro-
phic and hemibiotrophic pathogens. The genetic background of both pathogen and
host crop plants is quite specific. This specificity is also essential for R gene-based
recognition of effector molecules, suggesting the availability of highly conserved
incompatible or compatible interactions between two players. The recognition of
pathogen effectors (disease development agents) by the host R proteins could occur
in a direct or indirect manner, mostly following the injection of the pathogen effec-
tors into host crop cytoplasm through type three secretion system (T3SS). For
instance, Magnaporthe grisea AvrPita effector protein is recognized by the rice
NBS-LRR protein Pita through direct interaction (Jia et  al. 2000). Pto protein
directly interacts with bacterial effector proteins AvrPto and AvrPtoB, both of which
trigger resistance (Ntoukakis et al. 2014).
The recognition of MAMPs, PAMPs, or DAMPs triggers rapid defense response
which promptly occurs in hours, generally known as nonhost PTI response. ETI
response, however, requires the recognition of highly conserved pathogen effectors
by the R gene products (R gene encoding proteins) or its guardee proteins, resulting
in an immediate HR, also known as programmed cell death (PCD), mainly aiming
to restrict the pathogen invasion in the host. HR is triggered through the direct or
indirect recognition of specific effectors (these could be an elicitor, an Avr protein,
or a small peptide) by the R proteins, and it is a well-established strategy of host
plants. By doing so, host organisms in fact sacrifice some of their cell groups to
combat with pathogen, restrict the pathogen attacks, limit the incidence of disease
development, minimize the pathogen-based costly damage, and maintain its
integrity.
Recently, a different approach to apoplastic necrotrophs resulted in a new
description of ETI, called as effector-triggered defense (ETD) (Stotz et al. 2014). In
this scenario, both ETI and ETD are mediated by R genes. Unlike the intracellular
detection of pathogen effectors with ETI, for pathogens localizing at intercellular
spaces, the crop plant recognition occurs extracellularly, with the aid of cell surface-­
localized receptor-like proteins (RLPs). Further, because the necrotrophic invaders
can proliferate and are eligible to complete their sexual cycles in intercellular
spaces, this recognition does not even provide immunity, but defense response at
certain levels.
The discovery of the first plant receptor, enable to recognize highly conserved
PAMPs, lasts to the 1990s; the rice PRR XA21 encoding a receptor-like kinase and a
member of a multigene family (confers resistance to Xanthomonas oryzae pv. oryzae
race 6) was posted in 1995 (Song et al. 1995). In 2000, the second PRR was identi-
fied in model plant Arabidopsis thaliana as FLS2 (flagellin-sensitive 2, responsible
168 U. Uzuner et al.

for the recognition of well-studied bacterial PAMP molecule flagellin) (Gomez-

Gomez and Boller 2000). FLS2 is a leucine-rich repeat receptor-like kinase (LRR-
RLK) membrane protein and responsible for the recognition of bacterial elicitor
flagellin during pathogen attack. In addition, several other PRRs were released with
no delay; EFR for bacterial EF-Tu (Zipfel et al. 2006), CERK1 (Miya et al. 2007) for
fungal chitin, the receptor-like proteins (RLPs), LeEix for fungal xylanase EIX (Ron
and Avni 2004), and CEBiP for fungal chitin (Kaku et al. 2006) exemplify some of
well-characterized PRRs/PAMPs (Dou and Zhou 2012; Gohre and Robatzek 2008).
The structural features of PRR proteins are also highly diverse, and they localize
on different sites in plant tissue but mostly in plasma membrane, as the second most
essential defense layer of plant mechanism after cuticle, cell wall, and stomata-like
primary physical barriers. On the other hand, some defense response-associated
PRR proteins function as intracellular immune receptors, having mostly the NOD-­
like receptor (NLR) type. This type of intracellular receptors can directly or indi-
rectly recognize virulence effectors secreted into host cells by pathogen secretion
systems (the well-studied example is T3SS) and enable inducing effector-triggered
immunity (ETI). The PRR recognition is then transferred to a plethora of intracel-
lular downstream signal transduction pathway-related key player proteins, most of
which are involved in the recognition-based signal regulatory mechanism and the
activation of transcription factors to induce defense response-related gene expres-
sion. A more comprehensive and recent update on PRR proteins and downstream
signaling workflow is also available elsewhere (Macho and Zipfel 2014).
Wan and his colleagues (2008) identified LysM receptor-like protein (LysM
RLK1) required for chitin signaling in Arabidopsis thaliana (Wan et al. 2008). They
have revealed that LysM RLK1 protein is responsible for the expression of almost
all chitooligosaccharide-responsive genes in plants to confer resistance against fun-
gal pathogens. Together, the results have indicated that LysM RLK1 is essential for
chitin signaling in plants and is involved in chitin-mediated plant innate immunity
(Wan et al. 2008).

9.2.3 Major Resistance (R) Genes

Pathogens aim to maintain their generations over time by expanding their popula-
tions around and trying to find the best niche for their survival, like every kind of
plant performs. Pathogens thus use astonishing strategies to circumvent or hijack
host plant immune systems to colonize. Bacteria, fungi, and oomycetes produce
specialized molecules and deliver onto host plant cells where they localize to gain
their nutrient needs by overcoming PAMP-triggered immunity (PTI) (Misra et al.
2016; Niu et al. 2016; Oliveira-Garcia and Deising 2016). These specialized mole-
cules are called as effectors which encoded by Avr genes. Effectors molecules are
employed by pathogens to manipulate host cellular mechanisms to favor pathogen
invasion. Host plants, however, respond to these kinds of attacks by using their
devoted genome potential and trigger the expression of R genes to neutralize the
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 169

adverse effects of pathogenicity agents at the infection site and defend themselves
against pathogen invasion for their survival (ETI) (Oh and Martin 2011). The recog-
nition of pathogen effectors by host plant cells is performed by R gene products or
by its guardee proteins, previously named as “gene-for-gene theory” (De Wit et al.
2009; Jones and Dangl 2006).
There are several different types of R proteins essential for various functions for
innate immunity of host plants. The first and largest class of resistance genes
described to date contains an intracellular NBS (nucleotide-binding site) LRR
(leucine-­rich repeat) structure, revealing high similarity to animal NOD-like recep-
tors (Zipfel et al. 2006). NBS-LRR proteins may contain an N-terminal TIR (Toll/
interleukin-1 receptor) domain or coiled-coil (CC) motif which are the two major
classes of NBS-LRR proteins. P. syringae RPS2 and RPM1 resistance genes of
Arabidopsis and the tomato Fusarium oxysporum resistance gene I2 are the two
examples to CC-NBS-LRR resistance genes. The recognition of pathogen effectors
by NBSLRR proteins occurs through the coordinated action of local and systemic
signaling pathways, which in turn the induction of hormonal networks (SA and
JA), HR, reactive oxygen species (ROS), and activation of WRKY and TGA tran-
scription factors. The third class of resistance genes consists of a transmembrane
domain (TrD) and extra cytoplasmic eLRR (Gururani et al. 2012). The Cf-9, Cf-4,
and Cf-2 resistance genes of Lycopersicum esculentum having TrD-eLRR structure
are functional for disease resistance to Cladosporium fulvum pathogen (Gururani
et al. 2012). The fourth class of R genes contains an extracellular LRR domain, a
membrane spanning TrD domain, and an intracellular serine-threonine kinase
(KIN) domain (LRR-TrD-KIN) (Song et  al. 1995). For instance, rice Xa21 and
Xa26 resistance genes conferring resistance against to Xanthomonas oryzae patho-
gen are in this group (Sun et al. 2004). The fifth class of R genes is more likely
responsible for protein degradation which includes an extracellular LRR, short
protein motifs (ECS), and PEST (Pro-Glu-Ser-Thr) domain essential for protein
degradation. Tomato Ve1 and Ve2 R gene products are classified into this group
(Gururani et  al., 2012). The sixth class of R genes consists of the two common
domains from other R genes, LRR and a putative CC domain (e.g., Arabidopsis
RPW8 gene). The seventh class of R genes is recently described within the TIR-
NBS-LRR resistance gene class with a C terminal extension, nuclear localization
signal, and WRKY binding domain (e.g., Arabidopsis RRS-1 gene conferring
resistance to Ralstonia solanacearum) (Deslandes et al. 2002). The eighth type of
oligogenic resistance genes is enzymatic R genes and mostly encodes protein
kinases which do not contain any NBS or LRR domain. It has been shown that
protein kinase Pto protein confers resistance in tomato to bacterial pathogen aviru-
lence gene product AvrPto of P. syringae (Martin et al. 1993). Maize Hm1 gene
encoding HC toxin reductase is another good example of protein kinases as R gene
product which confers resistance to corn leaf blight disease agent Cochliobolus
carbonum (Gururani et al. 2012).
170 U. Uzuner et al.

9.3 Signaling Molecules Involved in Plant Immunity

9.3.1 Systemic acquired resistance (SAR)

SAR is a broad-spectrum plant immune response against pathogen attack since it is

effective against a wide range of pathogens. SAR response is triggered throughout
entire plant tissues following an earlier exposure to pathogen at primary tissues.
This exposure is perceived by various PRRs at the site of pathogen attack, and this
perception initiates the production of several signaling molecules at primary infec-
tion site in order to warn other parts of infected plant. The threat is immediately
transferred to other parts of the plant through different sorts of signaling molecules
to combat against pathogen invasion (Dempsey and Klessig 2013).

9.3.1.1 Salicylic Acid (SA)

SA is one of the most crucial signaling molecules for disease resistance in plants.
Both PTI and ETI are eligible to induce SAR through various signaling pathways by
employing a plethora of different players. The typical signs of SAR could be identi-
fied with SA accumulation at both primary and secondary infection sites, expression
of PR genes (PR1, PR2, and PR5), and a broad-spectrum, long-lasting systemic
resistance to pathogen infection (Dempsey and Klessig 2013). There have been sev-
eral SAR signaling molecules identified over time with multifaceted roles and dif-
ferential effects on long-distance transfer of SAR signal through vascular systems.
In addition to SA, there are also several SAR signals identified recently: methyl
salicylate (MeSA) which is a putative long-distance SAR signal up to now identified
as a methylated derivative and inactive form of SA, a G3P (glycerol-3-phosphate
dehydrogenase)-dependent signal (generated by SFD1/GLY1, suppressor of fatty
acid desaturase deficiency 1, also known as gly1), lipid transfer protein (LTP)
Defective in Induced Resistance1 (DIR1), auxin hormone, the dicarboxylic azelaic
acid (AzA) and its induced gene AZI1 (Azelaic Acid Induced 1), the abietane diter-
penoid (DA), and the amino acid (aa)-derivative pipecolic acid (Pip) (Dempsey and
Klessig 2012). Some of these metabolites are systemically transported throughout
plant tissues and regulate the communication between primary infected tissue and
distal parts to initiate SAR development. Some of these metabolites also overcome
essential roles in SAR-associated rapid activation of defense responses over subse-
quent pathogen exposure, a mechanism termed priming (Shah et al. 2014).
Nonexpressor of Pathogenesis-Related genes1 (NPR1) is a well-characterized
signaling component and one of the central regulators of SA-mediated SAR induc-
tion. It is also strongly suggested that NPR1 is a transcription factor and one of the
most essential receptors of SA (Wu et al. 2012). The accumulated SA as response to
pathogen attack at local infection site is also induced at distal parts of infected plant,
leading to the activation of NPR1 protein to initiate the expression of WRKY-like
transcription factors (TFs) for systemic SAR induction.
As a phloem mobile-SAR signal, MeSA is accumulated at the site of primary
infection, and it is converted to SA with the activity of SA-Binding Protein 2
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 171

(SABP2) enzyme (Kumar and Klessig 2003). MeSA is a systematically transported

derivative of SA signal and accumulates at both primary pathogen infection site and
at distal untreated leaves. The treatment of local sites of potato plants with arachi-
donic acid induces the accumulation of SA, MeSA, and expression of PR1 gene at
both at application site and distal plant tissues which is essential to trigger enhanced
disease resistance to Phytophthora infestans (Shah et al. 2014).
The G3P, generated by the activity of SFD1/GLY1 enzyme, is another possible
SAR signal proposed (Navarova et al. 2012). G3P is one of the major key molecules
for the glycolysis and glycerolipid biosynthesis. As systemic defense response-­
related signal, G3P was firstly suggested by Nandi and his colleagues (2004), and
they revealed that the dihydroxyacetone phosphate reductase encoding Arabidopsis
SFD1 (Suppressor of Fatty Acid Desaturase Deficiency1, also known as GLY1)
gene, responsible for the biosynthesis of G3P, is required for SAR induction (Nandi
et al. 2004). The Arabidopsis sfd1 mutants failed to induce the accumulation of SA
and expression of PR genes, indicating that sfd1 mutants are defective to produce
and spread a long-distance signal for SAR response (Chaturvedi et al. 2008).
Beyond functioning as a lipid transfer protein, DIR1 is also involved in plant
immune response and enhances the accumulation and systemic transport of G3P dur-
ing SAR (Chanda et al. 2011). The Arabidopsis dir1-1 (defective in induced resis-
tance 1-1) mutants were impaired to induce PR gene expression and develop SAR
response but were eligible to accumulate both local and systemic SA to the compa-
rable levels with wild-type (wt) plants and develop both ETI and PTI response against
virulent and avirulent Pseudomonas syringae pv. tomato (Pst) pathogen (Maldonado
et al. 2002). The recently performed trials demonstrated that although DIR1 contrib-
utes the SAR induction by AzA and only required for AzA-induced SAR response,
AZI1 is required for both AzA- and G3P-induced SAR development (Yu et al. 2013).
AzA is a 9-carbon dicarboxylic acid and accumulates to elevated levels in
Arabidopsis infection sites. AzA is mobile and eligible to prime SA production
promptly upon bacterial infection in plants. It was recently identified that AzA is
mobile and eligible to prime SA production promptly upon bacterial infection in
plants. It was recently identified that AzA molecules function to induce SAR-
associated priming of SA accumulation, PR protein expression, and signaling to
confer pathogen resistance at local and systemic tissues. (Jung et al. 2009). Flavin-
Dependent Monooxygenase1 (FMO1, essential for the systemic accumulation of
SA) and Aminotransferase1 (ALD1, also required for pipecolic acid biosynthesis)
are the two enzymes also involved in the biosynthesis of AzA and AzA-based SAR
priming. FMO1 is also essential for Isochorismate1 (ICS1)-mediated systemic
accumulation of SA for SAR (Chaturvedi et al. 2012).
Dehydrobietinal (DA) is an abietane-type diterpenoid which is mostly produced
by conifers and also some angiosperms and purified as a SAR-inducing signal from
avirulent pathogen-infected leaves (AvrPex) of Arabidopsis plants. It was identified
that the exogenously applied DA in Arabidopsis is able to move throughout the
vascular system and enable to induce SAR. DA is an active molecule that is one of
the most effective inducer of SAR. Chaturvedi and his colleagues (2012) displayed
that the miniscule amount (at picomolar concentration levels) of exogenously
172 U. Uzuner et al.

applied DA on local foliar plant parts triggers SA accumulation and PR1 expression
at distal plant parts and it is thus sufficient to induce a strong and systemic SAR
response at distal organs of plants such as Arabidopsis, tobacco, and tomato
(Chaturvedi et  al. 2012). This recent finding gives the advantage to suggest that
naturally isolated and as an endogenous plant product DA, can be used by farmers
as an exogenous SAR inducer during the sensitive stages of crop plants for disease
development.
Pipecolic acid (Pip) is a cyclic nonprotein amino acid derivative of lysine. It was
revealed that the levels of Pip in plant tissues are augmented as response to several
stress conditions (Moulin et al. 2006). It was also demonstrated that Pip levels are
increased at both local and distal plant parts through SAR-inducible pathogen infec-
tion (Navarova et  al. 2012). Pip accumulates in leaves soon (8  h) after pathogen
infection, and supplying Pip with irrigating water can induce foliar resistance. ALD1
gene has a strong substrate tendency for lysine and is required for the accumulation
of Pip-induced SAR response in Arabidopsis. Arabidopsis ald1 mutants fail to accu-
mulate SA in the pathogen free distal leaves, therefore, defective to induce systemic
SAR response. The exogenously applied ALD1 with irrigating water, however, is
eligible to complement the induction of systemic SAR response (Chaturvedi et al.
2012). The recent studies revealed that introduction of exogenous Pip with irrigating
water induces a stronger SA accumulation and PR gene expression response follow-
ing treatment with a SAR-inducible pathogen, indicating the role of Pip in the prim-
ing of defense responses (Navarova et  al. 2012). SA biosynthesis-­related FMO1,
thus, is also indirectly essential for Pip-triggered SA accumulation contributing to
priming for enhanced SAR response, suggesting that FMO1 acts as downstream
component in both ALD1- and Pip-based SAR signaling.
Singh and his colleagues (2013) recently identified SAR signaling related another
key player in Arabidopsis thaliana. They suggested that the Flowering Locus D
(FLD) of A. thaliana is required for responding to the SAR signals at distal tissues
in order to develop whole plant disease resistance (Singh et al. 2013). Although the
fld mutants are able to accumulate SAR signals at infected tissues, they are not able
to respond to SAR signal at secondary plant tissues in order to initiate the induction
of immune response-related gene expressions. In other words, both WT and fld
mutants of A. thaliana could perform dehydroabietinal (DA) and azelaic acid (AA)-
promoted SA accumulation at primary tissues, yet fld mutants are not eligible to
perform SAR induction at distal tissues; and FLD gene product is thus required for
true SAR induction. The FLD gene product is also involved in flowering and regu-
lates the expression of flowering repressor Flowering Locus C (FLC). This task of
FLD, however, is independent of its role in SAR (Singh et al. 2013).
Luna and his fellows have also investigated the inheritance potential of SAR
signal in Arabidopsis by using Pseudomonas syringae pv. tomato DC3000
(PstDC3000) and identified that SAR signal produced by previously infected host
plant is epigenetically transferred throughout at least few generations (Luna et al.
2012). Transgenerational SAR induction is maintained at least over one stress-free
generation, indicating an epigenetic basis of the phenomenon. Furthermore, while
progeny plants display a reduced awareness of JA-inducible genes, SA-inducible
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 173

promoters of Pathogenesis-Related Gene1, WRKY6, and WRKY53 are enriched

with acetylated histone H3 at lysine 9, indicating the epigenetically transmitted
action of SAR from ancestor plants. The current findings also suggest that transgen-
erational SAR is directed by hypomethylated genes which are eligible to transmit
SA-dependent defense response in the following generations (Luna et al. 2012). It
was also identified that the transgenerational SAR response, however, is NPR1
dependent, indicating the key role of NPR1 for both the induction SAR response
and its transfer to following progenies.

9.3.1.2 SA Biosynthesis

SA is synthesized within plant plastids through shikimate pathway (Garcion et al.
2008; Kumar 2014). Chorismate precursor is utilized through two different path-
ways to produce SA. The major SA biosynthesis pathway is isochorismate synthase
(ICS) dependent. ICS is believed to be responsible for the production of >90% of
SA synthesized during initiation of defense response (Boatwright and Pajerowska-­
Mukhtar 2013; Wildermuth et al. 2001). The other SA biosynthetic pathway is phe-
nylalanine ammonia-lyase (PAL) mediated pathway (Dempsey et  al. 2011;
Sánchez-Rangel et  al. 2015). SA can be readily modified to its various inactive
derivatives (via glucosylation, methylations, amino acid conjugation, sulphonation,
and hydroxylation) through these two routes (Dempsey et al. 2011). SA produced in
plants is mainly stored as glucosylated SA (SAG). SAG is the main storage form
with the potential to be converted back to SA to induce SAR. This conversion is
catalyzed by SA β-glucosidase through enzymatic reactions (Chun et  al. 2012).
SAG is not only capable of forming SAG, but also glucosyl salicylate (GS) (Lee and
Raskin 1999). The phytotoxic effects of high concentrations of SA may induce the
rapid formation of GS a protective mechanism; instead the more stable SAG form
may be released slowly from the conversion of SA (Lee and Raskin 1999). A meth-
ylated derivative of SA is volatile methyl salicylate (MeSA). Volatilization of SA
through MeSA synthesis could help plants excrete SA outside of the cell. It is likely
that plants produce volatile MeSA as alternative SAR inducer when defense
response induction is needed at distant plant parts (Xu et al. 2006). Moreover, this
mechanism provides advantage to plants to reduce the toxic effects of highly accu-
mulated SA in tissues (Cronje et al. 2004; Kumar 2014; Qi et al. 2011).
In addition to plant defense signaling, SA also functions as an essential signaling
molecule in distinct physiological responses of plants such as stomatal closure,
drought, thermogenesis, seed germination, flowering, salt stress, ozone, and chilling
(Borsani et al. 2001; Chini et al. 2004; Dat et al. 1998; Hao et al. 2011; Jin et al.
2008; Koch et al. 2000; Lee et al. 2010; Mori et al. 2001; Scott et al. 2004). It was
also reported that either exogenous treatments or endogenously enhanced SA levels
are eligible to repress the endocytosis function of different plasma membrane pro-
teins, suggesting to the role of SA in clathrin-mediated endocytic protein trafficking
(Du et al. 2013). There are also a number of outstanding reviews focusing on the
other functions of SA signaling in detail (Dempsey et al. 2011; Kachroo and Robin
2013; Kumar 2014; Vlot et al. 2009).
174 U. Uzuner et al.

9.3.2 Induced Systemic Resistance (ISR)

ISR is another well-known and heavily applied defense strategy commonly used by
crop growers. ISR is promoted by the colonization of certain strains of plant growth-­
promoting rhizobacteria (PGPR) on plant roots, which results in the direct stimula-
tion of plant growth by improving nutrient uptake or photosynthesis, and this
induced response of plants is mediated by a interplay between JA and ET plant
hormones (Spoel and Dong 2012). In plants, a variety of biotic and abiotic agents
can trigger IR to pathogen infection (da Rocha and Hammerschmidt 2005; Dale
Walters and Lyon 2008). On the other hand, the symbiotic relationship between
arbuscular mycorrhizal fungi and plant enables the initiation of an improved level of
resistance to root-infected pathogens (Cordier et al. 1998; Pozo and Azcon-Aguilar
2007). By colonizing at the root systems of plants, the arbuscular mycorrhizal fungi
not only promote the plant growth and induce systemic resistance which will be
later on resulted in ISR response but also seal the root environment over infective
pathogens of crops and impede the severe pathogen invasions and/or further
outbreaks.
The induction of ISR is mainly conferred through the cross talk between JA and
ET plant hormones. JA and ET are positive regulators of defense response and coor-
dinately induce the development of IR to pests and root-infecting pathogens in
crops (Rakwal and Komatsu 2004). Jasmonate is a lipid-derived compound and
well-known key signaling molecule for plant stress responses and development. The
production of JA and its further biosynthesis-related downstream pathway building
blocks are essential for resistance against multiple pests and pathogens (Lyons et al.
2013). The evidences suggest that JA-mediated ISR development, however, is con-
ferred in a pathogen type-dependent manner. For instance, as one of the main key
molecules in the JA biosynthesis, CO1 (coronatine1), unlike acting as a well-known
ISR inducer against Botrytis cinerea- and Alternaria brassicola-like necrotrophic
pathogens, it seems to promote susceptibility to root-infecting fungal pathogens
Fusarium oxysporum and Verticillium longisporum (Lyons et al. 2013; Ralhan et al.
2012). Similarly, the differential disease resistance or susceptibility behavior of
crop plants to diverse pathogen types can be also observed when some other JA
biosynthesis pathway-related mutants were established such as maize lox3–4
(lipoxygenase) mutant (Gao et al. 2009).
Linolenic acid (18:2) and α-linolenic acids liberated from chloroplast mem-
branes are the two most probable precursor phospholipids essential for the biosyn-
thesis of plant jasmonates. The lipoxygenase gene family members (LOXs) are
involved in the production of JA biosynthesis-related enzymes by oxidizing liber-
ated phospholipid precursors (Lyons et  al. 2013). JA plant hormone is produced
rapidly in response to biotic and abiotic stresses such as wounding or herbivore
damage. The biologically active form of JA is JA-Ile (jasmonate amino acid isoleu-
cine), produced by the activation of JAR1 (jasmonate resistant 1) encoding a GH3
amido synthetase. GH3 is responsible for the conjugation of JA to JA-Ile, and this
active JA-Ile is recognized by COI1 (coronatine insensitive 1) and JAZ (jasmonate
ZIM domain) (Fonseca et al. 2009; Staswick and Tiryaki 2004). COI1 and MYC2
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 175

are the two most critical regulators for JA signaling and related defense responses
during ISR induction (Kazan and Manners 2013). JA signaling and downstream IR
response against stress conditions are triggered through the activation of mitogen-­
activated protein kinase (MAP kinase) cascade, which eventually initiates the
expression of WRKY TFs and results in the expression of PDF1.2 (jasmonate-­
inducible plant defensin protein) and chitinases like defense response-related genes.
COI1 and MYC2 are targeted by pathogens to repress JA-induced ISR develop-
ment. The basic helix-loop-helix (bHLH) TF MYC2 performs the regulation of JA
response by coordinating the two different JA biosynthesis pathways, eventually
resulting in induced resistance to pests and plant pathogens. In addition, MYC2 is
responsible for the regulation of cross talk between JA and other plant hormones
such as abscisic acid (ABA), SA, gibberellins (GAs), and auxin (IAA). The critical
JA signaling component MYC2 also synchronizes the interactions between JA sig-
naling and light, phytochrome signaling, and the circadian clock, indicating that
JA-based induced response regulators coordinate a plethora of defense-related regu-
lations against a variety of stress conditions by fine-tuning either every biotic or
abiotic stresses-related impulse (Kazan and Manners 2013).
JA and related plant signaling systems take advantage to suggest that in con-
trolled agricultural environments, it is possible to apply ISR by incorporating
PGPRs to promote the disease tolerance of crops, enhance crop development, and
increase the amount of yield produced.

9.3.3 Priming of Defense Response

Specialized plant compartments are able to recognize variety of external factors and
potential threats such as biotic (insects, herbivores, and microbial pathogens) and
abiotic (extreme temperature, inappropriate water supply, etc.) stress conditions.
Once the related threats are recognized, plants typically switch to a primed state of
systemic defense response. When plants triggered the primed defense state, they
enable reacting to biotic and abiotic threats as more aggressively and rapidly through
the induced systemic response (ISR). The term “priming” itself is originated from
“the sensitization of a cell or organism for enhanced defense; causes faster and more
robust activation of defense responses upon challenge” (Conrath et al. 2015). In other
words, priming is a physiological regulatory switch that provides advantages plants
to respond more rapidly and/or more rigorously after exposure to biotic or abiotic
stress. It is well known that the primed immune defense can be transferred to next
several generations. Thus, the defense components of the plant immune response are
eligible to convey the various learnings from past incidents. The priming agents in
plants could be highly diverse. The molecular patterns of microbes as MAMPs,
PAMPs of pathogens, herbivore-associated molecular patterns (HAMPs) of herbi-
vores, or damage-associated molecular patterns (DAMPs) of wound signals, patho-
gen effectors, certain xenobiotics (some pesticides or synthetic compounds).
Moreover, some natural or synthetic chemicals were also identified as potential
176 U. Uzuner et al.

inducers of defense priming. Upon the initiation of priming, which is normally

occurs locally but not systematically, through one or some of these agents, the primed
defense response is formed systemically in each part of the plant. In this case, the
primed plant can easily recognize the very low level of second stimuli and create a
faster and stronger defense response. The primed state thus results from the improved
perception and/or amplification of defense response-inducing signals, rather than
from the direct activation of defense responses (Aranega-Bou et al. 2014).
Priming is quite common in current agricultural applications because primed
plants reveal more rapid and robust defense responses when challenged by patho-
gens, insects, or abiotic stress agents. In other words, priming is a useful strategy to
induce local and systemic immunity and stress tolerance for agricultural crops.
Recent molecular and genetic studies provided comprehensive findings on the
defense priming of plants and revealed that priming is a key physiological process
in various types of systemic plant immunity. Among the different types of defense
priming inducers, SA- and PA-initiated SAR, JA- and ET-dependent ISR (mostly
activated by growth-promoting bacteria and fungi), arbuscular mycorrhizal fungi,
natural or synthetic chemicals (BTH, MeSA, JA-Ile, AZA, 2,6-dichloroisonicotinic
acid (INA), isotianil, imprimatinC1, systemin, BABA-IR, etc.), metabolic activa-
tors, volatile organic compounds (VOCs), and wound signal-induced resistance
stand as comprehensively elucidated major priming mechanisms (Aranega-Bou
et al. 2014; Jung et al. 2009; Noutoshi et al. 2012).
The molecular basis of priming is poorly understood and has recently started to
be unraveled. The accumulated amounts of inactive mitogen-activated protein
kinases (MAPKs), transcription factors, and certain epigenetic marks were reported
as remarkable indicators of defense priming (Conrath 2011; Conrath et al. 2015;
Kinoshita and Seki 2014). It was also reported that a functional SA analog, benzo-
thiadiazole (benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester or BTH), is
also eligible to induce defense priming through the mitogen-activated protein kinase
(MAPK) cascade, in particular through MPK3 and MPK6. The quantitatively
greater MPK3/6 activity was also revealed to be associated with enhanced PR1 and
PAL1 defense gene expression and development of SAR (Conrath et al. 2015).
It was also indicated that pathogenicity-related receptors (PRRs) are essential for
the recognition of various priming inducers such as MAMPs, PAMPs, DAMPs, and
HAMPs. For instance, FLS2 is a leucine-rich repeat receptor kinase (LRR-RK) that
can recognize the highly conserved N-terminal amino acid epitope flg22 in the bac-
terial MAMP flagellin. FLS2-flg22 recognition is rapidly notified through down-
stream signal transducers and plays a key role in defense priming and stress
tolerance. Another example is that Arabidopsis malectin-like LRR receptor-like
kinase IOS1, which is associated with FLS2 and EFR (the bacterial EF-Tu recep-
tor), is also critical for priming of pattern-triggered immunity. The BTH treatment
of Arabidopsis can also induce increases in the level of FLS2 and its co-receptor
BAK1 which fosters the responsiveness to flg22, indicating the potential induction
of defense priming through a synthetic compound.
The recent findings point out that the induction of priming in plants may also
require chromatin modifications which are essential for faster and stronger
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 177

transcription of defense genes to initiate priming (Kinoshita and Seki 2014). The plant
heat shock factor (Hsf) is a novel molecular component for the gene-based activation
of primed defense and SAR. Hsfs play key roles in defense priming such as regulating
genes for heat shock proteins and other chaperones (89). More than 20 Hsfs have been
identified in Arabidopsis, which are belong to the A, B, and C classes. They play
diverse essential roles in chromatin modifications as well (Conrath et al. 2015).
There are numerous amounts of natural and synthetic compounds identified with
the ability to prime systemic defense response. However, only few priming com-
pounds are currently useful and preferred in practice due to the high level of fitness
costs. Substantial amounts of natural compounds were identified with the activity
essential to induce plant growth and defense response or priming through a plethora
of physiological alterations. Majority of those natural compounds are derivatives of
oligosaccharides, glycosides, amides, vitamins, carboxylic acids, and aromatic
compounds. Even though they can be toxic to plants as concentration dependent,
natural priming inducers tend to be better tolerated by plants than most of the syn-
thetic compounds (Noutoshi et al. 2012). Both natural and synthetic compounds,
however, mimic one or some of the numerous types of plant-pathogen interactions
by acting as endogenous or exogenous elicitors to induce priming.
The first identified synthetic priming compounds are the two common SA ana-
logs: 2,6-dichloroisonicotinic acid (INA) and BTH. BABA is a nonproteinaceous
amino acid that can induce priming in plants against biotic and abiotic stresses.
BABA-triggered resistance to pathogens and insect pests in Arabidopsis is estab-
lished through accumulation of PA (Navarova et al. 2012).
It has been also identified that a great number of specific agricultural fungicides
(isotianil, pyraclostrobin), herbicides (bensulfuron-methyl, clomeprop) and pesti-
cides (tiadinil, imidacloprid) could trigger defense priming at certain levels.
Moreover, these types of compounds can further reveal synergistic effects with
priming activators. However, it does not change the reality that either natural or
synthetic inducers for priming mostly rely on the existing either SAR- or ISR-­
dependent plant defense mechanisms, in particular the key systemic defense
response inducer SA (Aranega-Bou et al. 2014; Conrath 2011).
There are great numbers of reports indicating the presence of “gain-gain”
approach among small ecosystem members even though they are dissimilar organ-
isms but associated through an intimate relationship (Pieterse et al. 2014). No mat-
ter whether it is either rhizosphere fungi or plant growth-promoting bacteria, the
beneficial microorganisms settled in a close environment with plants may also
induce priming through ISR at certain levels. For instance, when biocontrol strain
Pseudomonas fluorescens WCS374 was applied for radish plants against Fusarium
wilt disease, the bacterium was able to increase yield and induce ISR (Leeman et al.
1995). It was also reported that Piriformospora indica colonizing at roots was able
to induce priming in barley against biotic and abiotic stress systemically (Glandorf
et al. 2001). The beneficial effects of the same rhizobium bacteria were also con-
firmed on the medicinal plants Spilanthes calva and Withania somnifera as increased
growth and enhanced biomass yield through field tests (Rai et al. 2001). Hundreds
of similar examples revealing the use of beneficial microorganisms to cure
178 U. Uzuner et al.

agricultural applications, increase product yields, and avoid massive disease out-
breaks (no matter whether they are fungi, bacteria, or virus oriented) can be given
from the current literature (Contreras-Cornejo et  al. 2011; Kloepper et  al. 2004;
Martinez-­Medina et al. 2013; Mathys et al. 2012; Vargas et al. 2011). In addition,
recent scientific findings point out to the major advantage of using beneficial
microbes and natural priming activators on row crops because the synthetic com-
pounds could be toxic, not only to the plants applied for but also to the inhabiting
species in the surrounding environments. Furthermore, the excessive use of syn-
thetic compounds on crops for disease control or priming can accumulate in fields,
and this could adversely affect the next plantation efforts and can infiltrate into
drinking waters which may eventually be toxic to human health. On the other hand,
the priming inducer natural compounds can be easily breaking down in the soil as
compared to synthetic ones.
The recent findings point out to the use of natural priming activators together
with beneficial microorganisms which were resulted in a combinatory effect as bet-
ter control of disease development and increased yields (Rejeb et al. 2014; Ton et al.
2005; Trabelsi and Mhamdi 2013; Walters et al. 2014). Beyond the synthetic regula-
tory compounds and chemicals developed, the extensive use of natural priming acti-
vators and beneficial microorganisms may thus be more considered as healthier and
ecosystem-friendly favorable strategies in crop breeding systems.

9.4 Resistance-Related Hormonal Cross Talk in Crop Plants

Phytohormones in plants take over important roles for plant’s responses to biotic
and abiotic stresses. In particular, ABA, ET, JA, and SA have crucial functions in
stress responses of plants (Derksen et al. 2013). Here, we briefly evaluate the com-
mon cross talk among these phytohormones regarding their roles for plant disease
and defense.
Many studies revealed that the JA and SA pathways interact by a complex net-
work of regulatory interactions (Derksen et al. 2013; Thaler et al. 2012; Wei et al.
2014) including priming (De Vos et al. 2006), complementary additive or synergis-
tic effects (Devadas et al. 2002; Schenk et al. 2000), and mutual antagonism (Diezel
et al. 2009; Zhang et al. 2013). The SA and JA signaling sectors often act antagonis-
tically. For instance, coronatine, which is produced by virulent Pseudomonas syrin-
gae bacteria, is a JA-mimicking phytotoxin and promotes virulence by suppressing
effectual SA-dependent defenses in Arabidopsis and tomato (Solanum lycopersi-
cum) (Brooks et al. 2005; Zheng et al. 2012).
NPR1 is a positive regulator of SA signaling (Dong 2004). SA treatment leads
dissociation of the NPR1 complex and migration to nucleus. Then, NPR1 binds
to TGA (TGACG motif-binding) transcription factors, enhancing their binding to
SA-responsive promoters. The regulatory protein NPR1 is also responsible of the
antagonism between salicylate and jasmonate. If NPR1 is impaired through a
mutation, expression of LOX2 increases, and JA is synthetized, eventually, JA
pathway-related gene expression occurs predominantly. Ethylene formation from
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 179

1-aminocyclopropane-1-carboxylic-acid in pear cell suspension cultures is also

inhibited by SA (Leslie and Romani 1988). In addition, NPR1 is involved in SA
antagonism against the JA and/or ET pathways in the A. thaliana/A. brassicicola
interaction (Spoel et al. 2007). Activation of salicylate pathway-related defense
genes such as PR-1 requires localization of NPR1 protein within nucleus. In
contrast, the antagonism between SA and JA pathway occurring via NPR1 does
not need nuclear localization of this protein (Spoel et al. 2003). In addition to the
central role of NPR1, there are several other key players that function during SA/
JA cross talk, such as mitogen-activated protein kinase MPK4 (Petersen et  al.
2000), the lipase-like proteins Enhanced Disease Susceptibility1 (EDS1) and
Phytoalexin-­Deficient4 (PAD4) (Brodersen et al. 2006), the fatty acid desaturase
Suppressor of SA Insensitivity2 (Kachroo et  al. 2003), glutaredoxin480
(GRX480) (Ndamukong et al. 2007), and class II TGA and WRKY transcription
factors (Leon-Reyes et al. 2010a; Gao et al. 2011). Mutation or ectopic expres-
sion studies of the corresponding genes reveal that these proteins are important
regulators of SA/JA cross talk. Although several regulatory proteins of SA/JA
cross talk have been identified, the molecular mechanism by which SA exerts its
antagonistic effect on the JA pathway is still largely unknown. Here we describe
our current understanding of proteins that play a role in regulating SA-mediated
suppression of JA pathway.
Mitogen-activated protein (MAP) kinases play a central role in plant immune
signaling in all eukaryotes (Rodriguez et  al. 2010). Arabidopsis MAP Kinase
(MPK4) mutant plants displayed elevated SA levels and constitutive expression of
SA-responsive PR genes but failed to induce the expression of JA defense marker
genes (Petersen et al. 2000). Therefore, mpk4 is recognized as a negative regulator
of SA yet a positive regulator of JA signaling.
Glutaredoxins (GRXs) and thioredoxins (TRXs) are important in mediating
redox regulation of protein activity (Kneeshaw et al. 2014). Among of the cellular
redox buffer, glutathione is affected by both SA and JA. The ratio between reduced
and oxidized glutathione is increased by SA, whereas JA decreases the glutathione
pool (Spoel and Loake 2011). In Arabidopsis, the specific time frame in which SA
suppresses JA-responsive gene expression coincides with a transient increase in the
level of glutathione (Koornneef et al. 2008). Importance of SA-mediated modula-
tion of the cellular redox state for the attenuation of the JA pathway was shown by
glutathione biosynthesis inhibitor L-buthionine sulfoximine (BSO) (Koornneef
et al. 2008). On the other hand, SA-dependent responses were not affected by over-
expression of GRX480  in Arabidopsis. In addition, a grx480 knockout mutant
showed wild-type levels of SA-mediated suppression of the JA pathway, indicating
that GRX480 is not required for SA/JA cross talk.
The regulatory protein NPR1 is important for the transcriptional co-activation of
many SA-responsive genes (Wang et al. 2006). SA signaling and SA-mediated sup-
pression of JA pathway is impaired in Arabidopsis npr1 mutants (Leon-Reyes et al.
2010a). Nuclear localization of NPR1 is essential for SA-responsive defense gene
expression, but not for SA-mediated suppression of the JA pathway (Spoel et al.
2003). Stimulation of the SA pathway in Arabidopsis plants overexpressing a fusion
180 U. Uzuner et al.

protein of NPR1 that was retained in the cytosol resulted in wild-type levels of sup-
pression of JA responses, indicating that SA/JA cross talk is mediated by cytosolic
NPR1 (Spoel et al. 2003). The role of cytosolic NPR1 in SA/JA cross talk was also
confirmed in rice (Oryza sativa). JA-responsive gene transcription was suppressed
by overexpression of cytosolic OsNPR1. In addition, no antagonistic effect on the
JA response was observed when OsNPR1 was constitutively targeted to the nucleus
(Yuan et al. 2007).
For SA-mediated suppression of the JA pathway, cytosolic NPR1 is sufficient. In
addition, nuclear NPR1 regulates several SA-responsive transcriptional (co)factors
suppressing JA-responsive gene expression. These (co)factors include GRX480,
TGAs, and WRKYs.
Important regulators of SA-induced expression of PR genes are TGAs during
SAR response (Zhang et  al. 2003). Because both NPR1 and GRX480 have been
implicated in SA-mediated suppression of JA pathway, the role of TGAs in SA/JA
cross talk has been tested. Necessity of TGAs in SA/JA cross talk was shown by tga
mutants that are impaired in SA-responsive gene expression. Interestingly,
SA-mediated suppression of the JA pathway is also blocked in the tga256 triple and
tga2356 quadruple mutants (Leon-Reyes et  al. 2010b), indicating that TGAs are
essential for SA/JA cross talk. In addition, without any SA stimulus, TGAs are of
importance in the activation of JA- and ET-dependent defense genes. On the other
hand, when SA pathway is induced, negative regulatory role of TGAs in the JA
pathway dominates (Zander et al. 2010).
Other important regulators of SA-dependent defense responses are WRKY tran-
scription factors and many of them are up-regulated by SA (Bakshi and Oelmüller
2014; Rushton et al. 2010). It has been identified that several WRKY transcription
factors such as WRKY50 and WRKY51 play roles during SA-JA cross talk.
Exogenous application of SA did not suppress JA-inducible gene in wrky50 or
wrky51 mutant plants (Gao et  al. 2011). Therefore, it seems that WRKY50 and
WRKY51 are essential for SA-mediated suppression of JA signaling.
Overexpression of WRKY70  in Arabidopsis enhances the expression of
SA-responsive PR genes, whereas it suppresses JA-responsive genes (Ren et  al.
2008). SA-induced WRKY70 negatively affects transcription of JA-responsive
genes, which is supported by microarray data (Li et al. 2004).
Here we discuss recent developments about how other plant hormones such as
ABA, ET, and cytokinins antagonistically or synergistically interact with the SA/JA
cross talk. We summarize SA, JA/ET pathways, and their cross talk with the other
plant hormones and ROS in Fig. 9.1.

9.4.1 Ethylene (ET)

There is a mutual antagonism among SA, JA, and ET pathways. Expression of

PDF1.2 in Arabidopsis plants is suppressed by SA; thus JA and ET responses are
suppressed (Ellis et al. 2002). On the contrary, JA antagonizes SA by inhibiting the
induction of PR-1. In case of wounding or herbivore attacks, SA has also inhibitory
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 181

SA PATHWAY JA/ET PATHWAY

Signals Signals

CEV1
PAD4
EDS1
ET FAD 3/7

SID2
ETR1/ERS1 JA
EDS5
CTR1
MAPK4 COI1
SA

EIN2 JAR1
NPR1
EIN3
TGA ERF1 Thi2.1
WRKY 70
VSP1
TGA2 PDF1.2
PR genes
PR1 Defense responses
PR5

BGL2

Defense responses

ABA Signaling CK Signaling NO Signaling

Fig. 9.1  Cross talk among hormone signaling pathways. Dashed lines indicate induction by plant
hormones

effects on JA/ET pathways. AtVSP (Vegetative Storage Protein) expression induced

by JA in response to wounding is inhibited by SA (Anderson et al. 2004). Expression
of ACCs involved in the biosynthesis of ET in response to wounding is inhibited by
SA as well (Li et al. 1992).
As the effects of different plant hormones are contradictory, interaction of signal-
ing pathways is complex in plants’ responses to many pathogens. It is difficult to
predict which defense signaling pathways are actually involved or which ones
would be more efficient in reducing the disease development or further complete
resistance.
182 U. Uzuner et al.

9.4.2 Abscisic Acid (ABA)

It is widely accepted that major physiological processes such as seed dormancy,

seedling development, and lateral root elongation are regulated by ABA. In addi-
tion, it is also functional for responses to environmental stresses such as abiotic
drought stress (Adie et al. 2007; Schwartz et al. 2003). The roles of ABA in plant
responses to abiotic stresses have been evidenced recently. There is a pile of evi-
dence about its interaction with other hormonal defense responses and of its role in
plant defense signaling. Several previous studies point out that abiotic stress-­
induced ABA accumulation results in enhanced susceptibility to pathogens
(Asselbergh et  al. 2008; Mohr and Cahill 2003). For instance, drought-induced
ABA accumulation in Arabidopsis decreased resistance to avirulent Pseudomonas
syringae pv. tomato (Pst) (Mohr and Cahill 2003). However, a positive correlation
between ABA and disease resistance was also reported by other studies. For exam-
ple, ABA-treated Arabidopsis was eligible to develop resistance to Alternaria bras-
sicicola and Plectosphaerella cucumerina (Ton et  al. 2009). In addition, few
Arabidopsis mutants, which were ABA deficient (aba2–1, 2, aba3–2) and ABA
insensitive (abi4–1), became more susceptible to Pythium irregulare and Alternaria
solani infections (Adie et al. 2007).
ABA interferes with JA-SA interactions. ABA acts synergistically or antagonisti-
cally on JA response pathway through the ERF branch (Abe et al. 2003; Anderson
et  al. 2004). ABA reacts with MYC branch of JA pathway. AtMYC2, allelic to
Jasmonate Insensitive1 (JIN1), is a positive regulator of ABA signaling (Abe et al.
2003). AtMYC2 is an important player in hormone signaling in response to biotic
and abiotic stresses and is related with integration of JA and ET pathway signals
(Lorenzo et al. 2004). JA-mediated defense response against herbivores is positively
regulated by AtMYC2 (An et al. 2010; Lorenzo et al. 2004). In addition, suppres-
sion of SA defenses during Pst infection needs AtMYC2 (Laurie-Berry et al. 2006).
ABA signaling was affected by JA signaling-inducing genes such as Pyrabactin
Resistance Like 4, encoding proteins in ABA receptor family (Lackman et al. 2011).
This is proof of the necessity of ABA for JA-inducible responses. On the other hand,
the antagonistic interaction between pathogen-modulated ABA signaling and
SA-mediated defenses was also revealed in 2009 (De Torres Zabala et al. 2009).
Similarly, studies on mutant that accumulate high amount of ABA by overexpress-
ing ABA biosynthetic enzymes such as NCED5 reported that JA accumulation
increased in those mutants, while SA content was reduced (Fan et al. 2009), sug-
gesting that SA signaling is negatively affected by ABA and JA response pathway.
Indeed, interactions between ABA, SA, and JA play essential roles in the regulation
of defense responses (Flors et al. 2008), but the general biological significance and
molecular details of this tripartite hormone interaction are still poorly understood.

9.4.3 Cytokinins (CKs)

The relation between CKs and plant resistance was shown by using mutant and
transgenic Arabidopsis. Choi et al. (2010) reported that increased resistance to Pst,
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 183

increased SA biosynthesis, and PR1 expression were well correlated with high CK
levels (Choi et al. 2010). However, plants with low CK levels become more suscep-
tible to Pst (Choi et al. 2010). It was revealed that through possible hormonal cross
talk between CK and SA, CK-induced pathogen resistance is enhanced by SA.

9.4.4 Brassinosteroids (BRs)

As plant-specific steroids, brassinosteroids (BRs) that are synthesized in young

active tissues take part in plant growth responses such as promotion of cell elonga-
tion and division, differentiation, and biotic stress responses (Shin et al. 2016). In
addition to growth promotion, positive roles of BRs in pathogen defense responses
have been shown by induction of resistance against various viral, bacterial, and
fungal pathogens in tobacco and rice in an SA-independent manner after treatment
with BRs (Takatsuji and Jiang 2014). On the other hand, epibrassinolide application
to Cucurbita pepo induced antiviral immunity against cucumber mosaic virus by
SA-mediated way (Tao et  al. 2015). Plant activation of BR signaling mostly
increases plant resistance to pathogens. However, in a recent study, rice was reported
to be hypersensitive to Pythium graminicola, a root pathogen by BR signaling due
to negative cross talk of BR signaling pathway with the SA and GA signaling path-
ways (De Vleesschauwer et al. 2012). To inflict disease by antagonizing SA- and
GA-mediated defenses, plant BR pathway was used by P. graminicola. Similarly, a
negative cross talk between BR and JA pathways was also reported about BR-induced
susceptibility of rice to the root-knot nematode Meloidogyne graminicola (Nahar
et al. 2013). As it can be seen from above information, the signaling pathways of
these hormones influence each other through a complex network of synergistic and
antagonistic interactions. BR biosynthesis and signaling are well understood in
Arabidopsis. Brassinosteroid signaling begins at the cell surface, where the hor-
mone is perceived by a small family of plasma membrane-localized leucine-rich
repeat receptor kinases (LRR-RKs). Brassinosteroid Insensitive 1 (BRI1) is the
founding member of this small family of receptors and accounts for most of the
BR-binding activity in Arabidopsis (Cano-Delgado et al. 2004). Binding of BR to
BRI1 induces phosphorylation of BAK1 (BRI1-associated receptor kinase 1), a
cytoplasmic receptor kinase, thereby modulating BR signaling (Lin et  al. 2014).
Pathogen-associated molecular patterns (PAMP) such as a bacterial flagellin and an
elongation factor are recognized by plants (Newman et al. 2013). The signals pro-
duce ROS and activate the MAPK (mitogen-activated protein kinase) cascade after
pattern recognition receptors (PRRs) detect PAMPs. Annihilated responses of
PAMP-triggered immunity (PTI) in the bak1 (BRI1-Associated Receptor Kinase 1)
mutantreveals that BAK1 forms a receptor complex with FLS2 in addition to BRI1.
FLS2 and EFR (EF-Tu receptor) are PRRs with a leucine-rich repeat receptor-
kinase structure similar to that of BRI1 and transduce defense signals to induce
resistance against bacterial pathogens (Chinchilla et al. 2007). Thus, BAK1 can be
accepted as a common co-receptor in immunity. Actually, PTI is modulated by BRs
or synergistically by BAK1 (Albrecht et al. 2012; Belkhadir et al. 2012). A PRR
184 U. Uzuner et al.

with a leucine-rich repeat receptor-kinase structure, Xa21 provide rice with a resis-
tance against most Xoo strains (Song et al. 1995). BRI1–XA21 that is a chimeric
receptor kinase to induce the XA21-mediated defense response in rice cells is acti-
vated by binding of BRs to the BRI1 extracellular LRR domain (He et al. 2000).
Park and his fellows (2011) reported a decreased sensitivity to BR and resistance to
Magnaporthe oryzae in rice after knockdown of OsBAK1 by RNAi (Park et  al.
2010). Thus, it is suggested that BAK1 plays a dual role in plant development and
immunity (Shin et  al. 2016). On the other hand, susceptibility to hemibiotrophic
pathogens increased in transgenic plants overexpressing BRI1 (35S::BRI1)
(Belkhadir et  al. 2012). This is consistent with a report that disruption of BRI1
resulted in increased disease resistance against necrotrophic and hemibiotrophic
pathogens, but had no effect on biotrophic pathogens. The EBR-induced antiviral
immunity may be mediated by a SA-independent pathway, whereas ROS may play
positive roles. EBR will be promising in controlling CMV in susceptible crops.

9.4.5 Reactive Oxygen Species

Cross talk among plant hormones with regard to plant’s biotic stress responses is
mediated by plant molecules, mainly known as reactive oxygen species (Verma
et al. 2016). Hydrogen peroxide (H2O2) and nitric oxide (NO) interfere with salicy-
late and jasmonate responses. Thiol oxidation referred to as S-nitrosylation by NO
is also important in plant defense response (Farnese et  al. 2016). Large pools of
S-nitrosoglutathione (GSNO) are generated after this reaction. GSNO act as a nitro-
sylating agent, and thus effects of NO persist in systemic acquired resistance
(Espunya et al. 2012). One of the best known examples of NO involvement in plant
pathology is the nitrosylation of NPR1. As already stated, oligomerization of NPR1,
the oligomeric status, is necessary for its role in defense response (Saleh et al. 2015;
Tada et al. 2008; Withers and Dong 2016) SA-activated thioredoxin triggers mono-
mer formation by the reduction of this S-nitrosylated cysteine residue (Tada et al.
2008). Thus, NO could be seen to play a paradoxical role, on the one hand initiating
SA to promote NPR1 monomer formation and translocation from cytoplasm to
nucleus, but on the other hand favoring oligomerization by initiating nitrosylation.
These opposing roles are reinforced at other steps in SA signaling pathways.

9.4.6 N
 itric Oxide and Ethylene/Jasmonic Acid/Salicylic Acid
Signaling

The suppressive effect of NO on ET signaling is reported. Leshem and Pinchasov

(2000) reported that NO content decreased as ET hormone increased in ripening of
avocados and strawberry (Leshem and Pinchasov 2000). GSNO application reduced
methionine adenosyltransferase (MAT1; At1g02500) activity and ET content in
Arabidopsis (Lindermayr et al. 2006). On the other hand, in vitro application of NO
precursor-SNP in tobacco plants has been reported to cause generation of both NO
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 185

and ET (Mur et  al. 2008). ACC synthase (ACS) expression could be induced by
SNP and NO could be produced following ET production (Mur et  al. 2008).
Similarly, it was identified that the expression of mammalian NOs in transgenic
tobacco enhances ACC oxidase (the final enzyme in ET biosynthesis) and ethylene-­
responsive element-binding protein (EREBP) expression (Chun et al. 2012).
The induction of JA production by NO has been shown recently (Mur et al. 2012,
2013). Increases in the expression levels of LOX3 (At1g17420) and
12-­oxophytodienoate reductases (OPR1, 2, and 3) by NO have been reported by
Palmieri and his colleagues (Palmieri et al. 2008). These recent findings confer that
the expression of a range of JA biosynthetic genes is regulated by NO signaling.
Endogenous NO accumulation in Arabidopsis, resulting from loss-of-function
mutations in NO overexpression 1 (NOX1), was reported to cause to disabled resis-
tance (R) gene-mediated protection, basal resistance, and defense against non-
adapted pathogens. In nox1 plants, both SA synthesis and signaling were suppressed,
reducing SA-dependent defense gene expression (Yun et al. 2016).

9.5  iological Control Agents Providing Disease Resistance

B
in Cereal Crops

9.5.1 microRNAs (miRNAs)

As one of two types of short (20–24 nucleotides) endogenous noncoding small

RNA molecules, miRNAs play essential roles in plant cells; in particular they
function during the regulation of gene expression through RNA silencing at the
posttranscriptional level and also contribute to the plant defense response during
biotic and abiotic stresses (Gupta et  al. 2014). miRNAs are derived from the
miRNA gene transcripts by the function of RNA polymerase II and are well con-
served in eukaryotic systems evolutionarily, which are thus essential for the gene
expression-based regulation of sustainable plant defense responses to specific
pathogens (Molnar et  al. 2007). The biogenesis of miRNAs necessitates the
involvement of several key proteins, from primary miRNA (pri-miRNA) to pre-
cursor miRNA (pre-miRNA) through the function of Dawdle (DDL) protein,
DICER-like protein 1 (DCL1), Hyponastic Leaves1 (HYL1), and Serrate (SE)
proteins. The mature miRNAs are eventually incorporated into argonaute 1
(AGO1) to be functional in RNA-induced silencing complex (RISC), against a
variety of unwelcomed RNA molecules which could be abnormal RNAs, viral
RNAs, or definite endogenous RNAs, and will be silenced as specific regulatory
response of plant cell systems to various conditions.
Imminent efforts on developing better crop disease resistance have recently
started to focus on miRNA-based applications to interfere with pathogen disease
development strategies and empower crop resistance (Baldrich and San Segundo
2016). Compared to R gene-based defense endeavors, employing miRNA-based
immune responses is becoming more common due to its strength and efficacy in
field practices. For instance, silencing of the dominant allele of rice bacterial blast
186 U. Uzuner et al.

resistance gene Xa13 by using an artificial microRNA confers resistance to

Xanthomonas oryzae pv. oryzae (Li et al. 2012). In addition, the expression of rice
TaCPK2-A, a calcium-dependent protein kinase gene, function during resistance
response to wheat powdery mildew has also been revealed to enhance bacterial
blight resistance in transgenic rice (Geng et  al. 2013). Zhang and his colleagues
(2016) suggested a novel defense strategy about specific miRNAs exported by host
plants to induce gene silencing in pathogenic fungi (Zhang et  al. 2015). They
showed that cotton plants increased synthesis of microRNA 166 (miR166) and
miR159 and exported them to Verticillium dahliae hyphae for specific silencing.
They also proved that two V. dahliae genes encoding a Ca2+-dependent cysteine
protease (Clp-1) and an isotrichodermin C-15 hydroxylase (HiC-15) were both
essential for fungal virulence and targeted by miR166 and miR159, respectively.
Overall, plant immune response-related miRNAs could be suitable and safe tools
in agricultural applications to confer resistance as nonchemical and nontoxic agents.

9.5.2 Small Interfering RNAs (siRNAs)

Small interfering RNAs, at times also known as short interfering RNAs, are com-
posed of double-stranded RNA molecules having 20–25 base pairs in length. Unlike
miRNAs derived from imperfectly base-paired and fold-back structures, siRNAs
are produced from perfectly paired double-stranded RNA (dsRNA) precursors by
the action of RNA-dependent RNA polymerases (RDRP). Up to now, four different
types of siRNAs have been identified in plants: trans-acting siRNAs (ta-siRNAs),
natural antisense transcripts (NATs)-derived siRNAs (nat-siRNAs), heterochro-
matic siRNAs (hc-siRNAs) or repeat-associated siRNAs (ra-siRNAs), and long siR-
NAs (lsiRNAs) (Katiyar-Agarwal and Jin 2010). Regulation of plant growth and
development, adaptation to abiotic stresses, and defense responses to biotic stresses
are the most described key roles of siRNAs in plants (Liu et al. 2014). It is described
that overlapping cis-NAT genes encoded in most eukaryotic genomes have potential
to encode nat-siRNAs. Even though their plant metabolism-related roles are cur-
rently elusive, it has been identified that nat-siRNAs play essential roles in both
biotic and abiotic stress conditions (Liu et al. 2014).
The recent advances point out that siRNAs play key roles in directing cytosine
methylation through the process of RNA-directed DNA methylation (Kanno and
Habu 2011). It is proposed that siRNA-mediated methylation has diverse roles in
development as well as in plant responses to the environment; most likely the
supreme role is the major contribution to the epigenetically control of transgenera-
tional SAR response.
Beyond stemming from various biosynthesis mechanisms, both miRNAs and
siRNAs or siRNAs function through the regulatory RISC complex, which requires
concerted action of a variety of different proteins. Host endogenous small RNAs are
mainly known as another posttranscriptional regulator in cells as well as miRNAs.
In Arabidopsis thaliana, it was identified that microRNAs trigger the biogenesis of
epigenetically activated small interfering RNAs (easiRNAs) from reactivated trans-
posable elements, suggesting the existence of a new siRNA pathway in plant cells
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 187

(Creasey et al. 2014). It was also stressed that these easiRNAs are eligible to specifi-
cally target transposon transcripts in case of any intrusion in the genome by trans-
poson elements during reprogramming of germ lines, similar to the PIWI-interacting
RNA pathway in animals (Creasey et al. 2014).
Transcriptional gene silencing (TGS) of transposons is mediated by heterochro-
matic (het) siRNAs (24-nt), RDR2, DCL3, and AGO4 (Law and Jacobsen 2010).
Posttranscriptional gene silencing (PTGS), however, is mediated by DICER-LIKE
protein 1 (DCL1)-dependent miRNAs in plants. DCL1 is also responsible for the
production of secondary short interfering RNAs (siRNAs) via RNA-Dependent
RNA Polymerase 6 (RDRP6), DCL4, and AGO1. It was described that in Arabidopsis
thaliana, transposons trigger the production of epigenetically activated small inter-
fering RNAs (easiRNAs) (21-nt) in Decreased DNA Methylation1 (ddm1) and
DNA Methyltransferase 1 (met1) mutants, as well as in the vegetative nucleus of
pollen grains and in differentiated plant cell cultures. The majority of transposon
transcripts of A. thaliana are specifically targeted by more than 50 miRNAs for
cleavage and silencing by RDRP6. The same biosynthesis mechanism is also
responsible for the production of the transposon regulators easiRNAs, similar to
secondary siRNAs. The transposon-based plant genome anomalies are thus medi-
ated by miRNA-directed easiRNAs, which also need to the key regulators of RISC
complex such as RDRP6, DCL4, and DCL1 to be functional (Creasey et al. 2014).

9.5.3 Transcription Factors

With the better understanding of biological, physiological, and further exquisite

regulations in plant defense systems, it is now more confident for breeders develop-
ing resistant crops. The new trend in providing crop resistance most likely directs to
the integration of a variety of DNA or RNA products into host crop plant genomes
which is eligible to overcome pathogen disease agents (Girard et al. 2016). In addi-
tion to R genes, these DNA or RNA products could be highly assorted. Transcription
factors (TFs) are one of the central key regulators responsible for differential gene
expression patterns in both plant and animals.
Most prokaryotic genes responsible for the production of a specific molecule are
clustered sequentially or/and placed together at a single site in the genome. Several
situations were also identified in plant genome as well. Typically, plant defense-­
related genes having related functions mostly tend to localize in a single site of plant
genome as similar to pathogenicity islands (PI) common in plant pathogens. It is
most likely that this clustering incidence of plant defense genes gives advantage to
host plants with respect to instant regulation of the defense response-related genes
by TFs during pathogen attacks. New engineering strategies regarding the resis-
tance of crop plants thus also started to focus on the integration of specific TFs
disposing critical roles in certain defense response pathways besides R gene-­
mediated defense responses (Ishihama and Yoshioka 2012).
As parallel to this strategy, there is an increasing effort to co-express defense-­
related genes and/or TFs in crop plants in order to establish the best defense
188 U. Uzuner et al.

responses, by also not disregarding the crop fitness and yield conditions (Jirschitzka
et al. 2013; Sonderby et al. 2010; Todd et al. 2010; Yu et al. 2012). For instance, the
simultaneous expression of a targeted cluster of TFs could become an effective
approach to balance and coordinate the cellular equilibrium in response to every
stress-based physiologic alteration in crop plants.

9.5.4 Crop Resistance Provided by Algal Metabolites

The recent findings suggest that algal metabolites have great potential to confer
cereal crop resistance to abiotic and biotic stresses beyond enhancing plant growth
(Walters et al. 2013). With less popularity, marine green algae also provide elicitors
to reduce disease susceptibility of cereal crops. The recent studies has displayed that
some green algae species have potential to enhance crop disease resistance. For
instance, β-1,3-glucans (laminarin), β-1,3-sulphated fucans, carrageenans, and ulvan
heteropolysaccharides produced by green alga Ulva spp. exhibit potential to improve
plant resistance to pathogen attacks. The readily attainable ulvans, from the cell walls
of Ulva spp., have great potential to induce crop resistance. When applied with irri-
gating water, crude Ulva extracts are able to confer resistance in Medicago truncat-
ula plants against Colletotrichum trifolii pathogen (Cluzet et  al. 2004), and for
Phaseolus vulgaris to Colletotrichum lindemuthianum infection (Paulert et al. 2009).
The carrageenans from red algae have also great potential to induce crop disease
resistance, but it is notably expensive to be used in plant protection (Stadnik and
Freitas 2014). In addition, the crude extracts of green macroalga Ulva armoricana
have ability to confer crop resistance in bean, grapevine, and cucumber against pow-
dery mildew (Jaulneau et al. 2011). Similarly, application of a seaweed, Ascophyllum
nodosum, extract to tomato results in a reduction in the severity of infections caused
by Phytophthora capsici through enhanced activities of various defense-related
enzymes such as β-1,3-glucanase, peroxidase, and polyphenol oxidase and some
upregulated genes, including lipoxygenase (LOX), chitinase (Chi), galactinol syn-
thase (GolS), and phenylalanine ammonia lyase (PAL) (Ali et al. 2016).
Most algal polysaccharides are eligible to induce multiple crop defense mecha-
nisms against to a plethora of plant pathogens. Eventually, instead of chemical and
synthetic compounds, using naturally produced algal metabolites could be a more
environmentally friendly strategy for agricultural applications to confer crop
defense responses to broad-spectrum pathogens due to the increase on the number
of inexpensive and facilitated algae-growing farms.

9.6 Concluding Remarks

The recent studies present useful findings about the establishment of field resistance,
in particular agriculturally essential cereal crops. Creating disease resistance not only
by using genetic and molecular strategies but also applying defense response induc-
ible chemical analogs is also possible. Furthermore, either genetically established or
9  Salicylic Acid: Molecular Basis of Stress Resistance in Plants 189

the chemically initiated immune response on parental crops could maintain its defen-
sive effects on next progeny. It was also identified that no matter whether it is patho-
gen based or chemically triggered, induced immunity of targeted crops could also
initiate immune responses on the neighboring plants through defense related volatile
compounds (sesquiterpenes like β-caryophyllene, lipoxygenase derivatives). In addi-
tion, due to its strength to induce in miniscule amounts, naturally producible, as an
endogenous plant product and some of the key SAR signals, DA, for instance, and
similar other SAR signals can be used by farmers as an exogenous SAR inducer dur-
ing the sensitive stages of crop plants for disease development.
Multifaceted gene clusters aiming to channel the specific defense pathways and
having potential to induce crop disease resistances are currently being used compre-
hensively by farmers and plant biologists. It is more likely that multifaceted induc-
tion and balanced regulation of SAR response are indispensable for plants in order
to ensure appropriate induction under various biotic and/or abiotic stress conditions.
In addition to integrating R gene or bunch of R genes into crop genomes, when the
defense-related response pathways are unrevealed, future strategies target the
expression of TFs, miRNAs, siRNAs, or RNAi agents to overcome pathogen aggres-
siveness. Defense response-related key regulators identified recently are becoming
more preferable choice to confer resistance in cereal crops due to their powerful
effects in order to control several mechanisms during pathogen attacks. Moreover,
getting benefits from rhizobacteria and beneficial fungi such as Trichoderma virens
and T. atroviride, even microalgae to provide better environmental conditions to
crops, is becoming powerful strategies in agricultural applications.
Molecular findings currently lead to farmers and commercial companies for
large-scale agricultural applications due to its power to provide rapid and sturdy
outcomes compared to conventional breeding efforts. To enhance the crop defense
responses as future approaches, considering the recent findings, promising strate-
gies, and most importantly the continuous cooperation between plant breeders and
researchers should be the foremost goal of agricultural efforts; in particular the most
common cereal crops should be targeted as major human food supplies such as rice,
wheat, maize, and soybean. Deciding on the best defense strategy for a specific
cereal crop most likely requires testing the performance of R genes used to confer
disease resistance through comprehensive experiments with common pathogens.

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Interaction of Salicylic Acid with Plant
Hormones in Plants Under Abiotic Stress 10
Shagun Bali, Poonam, Vandana Gautam, Parminder Kaur,
Kanika Khanna, Rupinder Kaur, Adarsh Pal Vig, Puja Ohri,
and Renu Bhardwaj

Abstract
Plants are exposed to a number of abiotic stresses like salinity, heavy metals,
temperature, drought, etc. which have adverse effects on their growth and yield.
They have well-developed mechanisms which recognize various stress signals
and manage the plants to grow under these stresses. Phytohormones play a major
role in stress protection in plants by intervening growth, nutrient distribution,
development, and source/sink transitions. In plants, interaction between various
phytohormones results in positive and negative cross talk that play an essential
role in response to abiotic stresses. Their biosynthetic pathways and mechanisms
of action are interlinked. A complex hormone signaling and their ability to
interact with each other make them optimal candidates for negotiating defense
responses. Salicylic acid (SA) is an important plant growth regulator which
regulates various physiological processes such as seed development, seed
establishment, cell growth, senescence etc. in plants. The interaction of SA with
other hormones like auxins, gibberellins, abscisic acid, ethylene, cytokinin,
polyamines, jasmonic acid, and brassinosteroids play an important role in fine-
tuning the network of immune response against abiotic stress.

Keywords
Plant hormones • Salicylic acid • Abiotic stress • Crosstalk

S. Bali • Poonam • V. Gautam • P. Kaur • K. Khanna • A.P. Vig • R. Bhardwaj (*)

Department of Botanical and Environmental Sciences, Guru Nanak Dev University,
Amritsar 143005, Punjab, India
e-mail: [email protected]
R. Kaur
Biotechnology Department, DAV College, Amritsar, India
P. Ohri
Department of Zoology, Guru Nanak Dev University, Amritsar, Punjab, India

© Springer Nature Singapore Pte Ltd. 2017 201

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_10
202 S. Bali et al.

10.1 Introduction

Various abiotic stresses have been considered as the major problem for agricultural
productivity throughout the world. These stresses like heavy metal, azone, tempera-
ture, drought, UV-B radiation, etc. have been aggravated through anthropogenic
activities (Wani et al. 2016). These stresses lead to negative impact on developmen-
tal processes in plants from seed germination to maturity which ultimately causes
severe damage to the yield of economical important plants. Plants have a wide range
of mechanisms to deal with these stresses through physiological, metabolic, and
molecular approaches (Kazan 2015). Alterations in stress-responsive phytohor-
mones help in modifying cellular dynamics and therefore play an imperative role in
symmetrically regulating various responses under stress (Kohli et al. 2013). Plant
hormones regulate developmental processes and signaling plexus as they are
connected directly or indirectly in an extensive spectrum of abiotic and biotic stress
responses in plants (Khan et  al. 2012a; Asgher et  al. 2015). The junction points
among signaling cascades of hormones are recognized as cross talk and form com-
plex signaling network (Harrison 2012). The antagonist and synergistic interactions
between phytohormones play crucial role in defense signaling pathways in plants.
Plant hormones like jasmonic acid, ethylene, abscisic acid, and salicylic acid are the
essential components of various signaling pathways engrossed in plant defense
(War et al. 2011; Vicent et al. 2011; Lu 2009). SA acts as signaling molecule and
regulates plant growth and development (Miura and Tada 2014). It is biosynthesized
from phenylalanine with the help of enzyme phenylalanine ammonia lyase in the
shikimate-phenylpropanoid pathway (Raskin 1992). Signaling cascades interplay
with the pathways transduced by plant hormones get stimulated by abiotic stress
(Harrison 2012). SA plays a crucial role in different plant processes like cell growth
(Vanacker 2001), thermotolerance (Clarke et  al. 2004), nodulation (Stacey et  al.
2006), seed germination (Rajou et al. 2006), respiration (Norman et al. 2004), and
senescence (Rao et al. 2002). In plants, the application of SA stimulates stress toler-
ance against salinity and temperature, nutrient contents, and pathogen attack
(Guzmán-Téllez et al. 2014; Wildermuth et al. 2002). It has been reported that treat-
ment of SA to Arabidopsis auxin receptor (TIR1/AFB2), mutants showed increase
in the expression of pathogenesis-related gene (PR1). Auxin signaling was down-
regulated by SA in TIR1/AFB2 mutants which showed antagonistic interaction
between SA and auxin (Iglesias et al. 2011). In Arabidopsis thaliana, endogenous
SA levels were enhanced by gibberellins and stimulated GASA (gibberellic acid-­
stimulated Arabidopsis) gene family that helped the plant to counteract the adverse
effects caused by abiotic stress (Alonso-Ramírez et al. 2009). Exogenous applica-
tion of SA inhibited the production of ethylene in Triticum aestivum under heat
stress which showed negative interaction between SA and auxin (Khan et al. 2013).
In rice plants, cytokinin has synergistic effect with SA and activated defense
response against stress (Jiang et al. 2013). Application of SA at low concentrations
may stimulate the polyamines synthesis, and alterations in PA catabolism may
involve in stress acclimation mechanisms in tomato plants (Szepesi et  al. 2009,
2011). SA may induce negative regulators that can directly or indirectly repress the
10  Interaction of Salicylic Acid with Plant Hormones in Plants Under Abiotic Stress 203

JA-responsive gene expression (Caarls et  al. 2015). The nonexpressor of

pathogenesis-­related genes1 (NPR1) is a master regulator of SA-mediated defense
response gene which is critical component of brassinosteroid-mediated elevation in
thermo and salt tolerance in A. thaliana mutants (Divi et al. 2010). These interac-
tions may play a major role in abiotic stress tolerance in order to develop stress
resistant varieties for sustainable agriculture. Keeping this in mind, the chapter was
focused on the interaction of SA with phytohormones under abiotic stress in plants.

10.2 I nteraction of Salicylic Acid with Auxins Under Abiotic

Stress

Auxins play a vital role in growth and development of plants. They respond to envi-
ronmental stimuli like root plastic growth and different tropic responses (Davies
2004). Auxins actively participate in many plant metabolic processes as well as root
differentiation (Agtuca et al. 2014). Apart from their role in normal plant growth and
development, they are also involved in abiotic stress tolerance. It has been found that
IAA plays an important role in salinity stress tolerance in some plants (Fahad et al.
2015). They also promote root and shoot growth in wheat under heavy metal or salin-
ity stress and alleviates salinity-induced dormancy of wheat (Egamberdieva 2009). It
has been reported that SA interferes with IAA signaling, and this integration enables
the plant to respond to many environmental stresses (Agtuca et al. 2014).
IAA and SA antagonistically affected each other in case of maize where exoge-
nous application of IAA enhanced lateral root formation rather than primary roots
formation, and SA enhanced total root biomass (Agtuca et al. 2014). SA function-
ally interacts with auxins in plant defensive mechanisms and by acting as an essen-
tial plant growth regulator in Arabidopsis. GH3 family belonging to auxin-responsive
genes is well known. This family encodes IAA-conjugating enzymes and increase
auxin levels endogenously which enables plant defense mechanism (Iglesias et al.
2011). One of the active members of this family of genes is GH3.5 that encodes
protein possessing in vitro adenylation activity on IAA and SA. Under stress condi-
tions, GH3.5 acts as stimulator in SA and auxin signaling. Overexpression of
GH3.5 in gh3.5-D mutant caused SA and IAA accumulation in Arabidopsis thali-
ana showing its active participation in defense mechanisms (Zhang et al. 2007). In
addition, Wes1-D gene of GH3.5 family in Arabidopsis thaliana induced tolerance
toward drought, freezing, and salinity stress via SA-auxin signaling (Park et  al.
2007). Studies reported that auxin receptors (TIR1 and AFB2) in Arabidopsis are
involved in salinity stress tolerance by improving germination rate, chlorophyll con-
tents, and root elongation. Furthermore, auxin mutants (tir1 afb2) showed elevated
transcript levels of pathogenesis-related gene PR1 when treated with salicylic acid.
Overexpression of this PR1 gene via SA application in auxin tir1 afb2 mutants give
insights of SA-induced auxin signaling in salinity stressed plants (Iglesias et  al.
2011). One of the common approaches of plants to deal with stress condition is
SA-mediated suppression of auxins studied in Arabidopsis. In this, microRNA
miR393, the main regulator of TIR1, AFB2, and AFB (auxin receptors) is stimulated
204 S. Bali et al.

under cold and salinity stress (Sunkar et  al. 2007). Similar studies were found
Navarro et  al. (2006) in which miR393-triggered repression of auxin receptors
occurs in plant so as to alleviate stress conditions. Exogenous supplementation of
SA caused repression of auxin-related genes and resulted in modulation of Aux/
IAA proteins and inhibition of auxin responses in Arabidopsis thaliana (Wang et al.
2007). Cross talk of salicylic acid with auxin under abiotic stress in different plants
is mentioned in Table 10.1.

10.3 I nteraction of Salicylic Acid with Gibberellins

Under Abiotic Stress

Gibberellins (GAs) are group of tetracyclic diterpenoid carboxylic acids that posi-
tively affects flowering, fruit development, seed germination, trichome initiation,
and leaf expansion (Yamaguchi et al. 2008). They stimulate growth and promote
phase transitions and actively participate in abiotic stress tolerance and adaptation
(Colebrook et al. 2014). Additionally, they regulate source-sink relationship within
plants under stressed and nonstressed conditions by altering sink formation, resource
mobilization, and photosynthesis (Iqbal et al. 2011). It has been reported that GAs
improve seed germination under salinity (Kaya et al. 2009), as well as alleviation of
salt stress from pigments, hill activity, and water use efficiency (WUE) (Shah 2007).
GA signaling enables plants to adapt to limiting environment conditions, and this
can be mediated through SA regulation. GAs and SA together are involved in regu-
lation of many plant responses. They interact and help in modulation of pathogenesis-­
associated protein expression (Miura et  al. 2014) and involved in plant defense
against abiotic stresses (Fayez et al. 2014; Miura et al. 2013).
It was reported by Xie et al. (2007) that SA decreased GA-stimulated amylase
production, seed germination, and post-germination growth in barley plants. They
found that this suppression of seed germination by SA occurs due to GA suppress-
ible WRKY genes that block GA-induced α- amylase expression (Xie et al. 2007).
The antagonistic relationship between SA and GA was studied in Arabidopsis plant
in regulation of trichome development. Exogenous SA suppresses GA-initiated tri-
chome development through downregulation of Npr1/Nim2 gene (Traw and
Bergelson 2003).
GAs induce degradation of well-known DELLA proteins and promote plant
growth (Alonso-Ramirez 2009). DELLA proteins stimulate gene expression of
ROS-detoxifying enzymes, thus modulating ROS levels after abiotic/biotic stresses
(Achard et al. 2008). They act as functional components in GA-SA interaction dur-
ing plant defense mechanisms (An and Mou 2011). Moreover, when exogenous
levels of GA3 are applied, they alter the inhibitory effects of abiotic stresses in seed
germination and seedling establishment. They also upregulate SA biosynthesis and
improve plant responses toward abiotic stress by increasing SA levels. However,
they prevent inhibitory effects of stressed conditions on seedling growth and germi-
nation by stimulating SA biosynthesis (Alonso-Ramirez et al. 2009). Another study
demonstrated that upregulation of GA3ox1 (GA biosynthetic gene) occurred after
10  Interaction of Salicylic Acid with Plant Hormones in Plants Under Abiotic Stress 205

Table 10.1  Cross talk of salicylic acid with gibberellins and auxins under abiotic stress
S. no Phytohormone Plant Stress Response References
1. Gibberellins Tomato Chilling Decreased chilling Ding et al. (2016)
injury index (CI)
and oxidative
damage by GA
metabolism, CBF1
gene expression,
and antioxidant
enzymatic activities
Upregulation of
GA biosynthetic
gene (GA3ox1)
through SA-GA
signaling
DELLA protein
degradation
2. Gibberellins Tomato Chilling Enhanced SA Zhu et al. (2016)
biosynthesis by
isochorismate
synthase (ICS)
pathway
Elevated CBF1
gene expression
through GA2ox1
upregulation
Downregulation of
GA biosynthetic
genes (GA20ox1
and GA3ox1)
3 Gibberellins Tomato Cold Activation of SA Ding et al. (2015)
(GA3) stress production through
GA3-SA signaling
4. Gibberellins Sorghum Drought SA-GA interaction Sheykhbaglou
bicolor L. increased catalase, et al. (2014)
ascorbate
peroxidase
activities,
germination
percentage, and
germination index
5. Gibberellins Arabidopsis Chilling Upregulation of Kim et al. (2013)
(GA3) ICS1 gene
(isochorismate
synthase)
Stimulation of SA
biosynthesis
(continued)
206 S. Bali et al.

Table 10.1 (continued)
S. no Phytohormone Plant Stress Response References
6. Gibberellins Zoysiagrass Salt stress Increased Dai et al. (2012)
(GAs) (Zoysia endogenous SA
japonica) levels and
alleviation of
growth inhibition
and plant death
Modulation of SA
biosynthesis
7. Gibberellins Arabidopsis Salinity Modulation of Lee et al. (2010)
(GAs) biochemical and
molecular GA-SA
signaling
mechanism
GA-SA signaling
improved seed
germination
8. Gibberellins Soyabean Salt stress Decreased SA Hamayun et al.
(GAs) content, under (2010)
increased GA3 and
elevated SA under
NaCl stress
Upregulation of SA
biosynthesis
9. Gibberellins Arabidopsis Salt stress Stimulation of SA Lee and Park
(GAs) thaliana biosynthesis by (2010)
inducing SID2
gene and genes
encoding GA
biosynthetic
enzymes
Increased seed
germination by
nodulation of
antioxidant
activities
10. Gibberellins Arabidopsis NaCl Improved Alonso-Ramirez
thaliana (salinity) germination of (2009)
SA-deficient sid2
mutants via SA-GA
signaling
11. Gibberellins Arabidopsis Heat Triggering Alonso-Ramirez
thaliana stress isochorismate (2009)
synthase1 and
nonexpressor PR1
genes of SA
biosynthesis
(continued)
10  Interaction of Salicylic Acid with Plant Hormones in Plants Under Abiotic Stress 207

Table 10.1 (continued)
S. no Phytohormone Plant Stress Response References
12. Gibberellins Arabidopsis Heat Overexpression of Horvath et al.
(GAs) thaliana stress FsGASA4 (2007)
(GA-induced gene
from beechnut)
modulates SA
biosynthesis
13. Gibberellins Arabidopsis Salinity SA enhanced Yamaguchi et al.
thaliana GA3-oxidase1 (2001)
(GA3ox1) gene
expression in
wild-type seeds
Improved seed
germination
through SA-GA
action
14. Auxins (IAA) Okra Salinity Stimulation of salt Esan et al. (2017)
tolerance,
antioxidant
activities, and
DPPH
(2,2-diphenyl
radical scavenging
capacity) via
IAA-SA interaction
15. Auxins (IAA) Barley Heavy Inhibition of Tamás et al.
metals Cd-induced IAA (2015)
(Cd) mediated ROS
generation through
IAA-SA signaling
Activation of
lipoxygenase or
glutathione
peroxidases along
with stimulated
root growth
inhibition,
swelling, and ROS
generation
16. Auxins (IAA) Maize Salinity Elevated Fahad and Bano
micronutrients (2012)
CO3+,Mn2+, Cu3+,
Fe2+ in roots
Increased chl a, chl
b, carotenoids,
sugars, proline, and
protein content
through SA-IAA
communication
(continued)
208 S. Bali et al.

Table 10.1 (continued)
S. no Phytohormone Plant Stress Response References
17. Auxins (IAA) Maize Heavy IAA-SA action Fahad and Bano
metals decreased Na+, (2012)
Ni3+, Pb4+, Zn2+,
Na+/K+ content in
soil and roots
18. Auxins (IAA) Arabidopsis Salt stress SA increased Iglesias et al.
thaliana transcript levels of (2011)
pathogenesis-­
related gene (PR1)
in tir1 afb2 mutant
SA-mediated
repression of auxin
signaling
Increased tolerance
toward salinity in
double-mutant
TIR1and AFB2
receptors tir1 afb2

SA supplementation followed by DELLA protein degradation (Ding et al. 2016).

C-repeat/dehydration-responsive element binding factor (CBF) plays a vital role in
cold stress (Oakenfull et  al. 2013). SA, along with GA3, elevated CBF1 gene
expression in tomato under cold stress (Zhu et al. 2016). Moreover, overexpression
of GA-responsive genes is involved in increasing endogenous levels of SA and
SA-biosynthetic genes ics1 and npr1 (Alonso-Ramirez 2009). It was reported in A.
thaliana that GA-activated isocitrate lyase enzyme required in lipid metabolism
during seed germination was induced by SA (de los Reyes et  al. 2003). Various
reports of cross talk of salicylic acid with gibberellins under abiotic stress in differ-
ent plants are mentioned in Table 10.1.

10.4 I nteraction of Salicylic Acid with Abscisic Acid

Under Abiotic Stress

Abscisic acid (ABA) is one of the most important hormones associated with the
discernment of several abiotic stresses, especially cold, osmotic, drought, etc.
(Cramer et al. 2011; Xiong et al. 2002; Vlad et al. 2008). The ABA-responsive ele-
ment (ABRE) endorses several defense genes which indicate the involvement of
ABA in the commencement of stress protection via transcriptional reprogramming
of plant cell metabolism (Wasilewska et al. 2008). ABA gets accumulated after the
stress symptoms appear and inhibits the former protection reaction of the plants (De
Torres-Zabala et al. 2007). SA and ABA both play an important role in plant defense
10  Interaction of Salicylic Acid with Plant Hormones in Plants Under Abiotic Stress 209

against water deficit or drought stress (Chinchilla et  al. 2007; Bandurska et  al.
2005). SA and ABA act antagonistically as SA-induced stress responses under the
combination of abiotic and biotic stresses are negatively managed by ABA (Mosher
et al. 2010). SA activates the accumulation of ABA in Solanum lycopersicum plants
under salinity stress, which ultimately facilitated the osmotic adjustment and better-
ment in growth quality and photosynthetic pigments (Szepesi et  al. 2009).
SA-associated abiotic stress response in plants during cold stress is modified by
ABA. The endogenous SA levels in the cold-stressed Zea mays were changed by the
ABA treatment (Szalai et al. 2011). The study was conducted by Yasuda et al. (2008)
to investigate the antagonistic behavior of ABA and SA during combination of abi-
otic and biotic stresses. The study shows that ABA holds back the SA-mediated
stress defense mechanisms as exogenous application of SA protected the plants
from salt stress, whereas this protection was observed to be hindered when ABA
was applied exogenously. The investigation on the interaction flanked by these two
hormones during abiotic stresses is still in its formative years. The biosynthetic
mechanisms of the ABA and SA in plants exposed to different types of abiotic
stresses can be customized to redirect the metabolites assembly to convene the
needs for abiotic stress defense.

10.5 I nteraction of Salicylic Acid with Ethylene Under Abiotic

Stress

Salicylic acid is a well-known key player in plant defense against the symptoms of
stress, whereas along with controlling several phases of plant growth and develop-
ment, sometimes, enhanced ethylene production also results in causing oxidative
stress to the plants under abiotic stress (Khan and Khan 2014; Khan et al. 2015).
Under abiotic stress, biosynthesis of ethylene is restrained by the salicylic acid by
confining the transformation of 1-aminocyclopropane-l-carboxylic acid to ethylene
(Leslie and Romani 1986). During heat stress, exogenous application of salicylic
acid helps to alleviate the damage to plants by limiting ethylene generation and
enhancing pro metabolism to such an extent so that activity of 1-aminocyclopropane-
l-carboxylic acid synthase enzyme is held back (Khan et al. 2013). Salicylic acid-­
induced inhibition of 1-aminocyclopropane-l-carboxylic acid in Vigna radiata
plants under salinity stress leads to the repression of ethylene development and
stepped up plant growth and photosynthesis (Khan et al. 2014). According to Poór
et  al. (2013), ethylene is responsible for cell death under salinity stress in
Lycopersicon esculentum cell suspension due to the production of reactive oxygen
species, whereas SA-induced reactive oxygen species generation was not found to
be directly related to ethylene. However, some reports show the synergistic cross
talk flanked by salicylic acid and ethylene under abiotic stress conditions in plants
(Ghanta et  al. 2014). In root stock of Prunus persica ethylene generation was
increased by addition of salicylic acid in MS medium, but under AgNO3 stress
210 S. Bali et al.

ethylene generation decreased (Molassiotis et al. 2005). It has been elucidated that
the effects of ethylene on chlorophyll, anthocyanin, carotenoid, ascorbic acid, flavo-
noids, dehydroascorbic acid, lipid peroxidation, total ascorbate, and ethylene pro-
duction in leaves of canola are pretreated with SA. The results demonstrated that the
ethylene treatments induced lipid peroxidation, while SA mitigated this effect. The
chlorophyll, anthocyanin, and carotenoid contents were decreased considerably by
ethylene treatment, whereas SA alleviated these effects (Tirani et  al. 2013). SA
application may alleviate the damages originated by stressful environment.

10.6 I nteraction of Salicylic Acid with Cytokinins

Under Abiotic Stress

Cytokinins (CKs) are a class of phytohormones known to be master regulators in

controlling plant growth and cell division (Nishiyama et al. 2011; Kang et al. 2012).
They are adenine derivatives with aromatic or hydroxyl side chain commonly
known as isoprenoid. Out of the major classes of CKs (depending upon the hydrox-
ylation and reduction of side chain) isopentenyladenine (iP), trans-zeatin (tz), and
cis-zeatin (cZ) types are found in abundance in plants (Strnad et al. 1997; Sakakibara
2006; Ha et al. 2012). Under abiotic stress conditions decline in the content of cyto-
kinins and reduced transportation in plants was observed. Treatment of plants with
exogenous CKs to plant can increase stomatal size and transpiration rate resulting
in increase of photosynthetic activity hence showing the important role of CKs in
stress management (Farber et al. 2016). Various kinds of stresses lead to activation
of CK pathway and lead to modulation of various responses. Davies et al. (2005)
observed tremendous reduction in trans-zeatin riboside contents under drought con-
ditions. Similar results were obtained in grapevines when roots were partially dried
(Stoll et al. 2000). Decline in CK content and accumulation of ABA was observed
in seedlings of wheat grown under 2% salinity stress. However, treatment of seeds
with SA before sowing prevented the reduction in the content of CKs in wheat seed-
lings (Shakirova et al. 2003). The role of SA and CKs in inhibiting normal cell cycle
was studied by Xia et al. (2009) in Arabidopsis thaliana grown under chilling stress.
Arabidopsis thaliana (amp1 type) plant which is a screened mutant was grown at
23, 16, 10, and 4 °C. It was observed that the amp1 plants increased level of cytoki-
nin and the wild-type plants given exogenous treatment of cytokinin at 4 °C had
relatively high growth and cell division rate in comparison to control. However, its
transgenic plants NahG having low levels of SA grew faster than its wild type and
has large cell size. It is assumed that there is an increase in SA and decrease in CK
in plants at low temperature which may negatively regulate the plant growth via
controlling the expression level of CYCD3;1 D-type cyclin and motivating the G1/S
phase transition (Riou-Khamlichi et al. 1999; Menges et al. 2006).
10  Interaction of Salicylic Acid with Plant Hormones in Plants Under Abiotic Stress 211

10.7 I nteraction of Salicylic Acid with Polyamines

Under Abiotic Stress

Polyamines (PAs) are small organic polycationic compounds having low molecular
weight. The most common types of PAs found in plants are putrescine (Put), sper-
midine (Sdp), spermine (Spm), and thermospermine (tSpm). These PAs are either
found in free or in conjugated forms and known for their simple structure, ubiqui-
tous distribution in cell compartments, and important physiological and biological
roles for various metabolites. Their accumulation in cells is involved in reducing
NH3 toxicity and sequestration of extra N that change the responses of plants by
changing the concentrations of PAs (Hussain et  al. 2011; Shi and Chan 2014;
Minocha et al. 2014). Enhanced levels of PAs lead to increased tolerance of plants
under abiotic stresses resulting in overexpression of genes ODC (encodes for putres-
cine biosynthesis) in tobacco, ADC (encodes for putrescine biosynthesis) and
SAMDC (encodes for putrescine and spermine) in rice, SPDS (encodes for spermi-
dine biosynthesis) in tomato, and ADC2 (encodes for putrescine) in Arabidopsis
(Kurima and Rajam 2002; Capell et al. 2004; Roy and Wu 2002; Wen et al. 2009).
Increase in content of Put, Spd, and Spm under drought stress has been observed in
rice plants which showed direct relationship between the PAs content and drought
resistance of rice plants (Zhou and Yu 2010). Exogenous application of 0.5 mM of
SA to maize under low temperature in hydroponic culture significantly enhanced
polyamines level (putrescine, spermidine) and improves plant tolerance to low tem-
perature and sensitivity to drought in zea mays plants (Németh et  al. 2002). The
increased level of polyamines protect plants under low temperature by reducing the
lipid peroxidation and conserving the membrane integrity which partially add to the
protective role of SA i.e., enhanced antioxidants under chilling stress (Janda et al.
1999; Yuan and Lin 2008). Priming of seeds of maize plants with SA and subse-
quent exposure to low temperatures also results in significant incline in putrescine
content and minor decrease in spermidine content in seeds after 5 days of sowing.
Increase in the content of putrescine, spermidine, and spermine in roots was
observed which may account for the normal growth of plants which suggest the role
of polyamines in stress tolerance after priming with SA (Szalai et al. 2016). Change
in the contents of Put, Spd, and Spm could be due to their interconversion in the
polyamine cycle and the degradation of polyamines catalyzed by polyamine oxi-
dases (Pàl et al. 2014). However antagonistic reports were reported in root nodules
of Medicago sativa pretreated with 0.1 and 0.5 mM. SA induces resistance under
salt stress. The SA treatment induces accumulation of H2O2 and inhibition of PAs in
root nodules of Medicago sativa. It was observed that decline in the activity of anti-
oxidative enzymes resulted in the accumulation of H2O2. Treatment of SA prevents
the accumulation of PAs in response to salinity stress and induction of 1-­aminocycl
opropane-­l-carboxylic acid (ACC) content by activating the ethylene signaling
pathway. Inhibition of PAs by SA may be responsible for activating ethylene depen-
dent hypersensitive response under salinity stress (Palma et al. 2013).
212 S. Bali et al.

10.8 I nteraction of Salicylic Acid with Jasmonic Acid

Under Abiotic Stress in Plants

Jasmonic acid, the lipid-derived compounds, is mainly associated with stress man-
agement. It is reported to interact with salicylic acid during stress protection in
plants. A combined study of proteomics and transcriptomics showed up-regulation
of common proteins by JA and SA under oxidative stress responses (Proietti et al.
2013). The role of AtMPK4 under abiotic stress is unclear, but it is well known that
AtMPK4 is activated under abiotic stress (Ichimura et al. 2000). The combination of
chlorothalonil and metalaxyl induced the production of SA and JA in potato plants
which show an unclear synergistic cross talk between these two hormones
(Gonzalez-Gallegos et al. 2015). The exogenous application of SA and JA enhanced
the total antioxidant capacity of chamomile plants against drought stress (Nazarli
et al. 2014). Redox controlled transcriptional cofactor NPR1 genes (Nonexpressor
of pathogenesis-related genes1) help in mediating cross talk of SA and JA (Backer
et al. 2015).
SA and JA show antagonistic interaction at MAPKs signaling and biosynthesis
(Khan et al. 2012a, b). The negative interaction of SA and JA also modulated the
expression of PR protein genes, whereas SA induces PR genes (Thaler et al. 1999;
Wang et al. 2001).

10.9 I nteraction of Salicylic Acid with Brassinosteroids

Under Abiotic Stress in Plants

Brassinosteroids (BRs) are the plant steroidal hormones which have gained atten-
tion for their role in conferring stress tolerance to plants under a wide array of
stresses. The interaction of BRs with other plant hormones like salicylic acid (SA)
has been reported to enhance tolerance of plants (Divi et  al. 2010). Arabidopsis
seeds with mutant NPR1-1 genes were treated with 1 μM EBR and grown under
controlled conditions for 21 days. The seedlings were exposed to 43 °C for 4 h to
give heat stress. The seeds were also treated with 1 μM EBR and salt treatment add-
ing 150 mM NaCl in growth medium. The EBR-mediated increase in thermal and
salinity stress is controlled by NPR1 genes, which are associated with regulation of
SA-mediated defense genes. Another transcription factor WRKY70 which partici-
pates in the expression of SA-induced PR genes has been found to upregulated by
BR (Divi et al. 2010). It has been reported that combined application of SA and BRs
was more effective in mitigating the adverse effects under salinity stress (Ding et al.
2012). It shows a positive regulatory relationship between SA and BR. It has been
observed that SA-insensitive NPR1-1 mutant plants showed less tolerance toward
exogenous application of BRs (Divi et al. 2010). Brassica juncea plants were grown
under salinity stress (NaCl, 4.2 dsm−1). Application of BRs and SA showed enhanced
stress tolerance. Twenty-nine-day-old plants were sprayed with SA (10−5 M) and
HBL (10−8 M). The combination of SA and BRs was found most effective in increas-
ing salt stress tolerance (Hayat et al. 2012). It has been found that a synthesized
10  Interaction of Salicylic Acid with Plant Hormones in Plants Under Abiotic Stress 213

conjugate of salicylic acid and 24-EBL and 24-epicastasterone improved the sur-
vival of millet seedlings under heat and salt stress. The conjugate was synthesized
by the reaction of brassinosteroids with 2-O-benzylsalicylic acid followed by
hydrogenolysis of O-benzyl groups. The conjugates also reduced the lipid peroxida-
tion (Litvinovskaya et al. 2016).

10.10 Conclusion

Plant growth is adversely affected by abiotic stresses such as heavy metal, extreme
temperatures, high salinity, drought, etc. which ultimately affect the crop productiv-
ity and sustainable agriculture. Phytohormones are synthesized in very low amount
and able to regulate various developmental processes in plants. They play a vital
role in coordinating different signaling pathways during stress conditions. SA can
be used as potential compound because of its critical role in adapting and improving
growth under stress. Cross talk of phytohormones triggers various signaling path-
ways which modify particular stress and developmental responses. Various tran-
scription factors and hormone inducible genes may help in the designing of
promising techniques to provide resistance against abiotic stress and improve the
crop yield. Plant hormone engineering manifests an important platform for abiotic
stress tolerance and provides a novel approach for sustainable agriculture.

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Salicylic Acid and Nutrients Interplay
in Abiotic Stress Tolerance 11
Tasir S. Per, Mehar Fatma, Mohd. Asgher, Sofi Javied,
and Nafees A. Khan

Abstract
Abiotic stress factors, such as cold, heat, drought, flood, salinity, or oxidizing
agents, are the major threats to agricultural system that affect the economic yield
of crop plants. Phytohormones, the chemical messengers, play a vital role in
resistance of plants to the changing environments by regulating physiological
and molecular processes. Salicylic acid (SA) regulates photosynthetic events,
nutrient metabolism, osmotic relations, and defense mechanisms in plants grow-
ing under optimal and changing environmental conditions. The role of SA in the
regulation of nutrients metabolism and their interplay for abiotic stress tolerance
is in infancy stage. Few reports are available on the interaction between SA and
macro- and micronutrients and the influence of nutrients on SA biosynthesis and

T.S. Per (*)

Plant Physiology and Biochemistry Division, Department of Botany, Aligarh Muslim
University, Aligarh 202002, UP, India
Govt Degree College Doda, Doda 182202, Jammu and Kashmir, India
e-mail: [email protected]
M. Fatma (*)
Plant Physiology and Biochemistry Division, Department of Botany, Aligarh Muslim
University, Aligarh 202002, UP, India
Plant Molecular Biology, School of Life Sciences Jawaharlal Nehru University,
New Delhi, 110067 Delhi, India
e-mail: [email protected]
M. Asgher
Plant Physiology and Biochemistry Division, Department of Botany, Aligarh Muslim
University, Aligarh 202002, UP, India
Molecular and Conservation Lab, School of Biosciences and Biotechnology,
Baba Ghulam Shah Badshah University, Rajouri 185234, India
S. Javied • N.A. Khan
Plant Physiology and Biochemistry Division, Department of Botany, Aligarh Muslim
University, Aligarh 202002, UP, India

© Springer Nature Singapore Pte Ltd. 2017 221

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7_11
222 T.S. Per et al.

perception. The present chapter discusses the role of SA in macro-, micro-, and
beneficial elements in the alleviation of adverse effects of abiotic stresses in
plants. Moreover, it also covers the effect of deficiency or excess of the mineral
nutrients on SA-induced abiotic stress tolerance mechanisms. The topics covered
in the chapter are (a) biosynthesis and signaling of SA in plants exposed to major
abiotic stresses (b) and understanding the mechanisms underlying between SA
and nutrient signal transduction pathway in plants for abiotic stress tolerance.

Keywords
Abiotic stress • Salicylic acid • Mineral nutrients

11.1 Introduction

The global agricultural aim for increasing the production of food by 70% by the
year 2050 for approximately 2.3 billion newborn individuals is facing dire obstruc-
tions in the figure of poverty, hunger, and minor change with the environmental
modification and exploitation of rare natural resources more economically (FAO
2009). These efforts are not going to meet success because of the abiotic stress fac-
tors (Mian et  al. 2011) and impact of climate change on ecosystems (Dimeyeva
et al. 2015) that has given severe negative impact on the gross agronomy. Abiotic
stress factors such as salinity, drought, heavy metals, UV-B radiation, air pollution,
and heat stress hamper plant growth. These stresses pose major threat to agricultural
system which affects growth and physiological and biochemical processes of plants.
Efforts have been made to know potential tolerance mechanisms and plant response
under such stressed conditions.
Several plant growth regulators are well-characterized signaling molecules in
plant defense reactions that work out for growth and development of plants under
optimal and stressful conditions. In particular, salicylic acid (SA), a phenolic com-
pound synthesized in plant cell system, regulates the growth and development of
plants in response to biotic and abiotic stresses (Khan et al. 2012, 2015; Miura and
Tada 2014). SA regulates photosynthesis, nitrogen (N) metabolism, and proline
synthesis and is responsible for synthesizing other plant growth regulators which
protect the plants against abiotic stresses (Khan et al. 2012, 2015; Nazar et al. 2011,
2015; Miura and Tada 2014). SA improves plant tolerance to almost all types of
abiotic stresses such as salinity (Khan et al. 2014; Nazar et al. 2015), osmotic (Naser
Alavi et al. 2014), heavy metals (Zhang et al. 2015), drought (Fayez and Bazaid
2014), and heat (Khan et al. 2013).
In order to perform normal physiological and biochemical functions, all plants
need an adequate supply of macro- and microelements. In addition to basic mineral
nutrients requirements such as N, phosphorus (P), and potassium (K), some other
elements like magnesium (Mg), sulfur (S), iron (Fe), zinc (Zn), boron (B), etc. are
considered to be essential for plant metabolism and growth and developmental pro-
cesses because they are cofactors and/or activators of many metabolic enzymes
11  Salicylic Acid and Nutrients Interplay in Abiotic Stress Tolerance 223

(Ashley 2011). The application of nutrients contributes to manipulate environmen-

tal variables when properly integrated into a soil management program. It can be
used as a supplement to compensate for shortcomings of soils, that is, to provide
adequate nutrients when nature does not supply them during the critical stages of
the seasonal growth cycle (Keller 2005). These nutrients are important for synthesis
of antioxidants and help in alleviating various abiotic stresses. Mineral nutrients like
Fe, calcium (Ca), copper (Cu), P, manganese (Mn), and Zn are also beneficial for
reducing the oxidative stress caused by abiotic stress conditions (Wang et al. 2011).
For example, S is one of the major nutrients for metabolism and plant growth and
development. It is a building block of protein and affects photosynthesis because S
is a major constituent of chlorophyll and many enzymes of the photosynthetic car-
bon reduction cycle (Marschner 1995). Sulfur plays an imperative role in the forma-
tion of amino acids, methionine (Met), and cysteine (Cys) or incorporated into
proteins or Cys-containing peptides such as glutathione (GSH) (Iqbal et al. 2013).
Methionine is the precursor for S-adenosylmethionine (SAM), ethylene, poly-
amines, and nicotinamine which is important for Fe nutrition in plants (Bürstenbinder
et al. 2007). Iron behaves as a cofactor for various enzymatic proteins. Its deficiency
affects chlorophyll biosynthesis (Graziano et al. 2002) and thylakoid synthesis and
also develops chlorosis symptoms (Jin et al. 2007).
Salicylic acid interacts with nutrients synergistically or antagonistically and reg-
ulates plant growth and development under optimal and stressful environments. For
instance, SA alleviates salt stress by inducing enzymes of S and N assimilation
(Nazar et al. 2011). It shows that the interaction of SA with S and N assimilation is
crucial in stress tolerance. Major progress has been made in identifying the key
components and understanding the role of SA and nutrients implicated in plant
defense pathways, but their role in abiotic stress defense is less studied. It is, there-
fore, important to identify and scrutinize how mineral nutrient balance is induced by
SA to counteract the deleterious effects of abiotic stress. The present chapter focuses
mainly to improve our knowledge on the area of regulation of abiotic stress toler-
ance through mineral nutrition employing physiological mechanisms and molecular
tools with special emphasis on the involvement of SA.

11.2 Biosynthesis of Salicylic Acid

The naturally synthesized endogenous phytohormone SA (2-hydroxybenzoic acid)

found in plants is a phenolic compound with seven-carbon (C) skeleton. These plant
phenolic compounds are produced by two main secondary metabolite-producing
pathways; one is shikimic acid pathway and another is malonic acid pathway.
However most of the phenolic compounds in plants are produced by shikimic acid
pathway by converting the precursors of carbohydrate obtained from glycolysis and
pentose phosphate pathway into aromatic amino acids like phenylalanine which is
the precursor of SA (Herrmann and Weaver 1999; Dempsey et al. 2011). Recently,
due to the presumption of nonessentiality of these aromatic compounds for critical
processes, these are categorized into “secondary metabolites” (Hadacek et al. 2011).
224 T.S. Per et al.

An alternative pathway also accumulates SA in plants called the isochorismate

pathway, but most of the SA in plants is synthesized through phenylalanine pathway
(Kawano et  al. 2004; Mustafa et  al. 2009). In phenylalanine pathway the trans-­
cinnamic acid is formed due to the elimination of ammonia from the phenylalanine
by the action of an enzyme phenylalanine ammonia-lyase (PAL). The cinnamic acid
acts as a precursor for two compounds, one is coumaric acid, produced by hydrox-
ylation of cinnamic acid at C4 position by the action of an enzyme cinnamate-4-­
hydroxylase (C4H). Later coumaric acid is followed by oxidation of its side chain
and hydroxylation leading to biosynthesis of SA. Second compound produced from
cinnamic acid is benzoic acid, which is followed by hydroxylation at ortho-position
leading to the formation of SA by the action of an enzyme benzoic acid 2-­hydroxylase
(Verberne et al. 1999; Mustafa et al. 2009).
In isochorismate pathway, chorismate acts as a precursor for isochorismate by
the activity of an enzyme isochorismate synthase (ICS). Isochorismate gets con-
verted into SA by the action of an enzyme isochorismate pyruvate lyase (Mustafa
et al. 2009). When the plant is subjected to any biotic or abiotic stresses, the ICS
level rises in the plant cells leading to high level of SA in plant defense processes
(Wildermuth et al. 2001). Most of the SA in plants remains in a glycosylated form
which is its inactive form. UDP-glucose SA-glucosyltransferase converts SA into
glycosylated form. However, SA glycosidase converts inactive form of SA into
active form. These two enzymes also act as regulatory enzymes which confer the
level of SA in plant cells in different environments. Primarily the biosynthesis of SA
in stressed Arabidopsis occurs in chloroplast via isochorismate utilizing pathway.
The schematic representation of the biosynthesis of SA is shown in Fig. 11.1.

11.3 Signaling of Salicylic Acid Under Abiotic Stresses

Salicylic acid acts as an endogenous natural signal molecule and plays a pivotal role
in defense mechanisms by regulating physiological and biochemical processes
(Gunes et al. 2007; Joseph et al. 2010). Various findings suggest that SA reduces the
effects of metal stress (Al-Whaibi et al. 2012), salt stress (Nazar et al. 2011, 2015;
Palma et al. 2013), drought stress (Yazdanpanah et al. 2011), and heat stress (Khan
et al. 2013). It has been suggested that SA plays a pivotal role in the regulation of
photosynthesis in plants under metal stress (Al-Whaibi et al. 2012; Noriega et al.
2012; Zhang et al. 2015). SA enhanced tolerance in Linum usitatissimum against
cadmium (Cd) by reducing reactive oxygen species and accelerated the activity of
an enzyme serine acetyltransferase (SAT) which is a key enzyme of S assimilatory
pathway and also increased the GSH content and eventually increased the resistance
of plants to Ni stress (Belkadhi et al. 2014). Exogenous application of SA (3.0 mM)
enhanced the expression of OSWRKY45 gene leading to the rise of endogenous SA
level. However, endogenous level of SA prevents the membrane damage by over-
coming the H2O2-like peroxidizing agents when O. sativa was exposed to Cd stress
(Chao et al. 2010). It has been found that application of 0.5 mM SA enhanced the
level of phytochelatins in Zea mays when exposed to Cd stress (Szalai et al. 2013).
11  Salicylic Acid and Nutrients Interplay in Abiotic Stress Tolerance 225

Shikimate pathway

Chorismate

Chorismic-acid
Isochorismate synthase chorismate Mutase

Isochorismate
Arogenic acid

IPL
Phenylalanine
PAL
Salicylic acid Trans-cinnamic acid
BALDH C 4H

Coumaric acid
Benzoic acid
BA2H
SA Glucosidase
Glycosylated salicylic acid Salicylic acid
UDP-glucose-SA glucosyltransferase

Fig. 11.1  A schematic representation of biosynthesis of salicylic acid

Salicylic acid accelerates the expression of most salt-responsive genes and sig-
naling pathway which are responsible for cell apoptosis under salt stress. These
responsive genes are encoding chaperones, heat shock proteins (HSPs), and enzymes
involved in secondary metabolite pathways that are sinapyl alcohol dehydrogenase
(SAD), cinnamyl alcohol dehydrogenase (CAD), and cytochrome P450 (Jumali et al.
2011). Under salt stress SA plays a pivotal role in plants by improving the mecha-
nism of photosynthetic characteristics of leguminous plants such as Vigna radiata
(Nazar et al. 2011) and Medicago sativa (Palma et al. 2013) and also accelerates the
antioxidant metabolism (Nazar et al. 2011; Palma et al. 2013). SA decreased the
accumulation of Na+ and Cl− in salt-treated Z. mays and Brassica (Gunes et  al.
2007) and alleviated the effect of salt stress by lowering the lipid peroxidation and
membrane permeability (Horváth et al. 2007). It has been suggested that salt stress
in O. sativa seedlings induced the expression of benzoic-acid-2-hydroxylase gene
which is a key enzyme of SA biosynthetic pathway (Sawada et al. 2006). Studies
have found that SA also induced the ABA and IAA pathway in Triticum aestivum
seedlings under salinity while as no effect was found on cytokinin (Sakhabutdinova
et al. 2003).Various studies have shown that SA acts as a cell stress protectant by
inducing antioxidant system which includes superoxide dismutase (SOD), glutathi-
one reductase (GR), and ascorbate peroxidase (APX) (Gémes et  al. 2011; Khan
226 T.S. Per et al.

et al. 2010). SA enhanced the expression of SlGST2, SlGSTT3, and SlGSTF4 mem-
bers of GST-gene family and alleviated the salinity stress injury in Solanum lycop-
ersicum (Csiszár et al. 2014). Exogenous application of 0.5 mM SA in T. aestivum
under salinity stress enhanced the transcription rate of antioxidant genes, i.e., GPX1,
GPX2, DHAR, GR, GST1, GST2, MDHAR, and GS (Li et al. 2013).
The exogenous application of SA (500  μM) alleviated the drought stress in
Hordeum vulgare by enhancing the rate of CO2 assimilation due to increasing stoma-
tal conductance (Habibi 2012). SA supplementation plays a pivotal role in the regu-
lation of enzymatic and nonenzymatic ingredients of AsA-GSH pathway and
overcomes the oxidative stress of drought-exposed plants (Alam et al. 2013). The
exogenous application of SA (5.0  μM) induced the expression of genes encoding
chaperones, HSPs, antioxidants (SOD, APX, GR), and secondary metabolite path-
way enzymes (Jumali et al. 2011). In Arabidopsis thaliana expression of SIZ1 gene
led to endogenous accumulation of SA which plays essential role in stomatal closure
under drought conditions (Miura et al. 2013). In addition, SA leads to expression of
over 76 proteins in drought-exposed T. aestivum; these proteins play a major role in
photosynthesis, carbohydrate metabolism, protein metabolism, stress and defense,
energy production, signal transduction, and toxin metabolism (Kang et al. 2012).
Exogenous application of 0.5 mM SA also modified the activity of antioxidant
enzymes such as APX, SOD, GPOX, GSH-reductase, and GR and enhanced the
chlorophyll fluorescence in Z. mays under low temperature stress (Janda et al. 1999).
It has been observed that 2.0 mM SA enhanced the phenolic accumulation and activ-
ity of PAL in Citrus fruits under cold stress (Siboza et al. 2014). However various
studies have found that exogenous application of SA in H. vulgare under cold stress
enhanced the antioxidant enzyme system, ice nucleation activity, and the motifs of
apoplastic proteins (Mutlu et al. 2013). SA developed chilling tolerance ability in
Musa acuminata by protecting its ultrastructures (Kang et al. 2012). Supplementation
of 1.0 mM SA overcame the electrolyte leakage and oxidative stress and increased
the yield of PSII, Fv/Fm, and the quantum yield of the PSII electron transport in
Cucumis sativus under heat stress (Shi et al. 2006). It has been reported that exoge-
nous application of 0.5 mM SA mitigated the adverse effects of heat stress due to
production and decrease in stress ethylene formation (Khan et al. 2013).

11.4 I nteraction Between Salicylic Acid and Mineral

Nutrients Under Abiotic Stresses

Inadequate and unbalanced supply of mineral nutrients is the main problem respon-
sible for a reduction in global food production especially in the developing coun-
tries. It is estimated that about 60% of cultivated soils have growth-limiting problems
associated with mineral-nutrient deficiencies and toxicities that is a major factor of
reduced crop productivity (Cakmak 2002; Guo et  al. 2016; Bagheri et  al. 2017).
Byrnes and Bumb (1998) suggested that in the coming 20 years fertilizer consump-
tion has to increase by about twofold to achieve the increased food production in
order to meet the food demands of large population. So this plant-nutrition-related
11  Salicylic Acid and Nutrients Interplay in Abiotic Stress Tolerance 227

research will be a high-priority research area contributing to crop production and

sustaining soil fertility in the coming decades. Survival and productivity of crop
plants exposed to environmental stresses are dependent on their ability to develop
adaptive mechanisms to avoid or tolerate stress. Literature suggests that the mineral
nutritional status of plants greatly affects their ability to adapt to adverse environ-
mental conditions. Reports have shown that mineral nutrient status in plants plays
an important role in the mitigation of abiotic stress (Nazar et al. 2012; Masood et al.
2012; Asgher et al. 2014; Khan et al. 2016). The importance of S as a plant nutrient
has been recognized for a long time, but active research started when widespread S
deficiencies were reported and the use of nutrients as fertilizers was ignored earlier.
The deficiency of S hinders plant metabolism (Honsel et al. 2012) and reduces chlo-
rophylls and photosynthesis (Kastori et al. 2000; Khan et al. 2016). S has now been
recognized to regulate photosynthesis under optimal and stressful environments and
plays an important role in mitigation of salt- and Cd-induced oxidative stress and is
used as a constituent of most of the defense compounds (Fatma et al. 2014; Asgher
et al. 2014, 2015a). Adequate nutrition is also essential for the maintenance of plant
structure such as N and Mg are important structural constituents of chlorophyll
involved in photosynthesis, P is a structural part of nucleic acids and is needed for
energy production and storage, and K is involved in osmotic regulation and activa-
tion of enzymes and plays a key role in the survival of plants under adverse environ-
mental stress conditions and is required for various physiological processes, such as
photosynthesis, translocation of photosynthates into sink organs, maintenance of
turgidity, and activation of enzymes under stress conditions (Marschner 1995;
Mengel and Kirkby 2001; Waraich et  al. 2011; Guo et  al. 2016; Ahanger and
Agarwal 2017). Deficiency of K causes severe reduction in photosynthetic CO2
fixation and impairment in partitioning and utilization of photosynthates. The role
of other nutrients in alleviating various abiotic stresses has also been traced, such as
silicon (Si) which has beneficial effects in increasing salinity tolerance (Tahir et al.
2011), Se in Cd tolerance in T. aestivum (Khan et al. 2015), and K in increasing
salinity tolerance (Munns 2005; Ahanger and Agarwal 2017).
Salicylic acid regulates the response of plants to the various abiotic stresses and
could be regarded as a plant growth regulator to enhance plant growth and stimulate
mineral nutrient concentrations such as P, K, Mg, and Mn under drought and boron
stress conditions in Z. mays (Gunes et al. 2005). Supplementation of SA in Cucumis
sativus reduced the Na+ uptake of plants by increasing the uptake of N, P, K, Ca, and
Mg under salt stress. This reduction of Na+ content might result in low membranes
injury, high water content, and dry matter production (Yildirim et al. 2008). SA is
involved in correcting iron chlorosis in Glycine max genotypes grown in calcareous
soils (Aly and Soliman 1998). Kong et al. (2014) have shown that in Arachis hypo-
gaea SA promoted Fe uptake and balanced mineral elements and protected Fe
deficiency-­induced oxidative stress indicating the strong SA response in removing
the oxidative stress. Nazar et al. (2015) found that SA increased S assimilation and
N assimilation which is linked to antioxidant system in plants and provided toler-
ance to salinity by inducing various physiological responses in plants. It has been
found in Arabidopsis that SA affects S uptake by controlling GSH biosynthesis
228 T.S. Per et al.

under ozone stress (Yoshida et al. 2009). High levels of salicylate have also been
implicated in tolerance to Ni, via inducing SAT resulting in increased GSH produc-
tion (Freeman et  al. 2005). SA at 0.5  mM concentration alleviated the negative
effects of salt stress and improved photosynthesis and growth through increase in
enzymes of AsA-GSH pathway which suggested that SA might participate in the
redox balance under salt stress. The increase in leaf S content resulting via higher
activity of enzymes of S assimilation such as ATP sulfurylase (ATPS) and SAT by
SA application was associated with the increased accumulation of GSH and lower
levels of oxidative stress that resulted in protection of photosynthesis and growth in
Brassica juncea (Nazar et al. 2015).
Literature is full of reports showing that exogenous application of SA minimizes
Na uptake while increasing concentrations of mineral nutrients such as K, Ca, and
Mg in tissue (Gunes et al. 2005; Yildirim et al. 2008; Ben et al. 2009; Khan et al.
2010; Syeed et al. 2011) and Fe, Mn, Cu (Gunes et al. 2005; Yildirim et al. 2008), P
(Gunes et al. 2005; Yildirim et al. 2008; Khan et al. 2010; Syeed et al. 2011), N
(Gunes et al. 2007; Yildirim et al. 2008; Khan et al. 2010; Nazar et al. 2011; Syeed
et al. 2011), and S (Nazar et al. 2011, 2015) in many plant species. However, there
are few conflicting results as well, showing that exogenous application of SA
decreased concentrations of K and P in shoot and root tissues of Z. mays (Gunes
et  al. 2007) and in H. vulgare shoots (El Tayeb 2005) under salinity stress.
Surprisingly in Spinacia oleracea roots and shoots, Na and Cl concentrations in
saline conditions were not affected by the application of SA (Eraslan et al. 2008). In
Solanum lycopersicum plants, application of SA inhibited K uptake and increased
Na uptake (Szepesi et al. 2009).
Application of SA maintained mineral status through increase in functioning of
mineral transporters and increase in mineral nutrient uptake resulting in the allevia-
tion of Cd toxicity by minimizing the root-to-shoot translocation of Cd and in
increase in antioxidant metabolism (Wang et al. 2013; Asgher et al. 2015b). Further
it has been shown that SA decreased both the uptake and transport of Cd and allevi-
ated Cd-induced inhibition of nutrient uptake and led to increase in contents of
chlorophyll and carotenoid (Saidi et al. 2013; Zhang et al. 2015). Wang et al. (2011)
suggested the involvement of SA in minimizing the lead (Pb)-induced oxidative
stress by regulating the uptake of several plant-beneficial elements like Mn, Ca, Cu,
Fe, P, and Zn. Syeed et al. (2011) attributed the SA-mediated changes in photosyn-
thesis to the nutrients content of N, P, K, and Ca in B. juncea cultivars differing in
salt tolerance. The increase in photosynthesis under salt stress by SA was particu-
larly due to decrease in cellular Na+ and Cl− ions and increase in the content of
nutrients. SA supplementation inhibited Na+ and Cl− accumulation and stimulated
N, Mg, Fe, Mn, and Cu concentrations in salt-stressed Z. mays plants as well (Gunes
et al. 2007). In another study, it was found that 0.5 mM SA maintained higher K+/
Na+ and Ca2+/Na+ ratios that were considered as a major factor underlying
SA-assisted improved growth, gas exchange, yield, and salinity tolerance in Z. mays
(Tufail et  al. 2013). SA supplementation enhanced the activities of the enzymes
involved in N assimilation in Cd-stressed plants (Hayat et al. 2012). Salicylic acid
has also been found to interact with N assimilation to influence Pro metabolism and
11  Salicylic Acid and Nutrients Interplay in Abiotic Stress Tolerance 229

protection of photosynthesis in T. aestivum subjected to water deficit (Singh and

Usha 2003) and in salinity-exposed B. juncea (Nazar et al. 2011). Cd suppressed the
uptake of macro- and micronutrients, but exogenous application of SA restored the
capability of plants to accumulate essential elements. Thus, the adverse effects of
Cd toxicity on growth, enzymes involved in the oxidative defense mechanism, and
nutrient uptake can be alleviated by foliar spray of SA in Brassica juncea (Ahmad
et  al. 2011). SA application prevented the uptake of Pb and partially reversed
Pb-induced alterations in Mn, Ca, and Fe content in leaves of Vallisneria natans
exposed to 50  μM Pb (Wang et  al. 2011). Al-Hakimi and Hamada (2001) also
observed similar effects of SA in Na, K, Ca, and Mg content of T. aestivum plants
grown under salinity. Calcium is one of the important nutrient elements and known
for various structural roles under both optimal and stressful conditions in plants
(White and Broadley 2003). It has been reported that a wide range of extracellular
signals such as cold and heat stresses cause transient elevation of cytosolic free Ca2+
level, and to maintain Ca2+ homeostasis, the concentration of Ca2+ returns to the
resting level, which requires active pumping of Ca2+ to the apoplast or organelles
(Knight 2000). There is, however, availability of little evidence that shows how Ca2+
homeostasis may be involved in SA-induced heat or cold tolerance. In a study of
SA-treated Vitis vinifera plants, Ca2+ homeostasis maintained under cold or heat
stress increased heat stress tolerance. The study showed that in SA-treated plants,
cytosolic Ca2+ in mesophyll cells was greater than that in controls at the normal
temperature. After cold or heat stress, in SA-treated plants, increased cytosolic Ca2+
in mesophyll cells was pumped back to vacuoles or intercellular spaces, and chloro-
plast structure was not destroyed. The chloroplast structure was destroyed in control
cells when increased cytosolic Ca2+ cells was not pumped back to vacuoles or inter-
cellular spaces (Wang and Li 2006). SA showed interaction with Ca2+ signaling (Du
et al. 2009), and the link between SA and Ca2+ signaling was further supported by
the study of Al-Whaibi et al. (2012), where the authors reported higher increase in
proline concentration with the combined treatment of SA and Ca2+. It has been stud-
ied that SA and Ca ameliorated the ill effects of salinity, by reducing the oxidative
stress and enhancing activities of antioxidant enzymes, thereby protecting photo-
synthetic pigments and growth in T. aestivum (Al-Whaibi et  al. 2012; Yücel and
Heybet 2016). A range of abiotic/environmental stresses (such as high temperature,
UV-B stress, or salt stress) has been reported to induce the expression of genes
encoding calmodulin-binding protein (Ca-containing protein). On the other hand,
SA has reported to induce calmodulin-binding proteins in abiotic-stressed A. thali-
ana and S. lycopersicum (Yang and Poovaiah 2002; Yang et  al. 2013). Calcium-­
dependent protein kinases (CDPKs) have been shown to be involved in abiotic
stress responses and may also be induced by SA (Chung et al. 2004; Leclercq et al.
2005). Supplementation of SA significantly decreased the oxidative stress in C.
sativus leaves exposed to excess Mn by increasing AsA and GSH contents (Shi and
Zhu 2008). Selenium and SA application increased the temperature stress tolerance
by increasing antioxidant enzyme metabolism in plants by decreasing ROS content
(Waraich et al. 2011). Application of Se and SA in combined application resulted in
a significant decrease in Na/K ratios by inducing enzymatic and nonenzymatic
230 T.S. Per et al.

antioxidant system. On the basis of this finding, it can be concluded that the foliar
utilization of Se in combination with SA may be used to relieve the signs of salinity
stress in G. max. (Ardebili et al. 2014). SA increased the rate of Ca, Mg, and K in
the shoot and roots of T. aestivum cultivated in arid conditions and reduced the
adverse effects of drought stress (El Tayeb and Ahmed 2010), and it has also been
identified that SA application especially increased the intake of Mg and increased
the tolerance to various stress sources (Raskin et al. 1990). The foliar application of
SA and Zn individually has been found to reduce the effect of drought stress like
decrease in plant height, spike length, number of grains per spike, grain weight,
chlorophyll content, and relative water content in T. aestivum (Yavas and Unay
2016). The results of this experiment suggest a possible cross talk between SA and
Zn that could be used for improving Triticum growth under drought stress. The use
of micronutrients such as Fe, Zn, Mn, and Br as a mixture at a concentration of 50
and 100 mg/L and SA at 100 and 150 mg/L improved cluster weight; berry weight;
juice volume; total chlorophyll content; content of N, P, and K of leaves; total phe-
nols; and β-carotene by all treatments in comparison with the control plants (Abdel-­
Salam 2016). The research conducted to determine the effect of changes of certain
minerals (Ca, Mg, Mn, Fe, Cu, Zn) in SA doses on different T. aestivum varieties in
drought stress showed that 1.0 mM kg−1 dose of SA significantly increased the min-
eral content in flag leaf of all varieties as compared to 0.1 mM kg−1 SA (Arpali et al.
2016). The spraying of Gossypium barbadense plants with SA (200  ppm) and
potassium citrate (2.5 g/L) under salt conditions caused enhancement of growth and
yield characters and increase of leaf chemical composition (El-Beltagi et al. 2017).
Some of the studies showing response of plants to SA in the presence of mineral
nutrients under abiotic stress have been tabulated in Table 11.1.

11.5 Conclusion and Future Prospects

It can be concluded from the above description that SA plays a promising role in
alleviating damaging effects of abiotic stress in plants. Besides regulating various
plant metabolic processes, SA interacts with mineral nutrients to govern various
plant responses under both optimal and stressed environments. Literature appraised
herein confirmed the focus of the studies related with SA and mineral nutrients in
abiotic-stressed plants mainly on unveiling various physiological/biochemical pro-
cesses. A number of interesting questions are still awaiting further investigation.
The dependence of SA and mineral nutrients on each other can be exploited in
manipulating plants response in the changing environment and sustainable agricul-
ture development. For instance, low agricultural productivity due to limitation of
mineral nutrients and the shortage of nutrients can be maintained by targeting
endogenous accumulation of SA for increased nutrient use efficiency. If the mecha-
nisms of how SA induces the nutritional status and vice versa under each stress are
understood, this knowledge would contribute in improving crop vigor and develop-
ment under changing environment. Unveiling SA signaling in abiotic stresses and
the relationship between distinct facets of interaction of SA with mineral nutrients
11  Salicylic Acid and Nutrients Interplay in Abiotic Stress Tolerance 231

Table 11.1  Studies showing the response of plants to SA in the presence of mineral nutrients
under abiotic stress
Concentration Response of plant to
Plant Abiotic stress of SA mineral nutrients References
Cucumis sativus Salt stress 1.0 mM SA Increased the uptake of Yildirim
N, P, K, Ca, and Mg et al. (2008)
Zea mays Drought and 0.5 mM Stimulated mineral Gunes et al.
boron stress nutrient concentrations (2005)
such as P, K, Mg, and
Mn
Arachis Iron deficiency 1.0 mM SA SA promoted Fe uptake Kong et al.
hypogaea and balance of mineral (2014)
elements
Brassica juncea Salt stress 0.5 mM SA SA increased S Nazar et al.
assimilation and (2015)
alleviated salt-induced
decrease in
photosynthesis through
inducing the activity of
AsA-GSH pathway and
GSH production
Arabidopsis Ozone stress SA SA affected S uptake by Yoshida
thaliana controlling GSH et al. (2009)
biosynthesis
Thlaspi Ni stress 0.5 μM SA SA increased tolerance (Freeman
goesingense to nickel, via inducing et al. 2005)
SAT resulting to an
increase in GSH
Solanum Salt stress 10−7 or SA inhibited K uptake Szepesi et al.
lycopersicum 10−4 M SA and increased Na uptake (2009)
Vallisneria Pb stress 100 μM SA SA application inhibited Wang et al.
natans the uptake of Pb and (2011)
partially reversed
Pb-induced alterations
in Mn, Ca, and Fe
content
Zea mays Salt stress 0.5 mM SA SA maintained higher Tufail et al.
K+/Na+ and Ca2+/Na+ (2013)
ratios that were
considered as a major
factor underlying
SA-assisted improved
growth, gas exchange,
and yield
B. juncea Temperature 1.0 mM SA SA restored the Ahmad et al.
stress capability of plants to (2011)
accumulate essential
elements
(continued)
232 T.S. Per et al.

Table 11.1 (continued)
Concentration Response of plant to
Plant Abiotic stress of SA mineral nutrients References
Triticum Salt stress 10−5 M SA Increased proline Al-Whaibi
aestivum concentration with the et al. (2012)
combined treatment of
SA and Ca2+
T. aestivum Water stress 1, 2, 3 mM SA has also been found Singh and
SA to interact with N Usha (2003)
assimilation to influence
Pro metabolism and
protection of
photosynthesis
C. sativus Manganese 100 μM SA SA significantly Shi and Zhu
toxicity decreased oxidative (2008)
stress in leaves exposed
to excess Mn
Glycine max. Salt stress 0.5 mM SA Se and SA in Ardebili
combination, resulted in et al. (2014)
a significant decrease in
Na/K ratios
T. aestivum Drought stress SA SA increased the rate of El Tayeb and
Ca, Mg, and K in shoot Ahmed
and roots in arid (2010)
conditions and reduced
the adverse effects of
drought stress
Gossypium Salt stress 200 ppm SA SA and K citrate caused El-Beltagi
barbadense enhancement of growth et al. (2017)
and yield characters

in plant immunity and abiotic stresses responses would also be fascinating and
rewarding. Meanwhile, more genomics and proteomics studies are expected to
broadly reveal SA-responsive genes and proteins upon stresses. Overall, finer details
of SA-mediated defense networks/plant immunity as well as further insights into
the cross talk of SA with mineral nutrients in abiotic-stressed plants can be uncov-
ered through adopting an integrated approach incorporating genetics, molecular
biology, biochemistry, genomics, bioinformatics techniques, and computational
biology.

Acknowledgment  Research facilities in the lab of NAK under the DBT-BUILDER program (No.
BT/PR4872/INF/22/150/2012) of Department of Biotechnology, New Delhi, are gratefully
acknowledged.
11  Salicylic Acid and Nutrients Interplay in Abiotic Stress Tolerance 233

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Index

A Auxins, 78, 115, 125, 147, 149, 153, 170, 175,

ABA-responsive (ABRE)-cis-elements, 79 202–205, 207, 208
ABA-responsive element-binding/ABA-­ Azelaic acid (AzA), 62, 63, 98, 170, 172
binding factors (AREB/ABFs), 79
Abiotic stress, 18–20, 29, 35–44, 48, 57, 65,
79, 80, 85, 90, 98–105, 111, 114, 125, B
133, 134, 146, 149, 155, 165, 174–178, BABA, 176, 177
182, 185, 186, 189, 201–213, 221–232 β-aminobutyric acid (BABA, a non-protein
Acibenzolar-S-methyl (ASM), 23 amino acid), 149
Active oxygen scavenging (AOS), 149, 154 Barley, 25, 26, 28, 37, 50, 80, 90, 102, 125,
α-ketoglutarate dehydrogenase, 20, 106 177, 204, 207
Aluminum toxicity, 134 Basal defense, 165–166
Amino acids, 57, 58, 63, 79, 100, 149, 170, Benzoic acid-2-hydroxylase (BA2H), 100,
172–174, 176, 177, 223 126, 225
1-aminocyclopropane carboxylic acid, 54, Biotic stress, 18, 20–22, 58, 105, 114, 127,
179, 209 183, 184, 202
Aminooxyacetic acid (AOA), 54 BjDHN2/BjDHN3 dehydrin genes, 90
AMP-activated protein kinase (AMPK), 109 β-oxidation pathway, 126
Antioxidant capacity, 25, 48, 102, 212 Brassica napus, 113, 128, 129, 131, 133
Antioxidant genes, 226 Brassinosteroids (BRs), 115, 125, 147, 150,
Antioxidative enzymes, 22, 126, 131–133, 154, 183–184, 203, 212–213
137, 154, 211 Brosimum alicastrum, 3, 7–13
Arabidopsis, 20, 21, 24–29, 49, 50, 55, 56, 60, BTB/POZ domain, 107, 109
62–65, 92, 99, 101, 103–106, 109, 110, bZIP family, 79
112, 113, 125, 126, 149, 150, 152–155,
167–169, 171, 172, 176–180, 182–187,
202–208, 210–212, 224, 226, 227, 231 C
AsA-GSH pathway, 226, 228, 231 Cadmium, 3, 77–93, 124, 135, 224
Ascorbate (AsA), 20, 24, 26, 106, 131, 132, Calcium-dependent protein kinases (CDPKs),
148, 205, 210, 225 105, 229
Ascorbate peroxidase (APOX), 131 Calcium signalling, 105, 114
Ascorbate peroxidase (APX), 20, 106, 148, Calmodulin (CaM), 105
205, 225 Calmodulin-binding protein, 105, 229
Ascorbic acid, 24, 127, 131, 210 Calmodulin-binding protein 60 g
ATP sulfurylase (ATPS), 228 (CBP60g), 105
ATP-sulphurylase, 39 Calmodulin proteins, 229
Auxin mutants (tir1 afb2), 203 Calvin cycle, 127
Auxin receptor (TIR1 and AFB2), 202, 203 Carbon dioxide (CO2), 51, 124, 130, 131, 146,
Auxin response factors (ARFs), 149 147, 149, 226, 227

© Springer Nature Singapore Pte Ltd. 2017 239

R. Nazar et al. (eds.), Salicylic Acid: A Multifaceted Hormone,
https://doi.org/10.1007/978-981-10-6068-7
240 Index

Cassia tora L., 102 Drought stress, 19, 26, 28–29, 134, 149, 182,
Catalase (CAT), 20, 27, 29, 99, 106, 127, 131, 209, 211, 212, 224, 226, 230, 232
148, 152, 205 Durable defense, 166–168
Cell redox state, 20, 21, 179
Chaperones, 103, 155, 177, 225, 226
Chilling, 18, 39, 98, 205, 210, 211, 226 E
Chilling injury, 39, 173 Effector-triggered immunity (ETI), 166–168
Chitinases (Chi), 24, 165, 175, 188 Effector-triggered susceptibility (ETS), 100
Chitin oligomers, 79 Electrolyte leakage, 26, 28, 29, 90, 92, 93,
Chlorofluorocarbons (CFCs), 146 133, 152–155, 226
Chlorophyll, 3, 26, 28, 38, 50, 51, 55, 78, 102, Enhanced disease susceptibility 5 (eds5),
124, 127, 130, 131, 152, 203, 210, 223, 99, 104
226–228, 230 Enzymatic, 39, 48, 50, 114, 131, 132, 148,
Chloroplastic carbonic anhydrase, 106 169, 173, 205, 223, 229
Cinnamic acid, 36, 100, 104, 224 Enzyme, 18, 20, 22, 24, 27, 29, 36, 37, 39, 50,
Cinnamyl alcohol dehydrogenase (CAD), 225 54, 55, 58–60, 83, 100, 103, 104, 113,
cis-regulatory elements, 92 114, 124–127, 131–134, 136, 137, 148,
Commelina communis L, 37 152–155, 164, 171, 174, 184, 185, 188,
Constitutive expression of PR-1 gene (cep), 202–204, 206, 208, 209, 211, 222–229
22, 99 ERF1, 181
Copper (Cu), 4, 6, 106, 107, 124, 137, 223 Ethylene, 49, 56, 99, 114, 125, 147, 148, 150,
Cross talk, 55, 156, 165, 174, 175, 178–181, 152, 154, 165, 178, 180–181, 184–185,
183, 184, 202, 204, 205, 208, 209, 212, 202, 209–211, 223, 226
213, 230, 232
Cullin 3 (CUL3) ubiquitin E3 ligase, 106
Cyclophilins (Cyp), 103 F
Cys 521, 106, 107 Flavin-Dependent Monooxygenase1 (FMO1),
Cys 529, 106 12, 62, 171
Cytochrome P450, 225 Flavonoids, 37, 210
Cytokinin, 78, 115, 125, 147, 153, 180, Flowering, 2, 4–5, 52–55, 125, 172, 173, 204
182–183, 202, 210, 225 Fluridone (Fl), 82–89, 91–93

D G
Damage-Associated Molecular Patterns Germination, 18, 25, 26, 48–49, 92, 98, 102,
(DAMPs), 166, 167, 175, 176 128, 129, 153, 154, 173, 202–204
Dehydrins, 77–93, 103 γ-glutamyl kinase and pyrroline-5-carboxylate
Dehydrins (DHN) clusters, 90 reductase, 134
Dehydroabietinal (DA), 62, 63, 98, 172 Gibberellic acid stimulated Arabidopsis
Dehydroascorbate reductase (DHAR), 24, 131 (GASA), 202
Dehydrobietinal (DA), 171 Gibberellins (GAs), 49, 125, 175, 202,
DELLA proteins, 204, 205, 208 204–208
de novo ABA synthesis, 83 Global warming, 37, 147, 164
de-novo synthesis, 136 Glutathione, 20, 21, 113, 127, 131, 132, 136,
2,6-dichloroisonicotinic acid, 18, 23, 176, 177 148, 179, 207, 223
Disease resistance systems, 165–169 Glutathione peroxidase (GPOX), 131–133, 226
DREB2A, 149 Glutathione reductase (GR), 24, 131, 148, 225
DRE/CRT (dehydration responsive elements/C Glutathione S-transferase (GST), 24, 27,
repeats), 79 127, 131
Drought, 2, 3, 8, 18, 28, 29, 37, 41, 79, 87, 90, Glycinebetaine, 134
98, 134, 149, 173, 182, 202, 203, 205, Glycine max, 51, 113, 134, 154, 227, 232
208, 210, 211, 213, 222, 226, 227 Greenhouse gases (GHG), 145, 146
Index 241

GSH/GSSG, 113 Lipoxgenase gene family members (LOXs), 174

GSTF2, 106 Low temperature, 37, 39, 54, 79, 125, 134,
GSTF8, 106, 113 210, 211, 226
GSTF10, 106, 113
GSTF11, 106, 113
Guaiacol peroxidase (POD), 20, 25, 131 M
Macro nutrients, 4, 5, 41
Malondialdehyde, 89, 93, 155
H MBF1c, 148, 149
Heat, 2, 50, 99, 103, 145–156, 202, 206, 209, MDHAR, see Monodehydro ascorbate
212, 213, 222, 224, 226, 229 reductase (MDHAR)Mediators,
Heat shock proteins (HSPs), 103, 148, 149, 19, 64, 65, 98
151, 154, 155, 177, 225, 226 Membrane permeability, 147, 225
Heat stress factor (HSF), 148, 177 Metabolic imbalance, 148
Heavy metals, 37, 78, 81, 98, 123–137, 202, Metal nutrition, 133
203, 207, 208, 213, 222 Metalothionins (MTs), 136
Herniarin, 135 Metal phytochelatin complexes (M-PCs
Hg2+, 102 complexes), 136
Hsp17.6, 103 Metal pollution, 124
Hsp70, 103, 154 Methionine (Met), 184, 223
Hydrogen peroxide (H2O2), 19–21, 23–29, 99, Methy glyoxal (MG), 124
113, 114, 131–133, 148, 152–155, 184, Methyl salicylate (MeSA), 39, 62, 63, 98–101,
211, 224 106, 170, 171, 173, 176
Hydroxyl radical (OH·), 131, 148 Methyl salicylate esterase, 98, 106
Microbial-associated molecular patterns
(MAMPs), 165–167, 175, 176
I MicroRNAs (miRNAs), 165, 185–187, 189
Indole acetic acid (IAA), 54, 78, 149, 152, Mineral nutrition, 124, 223, 227
175, 203, 204, 207, 208, 225 Mitogen-activated protein kinase 6 (MPK6),
Indole-3-acetic acid (IAA), 54, 78 55, 64, 176
Induced systemic resistance (ISR), 165, Mitogen-activated protein kinases
174–177 (MAPKs), 64, 98, 102, 104, 175,
Isochorismate pathway, 99, 104, 114, 125, 176, 179, 183, 212
127, 224 Monodehydro ascorbate reductase (MDHAR),
Isochorismate pyruvate lyase (IPL), 37, 104, 24, 26, 28, 131, 132, 226
126, 224
Isochorismate synthase (ICS), 37, 62, 100,
126, 127, 173, 205, 206, 224, 225 N
Isochorismate synthase 1 (ICS1), 100, 104, N-acetyl-D-glucosamine, 79
105, 114, 171, 205 Net assimilation rate (NAR), 148
Nitric Oxide (NO), 54, 56, 114, 147, 154,
184–185, 187
J Nitrogen, 1, 4, 41, 222
JA biosynthesis, 174, 175 Nitrogen metabolism, 222
Jasmonic acid (JA), 18, 49, 55, 56, 99, 147, Non-enzymatic, 48
153, 165, 184–185, 202, 212 Non-enzymatic antioxidants, 48
Non-expressor of pathogenesis-related genes 1
(NPR1), 19, 21, 22, 37, 47, 55, 57, 64,
L 65, 97, 100, 103, 106, 112–114, 170,
Linolenic acid, 174 173, 178–181, 184, 204, 212
Lipid peroxidation, 24–26, 28, 89, 124, Nutrients, 4, 8, 13, 40, 41, 43, 133, 137, 146,
131–133, 137, 210, 211, 213, 225 149, 152, 155, 164, 168, 174, 202,
Lipid transfer protein (LTP), 170, 171 221–232
242 Index

O Plant growth regulators, 36, 65, 125, 203, 222

Ortho-hydroxybenzoic acid, 36, 98 Priming, 23, 35, 38, 39, 53, 64, 65, 98,
Oshsp18.0-CII, 103 170–172, 175–178, 211
Osmolytes, 125, 127, 134, 152, 153, 155 Programmed cell death (PCD), 101, 108, 167
Osmoprotectant, 134, 146, 156 Proline, 26, 28, 127, 134, 152, 154, 155, 207,
Osmotic stress, 29, 79, 134, 135 222, 229, 232
Osmotin gene, 103 Proteasomes, 57, 106, 107, 109, 111, 112
Oxidative damage, 24, 25, 29, 110, 131, Protein phosphorylation, 112–113
148, 205 Pseudomonas syringae, 24, 56, 63, 101, 171,
Oxidative stress, 25, 28, 38, 124, 133, 137, 172, 178, 182
151, 153, 209, 212, 223, 226–229, 232 Putresciene, 39, 54, 211
Oxidized glutathione, 179 PvSR3 gene, 80, 90, 92
Ozone, 19, 28, 101, 114, 152, 231
Ozone stress, 19, 101, 228, 231
R
Rauwolfia serpentine, 134
P Reactive oxygen species (ROS), 19–27, 29,
PAMP-triggered immunity (PTI), 100, 101, 49, 59, 78, 80, 101, 113, 124, 127, 129,
108, 166, 168, 170, 171, 183 131, 134, 137, 146, 148, 151, 155, 169,
Pathogen-associated (or microbe-associated) 180, 183, 184, 207, 229
molecular patterns (PAMPs), 59, 100, Reactive oxygen species (ROS) scavenger, 29
101, 165–168, 175, 176, 183 Reduced glutathione, 131
Pathogenesis-related (PR) proteins, 21, 24, 48, Resistance (R) genes, 166, 167, 169, 185,
98, 101, 102, 171, 212 187, 189
Pattern recognition receptors (PRRs), Rhizobacteria, 165, 174, 189
165–168, 170, 183 Ribulose-1,5-bisphosphate carboxylase/
Pb2+, 102 oxygenase (RuBisCo), 28, 50, 125,
Perception, 97–115, 146, 165, 170, 176, 222 134, 148, 152, 154
Peroxidase (POD), 127, 131–133 RING finger proteins, 58
Phosphatidic acid (PA), 113, 176, 177, 202 RNA polymerase, 57, 59, 60, 185–187
Phosphatidylinositol-4-phosphate (PI4P), 113 Root apical meristem, 79, 88, 92
Phospholipase D, 113 Root differentiation, 203
Phospholipid signaling, 113 ROS, see Reactive oxygen species (ROS)
Photosynthates, 227
Photosynthesis, 18, 26, 37, 39, 48, 50–52,
78, 124, 125, 127, 130, 133, 147, 151, S
152, 174, 204, 209, 222–224, 226–229, SA-auxin signaling, 203
231, 323 SA-binding protein 2 (SABP2), 19, 23, 62, 63,
Photosynthetic carbon reduction pathway, 127 100, 106, 171
Photosynthetic efficiency, 25, 50–52, 102, SA-binding proteins (SABPs), 20, 62, 100,
127–129 106, 108, 109, 114, 170
Photosynthetic machinery, 24, 51, 125, 127, 152 S-adenosylmethionine (SAM), 62, 223
Photosystem I (PSI), 127 SAGT SA glycosyltransferase, 100
Photosystem II (PSII), 148, 153 SA-hydroxylase gene (NahG), 25, 27, 29, 55,
Phytochelatins (PCs), 136, 224 98, 99, 101, 103, 114, 155, 210
Phytohormones, 2, 19, 42, 52, 98, 99, 114, SA-induced protein kinase (SIPK), 56, 112, 113
126, 134, 135, 146, 149–156, 165, 202, SA induction deficient 2 (Sid2) mutant, 99
203, 205–208, 210 Salicylic acid
Phytohormones signaling, 150 biosynthesis, 21, 37, 56, 62, 100,
Phytotoxicity, 124, 125 103–105, 125–127, 153, 172, 173,
Phytotoxins, 101, 178 183, 204–207
Pipecolic acid (Pip), 62–64, 98, 170–172 perception, 165
Plant activators, 22 signaling, 146, 184–185
Index 243

Salicylic Acid 2-O-β-d-glucoside (SAG), Thermogenesis, 18, 125, 173

100, 173 Thermospermine (tSpm), 211
Salicyloyl glucose ester (SGE), 100 Thermostability, 79
Salinity, 3, 18, 25, 49, 79, 87, 98, 102, 103, Thermotolerance, 98, 103, 146, 148, 149, 152,
108, 125, 134, 202–204, 206–213, 222, 154, 155, 202
225–230 Thiobarbituric acid reactive substances
stress, 49, 134, 203, 204, 209, 211, 212, (TBARS), 133, 152, 154
226, 228, 230 Thioredoxins (TRXs), 111, 179, 184
tolerance, 108, 227, 228 Thylakoid membrane, 50, 124, 148
Salt stress, 18, 25, 28, 49, 78, 173, 204, 206, TIR1/AFB2, see Transport inhibitor
208, 209, 211, 212, 223–229, 231, 232 response1/auxin-­related F-Box (TIR1/
Salt tolerance, 133, 203, 207, 228 AFB2)Tobacco mosaic virus (TMV),
SA methyltransferase 1 (SAMT1), 100, 106 21, 48, 59, 114
carboxyl methyltransferase, 100 Transcription factors, 22, 55–57, 64,
Secondary metabolites, 135, 137, 223, 225, 226 65, 79, 104, 105, 111–114, 152,
Senescence, 18, 48, 52, 55–56, 98, 148, 153, 202 165, 168–170, 175, 176, 178–180,
Serine acetyl transferase (SAT), 224, 228, 231 187–189, 212, 213
Shikimic acid pathway, 126, 223 Transcriptomics, 166, 212
Short protein motifs, 169 trans-factors, 92
Signal transduction pathways, 97, 103, Transport inhibitor response1/auxin-related
112–114, 149, 222 F-Box (TIR1/AFB2), 202, 203, 208
Sinapyl alcohol dehydrogenase, 225 Triticum aestivum, 49, 51, 52, 56, 129, 130,
Singlet oxygen (1O2), 131, 148 154, 202, 225, 232
SKn-type dehydrin, 80, 90 TRXs, see Thioredoxins (TRXs)
Small interfering NAs (siRNAs), 60, 61, 165, tSpm, see Thermospermine (tSpm)
186–187, 189
Snf1-related kinases (SnRKs), 109
S-nitrosoglutathione (GSNO), 184 U
Source-sink relation, 153, 204 Ubiquitination, 57–59
Spermidine, 39, 211 Ubiquitin ligase (E3), 57–59, 106, 110, 111,
Spermine, 39, 211 113
Stomatal closure, 29, 37, 50, 51, 78, 83, 125, Ubiquitous proteins, 103
127, 173, 226
Stress adaptation, 102
Sulfur (S), 136, 222, 223 V
Superoxide dismutase (SOD), 24, 26–29, 39, Vigna radiata, 54, 209, 225
126, 131–133, 148, 153, 225, 226
Superoxide radical (O2·–), 131, 148
Suppressor of npr1-1, constitutive (SNC1), 108 W
Suppressor of npr1-1, inducible (SNI1), 108 Water relation, 78, 82, 90, 124, 125, 130
Suppressors of NPR1, 108 Wheat germ agglutinin (WGA), 77–93
Systemic acquired resistance (SAR), 18, Wounding, 103, 174, 180, 181
20–23, 48, 62–65, 98, 99, 101, 105, WRKYs, 55, 56, 65, 104, 105, 169, 175, 179,
106, 108, 111, 114, 165, 170–173, 176, 180, 204
177, 180, 184, 186, 189

X
T Xanthomonas oryzae, 167, 169, 186
TADHN, 79, 80, 90, 92
TBARS, see Thiobarbituric acid reactive
substances (TBARS)TGA transcription Y
factors, 57, 112, 114, 169 YSK2-and Kn-type DHNs (Dehydrins), 90