Journal Pre-proof

A novel enzymatic method discriminating wheat pre-harvest sprouting from Late

Maturity alpha-amylase

D. Mangan, A. Draga, R. Ivory, C. Cornaggia, M. Blundell, C. Howitt, B. McCleary,

J.P. Ral

PII: S0733-5210(22)00069-8
DOI: https://doi.org/10.1016/j.jcs.2022.103480
Reference: YJCRS 103480

To appear in: Journal of Cereal Science

Received Date: 13 January 2022

Revised Date: 5 April 2022
Accepted Date: 12 April 2022

Please cite this article as: Mangan, D., Draga, A., Ivory, R., Cornaggia, C., Blundell, M., Howitt,
C., McCleary, B., Ral, J.P., A novel enzymatic method discriminating wheat pre-harvest sprouting
from Late Maturity alpha-amylase, Journal of Cereal Science (2022), doi: https://doi.org/10.1016/
j.jcs.2022.103480.

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© 2022 Published by Elsevier Ltd.

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 1 A novel enzymatic method discriminating wheat Pre-Harvest sprouting from Late Maturity

2 Alpha-amylase.

3 Mangan D.a, Draga A.a, Ivory R.a, Cornaggia C.a ,Blundell M.b, Howitt C.b, McCleary B.c and Ral J.P.b*

4 Addresses:

5 a. Neogen, Megazyme, IDA Business Park, Bray, Co. Wicklow, A98YV29, IRELAND

6 b. CSIRO Agriculture and Food, Canberra, ACT 2601, Australia

7 c. MGZ Consultants, Greystones, Co. Wicklow, A63YW01, Ireland.

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8 Corresponding author: David Mangan

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9 Contributions: -p
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10 DM, CC, CH BMC and JPR developed the project concept

11 MB, JPR provided the flour material

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12 MB, JPR helped with the Falling Number assessment

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13 AD, RI, DM, CC helped with enzymatic analysis

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14 DM, CC, JPR developed the manuscript

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16 Key words: wheat, grain, germination, LMA, Pre-Harvest sprouting, amylase, glucosidase

17

18 Abstract

19 The primary quality assessor of wheat grain is the Hagberg Falling Number (FN) method. This is a

20 viscometric test surrogate for α-amylase activity. Despite being used for over sixty years, FN has been

21 increasingly scrutinised due to its low throughput, poor reproducibility and inability to differentiate

22 between the causes of low FN including Pre-harvest Sprouting (PHS) and Late Maturity -Amylase

23 (LMA). Our study describes initial efforts to analyse a specific wheat flour set tailored for the
24 identification of enzymatic candidates that would allow discrimination between PHS and LMA affected

25 grains. Using the sensitive enzyme-coupled assay substrate R-AMGR3, results suggest that -

26 glucosidase (exo--glucosidase) is a potential enzyme marker candidate to specifically detect sprouted

27 but not LMA-affected grain.

28 Funding: This research did not receive any specific grant from funding agencies in the public or not-

29 for-profit sectors.

30

31 Introduction

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32 The primary quality assessor of wheat grain produced globally is the Hagberg Falling Number (FN)

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method which was created as an attempt to assist bakers to standardise their baking process. The

Falling Number test was created in 1960 by Sven Hagberg as a simple surrogate for measuring -
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35 amylase activity (Hagberg, 1960). Falling Number is a viscometric test in which wholemeal flour slurry
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36 is incubated in boiling water under agitation and is subsequently exposed to a plunger that, based on
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37 the viscosity of the slurry, will take varying times to fall to the bottom of the cylinder within the FN
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38 instrument. When elevated levels of -amylase are present in the slurry, starch is rapidly degraded
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39 and the gel offers limited resistance to the plunger, thus reducing the FN dramatically. Over time FN

40 has been created as a proxy for weather damage due to sprouting but does not discriminate between

41 the several causes of elevated levels of -amylase in the grain, including Pre-Harvest Sprouting (PHS),

42 Late Maturity -Amylase (LMA) or to a lesser extent, frost. These factors can have various impact on

43 end-product quality (Buchanan and Nicholas, 1980; Kiszonas et al., 2018; Newberry et al., 2018).

44 Indeed, α-amylase expression is elevated in both PHS and LMA affected samples and so the FN test is

45 unable to differentiate between these conditions (Mares and Mrva, 2008). Crucially, studies suggest

46 that unlike PHS, LMA affected wheat does not negatively impact wheat baking properties and

47 therefore LMA affected grains are erroneously downgraded to feed quality, thereby penalising

48 growers financially following the precautionary principle (Newberry et al., 2018; Delwiche et al., 2015).
49 Between 2014 and 2016, LMA events costed wheat growers in the Pacific North West region ~$300m

50 (Bettge, 2018).

51 Finding an alternative method that would complement or replace FN and provide an indication as to

52 whether LMA or PHS affects a given wheat sample would represent an appealing alternative to

53 improve wheat commodity pricing throughout the value chain.

54 Previous reports have described the increase in exo- and endo-acting glycosyl hydrolases (GHs) and

55 protease activities during sprouting (Barrero et al., 2013; Collins et al., 2021; Kranz et al., 2015) and

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56 the current study describes our initial efforts to identify a selective enzyme activity marker to allow

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57 differentiation of LMA and PHS.

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59 Materials and Methods
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60 Biological Material:
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61 Wheat grain generated from a tall variety taken from a MAGIC 4-parent described in Newberry et al.
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62 (2018) were used for this study. Plants were grown in glasshouses in two consecutive seasons at CSIRO
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63 Agriculture and Food, Canberra, Australia Capital Territory, Australia, under natural light with no

64 additional artificial lighting on a diurnal temperature cycle of 14/20°C and watered automatically at a

65 rate equivalent to 10mm of water every three days. Commercially purchased Australian variety Mace

66 as described in Whan et al. (2014) was also used.

67 Germination experiment

68 Germination assays were performed as described by Jacobsen et al. (2013). Grains were kept at 4oC

69 over two days to break any residual dormancy and imbibed on filter paper soaked with sterile MilliQ

70 water at 20oC. Grain samples were taken at appropriate time points, snap frozen and freeze dried

71 before milling.

72 Milling and Falling Number

73 The Falling Number (FN) of all samples was measured in triplicate on a Falling Number instrument

74 FN1000 (Perten Instruments, Hägersten, Sweden) using 7 g of wholemeal mixed with 25 mL Milli-Q

75 water according to the manufacturer's instructions and the AACC method 56‐81B.

76 Enzymatic assays

77 Enzyme extraction and Assay development are schematised in Supplemental Fig S1A.

78 In brief, wheat flour was milled to pass a 0.2 mm screen and enzymes were extracted using a modified

79 version of the ‘One extraction for all’ procedure as described by Cornaggia et al. (2019). A shorter 1-

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80 hour extraction time was employed and dithiothreitol was omitted from the extraction buffer.

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81 α-Amylase, β-amylase and casein-active protease activities were measured following the instructions

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supplied in the Megazyme Assay Kit protocols (Megazyme Cat. No. K-CERA, K-BETA3, and S-AZCAS
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83 respectively). Wheat extract was diluted in sodium maleate buffer (0.1M, pH 5.5) containing BSA (1
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84 mg/mL) where required and all analysis was carried out at 40°C. In the case of -amylase (adjusted),

85 the activity measured at 24 hours was arbitrarily set at 100% in order to allow for comparison at similar
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86 baseline activity levels across the three enzymes.

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87 α-Glucosidase and β-glucosidase activity were measured using 4-nitrophenyl-β-D-maltoside

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88 (Megazyme Cat. no R-AMGR3) and 4-nitrophenyl-β-D-glucopyranoside (O-PNPBG) respectively as

89 described in the Supporting Information.

90 The wheat cysteine protease, Triticain-α, was measured using acetyl-Pro-Leu-Val-Gln functionalised

91 with 7-amino-4-methylcoumarin (Ac-PLVQ-AMC) as substrate (obtained from Pepmic Co. Ltd) as

92 described by Gorokhovets et al. (2017).

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94 Results and Discussion

95 To screen for new potential enzymatic activities that would allow discrimination between PHS and

96 LMA, a set of 3 wheat samples were produced by CSIRO using a tall line from the MAGIC Population.
 97 This variety was grown in a glasshouse for two consecutive seasons and was impacted by LMA only

98 during the first growing season. The wheat set included 1) sprouted (FN = 62), 2) unsprouted but LMA

99 affected (FN = 64) and 3) sound (FN = 433) wheat. Measurable activities were screened from the

100 sprouted sample using a broad panel of enzyme substrates following a modified version of the recently

101 reported “one-for-all” extraction procedure (data not shown, Fig S1A, Supporting Information). This

102 first step allowed the identification of a focused enzyme substrate panel that was then used to screen

103 all 3 representative wheat samples, the results of which are shown in Table 1.

104 To allow for discrimination between PHS and LMA affected samples, promising enzyme activity

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105 candidates would exhibit a significant difference in expression between these conditions while also

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106 displaying a significant difference between PHS and sound wheat. -Amylase provided the expected

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profile including very strong response in the case of PHS, underlining the basic principle of the FN test,
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108 and a somewhat lower but greatly increased level for LMA relative to sound wheat, (Mares and Mrva,
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109 2014). -Glucosidase and endo-protease both exhibited the desirable attribute of increased
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110 expression in the case of PHS only but the magnitude of the increase was deemed insufficient to form

111 the basis for an analytical test. -Amylase did not appear to be expressed at an elevated level in
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112 sprouted grain relative to sound grain although the basal level was significant, a finding that somewhat
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113 supports Collins et al. (2021) description of the elevated presence of -amylase transcripts in barley

114 dry grain and Zhang et al. (2021) who also described carbohydrate degradation from the early stages

115 of wheat grain germination prior to the release of newly generated -amylase. The key finding

116 highlighted in Table 1 was the activity profile observed for -glucosidase (exo--glucosidase), which

117 showed increased activity only in the case of sprouting when compared to LMA affected and sound

118 wheat samples. Although the magnitude of the response for PHS versus sound samples (~10x increase)

119 did not provide the same resolution as that observed for -amylase (~50x increase), -glucosidase

120 was considered the lead activity candidate at this point. It is also noteworthy that the ubiquitous

121 substrate for the measurement of -glucosidase, namely 4-nitrophenyl--glucopyranoside (PNPAG),

122 lacked the sensitivity required to generate a significant assay response in this application. This issue
123 was overcome by the use of an enzyme coupled assay typically employed for the measurement of

124 amyloglucosidase. It was found that 4-nitrophenyl--maltoside was hydrolysed by wheat -

125 glucosidase at a rate of ~9x that observed for PNPAG and the presence of saturating levels of ancillary

126 -glucosidase in the assay afforded rapid hydrolysis of the 4-nitrophenyl--glucopyranoside

127 intermediate which allowed for the quantitative measurement of the target analyte within a 60-

128 minute timeframe (Fig S1B, Supporting Information).

129 Following the promising results obtained in the initial activity screen, an experiment was designed to

130 evaluate whether -glucosidsase activity would increase concomitantly with the FN decrease. A

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131 second sample set comprising a germination time course of the commercially available Australian

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132 variety Mace was produced. Flours generated from seven time points (6-48 hours) were analysed

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using the FN method and a range from 540 to 62 seconds was established. At ~18 hours post imbibition
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134 the FN value crossed the quality threshold point with a measured FN of 270 sec. In addition to -
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135 glucosidase, cystein protease (Triticain-), previously identified as an upregulated protein during
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136 germination (Barrero et al., 2013), was also brought forward to this second phase for further

137 investigation. Finally, -amylase was assayed in all samples as a control. Results are shown in Figure
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138 1.
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139

140 -Amylase displayed strong correlation with FN as expected (Kiszonas et al., 2018; Newberry et al.,

141 2018). However, it was interesting to note that FN dropped significantly before increasing -amylase

142 activity could be detected suggesting the involvement of other enzymes in the FN reduction. This

143 observation confirmed previous reports that several factors other than -amylase can impact FN (He

144 et al., 2019). While triticain- showed an interesting increase in activity, its onset appeared later than

145 the decrease in FN and therefore would not represent a suitable enzyme marker. However Figure 1

146 shows the detectable increase in -glucosidase activity occurs from 18 hours post germination which

147 aligns with the transition across the FN acceptability threshold. This activity corroborated the results
148 obtained by Collins et al. (2021) who described that -glucosidase transcript was detected from the

149 early stage of barley germination. Whether the differentiation afforded by this increased enzyme

150 activity post germination versus the baseline activity in sound wheat can compete with the FN method

151 for the purpose of grain quality assessment is a matter requiring further investigation.

152

153 Conclusion

154 Overall, and despite being widely used as an indicator of grain soundness and quality, Falling Number

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155 offers a non-discriminating and low throughput method to indirectly assess the cause of weather

156 damage in grain mainly due to its inability to discriminate between PHS and LMA. Using the sensitive

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enzyme-coupled assay substrate, R-AMGR3, our study demonstrated that -glucosidase was
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158 expressed in PHS but not in sound grain or LMA affected grains. This preliminary study is a step
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159 towards the development of a new generation of discriminating enzyme activity-based assays for grain

160 quality assessment.

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162
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163 References

164

165 Barrero, J. M., Mrva, K., Talbot, M. J., White, R. G., Taylor, J., Gubler, F., and Mares, D. J. (2013).

166 Genetic, hormonal, and physiological analysis of Late Maturity alpha-Amylase in wheat.

167 Plant Physiology 161, 1265-1277.

168 Bettge, A. (2018). Low Falling Numbers in the Pacific Northwest wheat growing region: Preharvest

169 Sprouting, Late Maturity Amylase, Falling Number instrument, or low protein? Cereal Foods

170 World 63, 12-16.

171 Buchanan, A. M., and Nicholas, E. M. (1980). Sprouting, alpha-amylase, and breadmaking quality.

172 Cereal Res. Commun. 8, 23-28.

173 Collins, H. M., Betts, N. S., Dockter, C., Berkowitz, O., Braumann, I., Cuesta-Seijo, J. A., Skadhauge, B.,

174 Whelan, J., Bulone, V., and Fincher, G. B. (2021). Genes That Mediate Starch Metabolism in

175 Developing and Germinated Barley Grain. Frontiers in Plant Science 12.

176 Cornaggia, C., Evans, D. E., Draga, A., Mangan, D., and McCleary, B. V. (2019). Prediction of potential

177 malt extract and beer filterability using conventional and novel malt assays. Journal of the

178 Institute of Brewing 125, 294-309.

179 Gorokhovets, N. V., Makarov, V. A., Petushkova, A. I., Prokopets, O. S., Rubtsov, M. A., Savvateeva, L.

180 V., Zernii, E. Y., and Zamyatnin, A. A. (2017). Rational Design of Recombinant Papain-Like

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181 Cysteine Protease: Optimal Domain Structure and Expression Conditions for Wheat-Derived

182 Enzyme Triticain-alpha. International Journal of Molecular Sciences 18.

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Hagberg, S. (1960). A rapid method for determining alpha-amylase activity. Cereal Chemistry 37, 218-
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184 222.
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185 He, Y. Z., Lin, Y. L., Chen, C., Tsai, M. H., and Lin, A. H. M. (2019). Impacts of Starch and the

186 Interactions Between Starch and Other Macromolecules on Wheat Falling Number.
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187 Comprehensive Reviews in Food Science and Food Safety 18, 641-654.
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188 Jacobsen, J. V., Barrero, J. M., Hughes, T., Julkowska, M., Taylor, J. M., Xu, Q., and Gubler, F. (2013).
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189 Roles for blue light, jasmonate and nitric oxide in the regulation of dormancy and

190 germination in wheat grain (Triticum aestivum L.). Planta 238, 121-38.

191 Kiszonas, A. M., Engle, D. A., Pierantoni, L. A., and Morris, C. F. (2018). Relationships between Falling

192 Number, alpha-amylase activity, milling, cookie, and sponge cake quality of soft white

193 wheat. Cereal Chemistry 95, 373-385.

194 Kranz, B., Koch, M., Schapfl, M., and Fischer, L. (2015). Investigation of the Germination of Barley

195 and Wheat Grains with a Design of Experiments for the Production of Hydrolases. Food

196 Technology and Biotechnology 53, 127-135.

197 Mares, D., and Mrva, K. (2008). Late-maturity alpha-amylase: Low falling number in wheat in the

198 absence of preharvest sprouting. Journal of Cereal Science 47, 6-17.

199 Mares, D. J., and Mrva, K. (2014). Wheat grain preharvest sprouting and late maturity alpha-amylase.

200 Planta 240, 1167-1178.

201 Newberry, M., Zwart, A. B., Whan, A., Mieog, J. C., Sun, M., Leyne, E., Pritchard, J., Daneri-Castro, S.

202 N., Ibrahim, K., Diepeveen, D., Howitt, C. A., and Ral, J.-P. F. (2018). Does Late Maturity

203 Alpha-Amylase Impact Wheat Baking Quality? Frontiers in Plant Science 9.

204 Whan, A., Dielen, A. S., Mieog, J., Bowerman, A. F., Robinson, H. M., Byrne, K., Colgrave, M., Larkin,

205 P. J., Howitt, C. A., Morell, M. K., and Ral, J. P. (2014). Engineering alpha-amylase levels in

206 wheat grain suggests a highly sophisticated level of carbohydrate regulation during

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207 development. Journal of Experimental Botany 65, 5443-5457.

208 Zhang, Q., Pritchard, J., Mieog, J., Byrne, K., Colgrave, M. L., Wang, J. R., and Ral, J. P. F. (2021).

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Overexpression of a wheat alpha-amylase type 2 impact on starch metabolism and abscisic
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210 acid sensitivity during grain germination. Plant Journal.
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211
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212 Figure and table

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213 Figure 1: Observed enzyme activity as a function of time following imbibition in MACE wheat line for -amylase,
214 (yellow), -glucosidase (blue) and triticain- (green) set at 100%. In the case of -amylase (adjusted) (red), the
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215 activity measured at 24 hours was arbitrarily set at 100% to allow for comparison at similar baseline activity
216 levels across the three enzymes. Falling Number (black) is represented in seconds on the second Y-axis.

217
218 Table 1: Enzyme activity screening to identify potential candidates to replace a-amylase as a selective PHS
219 determination tool. All substrates except Ac-PLVQ-AMC were obtained from Megazyme. Specific activities for the
220 non-specific endo-protease activity and triticain-a were not reported and the relative activites described are
221 based on absorbance at 440nm, and RFU measured at 440nm following excitation at 340nm, respectively.

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 Enzyme Activity Activity( Sprouted/
Activity Sample Substrate (U/g) Rel. %) Sound

Sprouted
(FN = 62) 32.9 100

α- LMA
K-CERA
Amylase affected
(FN = 64) 11.61 35 50
Sound
(FN =
433) 0.56 2

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Sprouted
(FN = 62) 31.08 100

β- LMA
K-BETA3 -p
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Amylase affected
(FN = 64) 26.3 85 1
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Glycosyl Hydrolase

Sound
(FN =
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433) 30.96 99.6

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Sprouted
(FN = 62) 0.33 100
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α-
LMA
Glucosid R-AMGR3
affected
ase
(FN = 64) 0.03 8 10
Sound
(FN =
433) 0.03 10

Sprouted
(FN = 62) 1.35 100
β-
LMA
Glucosida O-PNPBG
affected
se
(FN = 64) 0.73 54 1.5
Sound
(FN =
433) 0.89 66
 Sprouted
(FN = 62) 100

endo - LMA
S-AZCAS
Protease affected
(FN = 64) 62 1.7
Sound
(FN =
Protease

433) N/A 58

Sprouted
(FN = 62) 100

of
Triticain- LMA
Ac-PLVQ-AMC
a

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affected
(FN = 64) 40 7.1

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Sound
(FN =
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433) N/A 14
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 CSIRO
Black Mountain science and innovation park, Canberra ACT 2601
GPO Box 1700 Canberra ACT 2601 Australia

csiro.au | ABN 41 687 119 230

04 April 2022

Cover letter for Journal of Cereal Science article resubmission

The Editor in chief of JCS

Dear Pr Cristina M. Rosell

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Following your encouragement, we resubmit the manuscript entitled “A novel enzymatic method
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discriminating wheat Pre-Harvest sprouting from Late Maturity Alpha-amylase.” by Mangan D. , Draga A.,
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Ivory R., Cornaggia C. ,Blundell M., Howitt C., McCleary B. and Ral J.P.for review.

Contributions as follow:
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DM, CC, CH BMC and JPR developed the project concept

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MB, JPR provided the flour material

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MB, JPR helped with the Falling Number assessment

AD, RI, DM, CC helped with enzymatic analysis

DM, CC, JPR developed the manuscript

The authors declare no conflict of interest.

Yours sincerely and on behalf of all the authors

Dr Jean-Philippe Ral
Principal Research Scientist and Team Leader, CSIRO Agriculture and Food
[email protected]
+61 (0)2 6246 5245

CSIRO
Australia’s National Science Agency