Effect,of,high-molecular-weight,glutenin,subunit,Dy10,on,wheat,dough,properties,and,end-use,quality

WANG Yan ,GUO Zhen-ru ,CHEN Qing ,Ll Yang ,ZHAO Kan ,WAN Yong-fang ,Malcolm J.HAWKESFORD,JlANG Yun-feng,KONG Li,PU Zhi-en,DENG Mei,JlANG Qian-tao,LAN Xiu-jin,WANG Ji-rui,CHEN Guo-yue,MA Jian,ZHENG You-liang#,WEl Yu-ming,Ql Peng-fei#

1 State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Chengdu 611130, P.R.China

2 Triticeae Research Institute, Sichuan Agricultural University, Chengdu 611130, P.R.China

3 Rothamsted Research, Harpenden AL5 2JQ, UK

Abstract High-molecular-weight glutenin subunits (HMW-GSs) are the most critical grain storage proteins that determine the unique processing qualities of wheat.Although it is a part of the superior HMW-GS pair (Dx5+Dy10),the contribution of the Dy10 subunit to wheat processing quality remains unclear.In this study,we elucidated the effect of Dy10 on wheat processing quality by generating and analyzing a deletion mutant (with the Dy10-null allele),and by elucidating the changes to wheat flour following the incorporation of purified Dy10.The Dy10-null allele was transcribed normally,but the Dy10 subunit was lacking.These findings implied that the Dy10-null allele reduced the glutenin:gliadin ratio and negatively affected dough strength (i.e.,Zeleny sedimentation value,gluten index,and dough development and stability times) and the bread-making quality;however,it positively affected the biscuit-making quality.The incorporation of various amounts of purified Dy10 into wheat flour had a detrimental effect on biscuit-making quality.The results of this study demonstrate that the Dy10 subunit is essential for maintaining wheat dough strength.Furthermore,the Dy10-null allele may be exploited by soft wheat breeding programs.

Keywords: HMW-GS,nonsense mutation,Dy10-null allele,end-use quality

Among cereal crops,wheat is unique because the biomechanical properties of the gluten proteins when hydrated enable its flour to be used to make various food products,including bread,noodles,and biscuits.Gluten proteins are classically divided into gliadins and glutenins.Gliadins are usually monomeric and mainly influence the extensibility of wheat dough (Qiet al.2011;Baraket al.2015).Glutenins consist of high-molecular-weight glutenin subunits (HMW-GSs) and low-molecular-weight glutenin subunits (LMW-GSs),which can form glutenin macropolymers (GMPs) through intermolecular disulfide bonds,thereby enhancing dough strength and elasticity(Shewryet al.2003).

The HMW-GSs account for only 7–15% of the glutenins in common wheat (Triticumaestivum),but their composition and concentration have critical effects on gluten structure and determine the viscoelastic properties of dough (Shewryet al.2003;Rustgiet al.2019).Additionally,the HMW-GS genes are included in theGlu-A1,Glu-B1,andGlu-D1loci on the long arms of chromosomes 1A,1B,and 1D,respectively (Payneet al.1982).Each locus contains two closely linked genes that encode a small y-type subunit and a large x-type subunit.However,because of allelic variation and gene silencing,only 3–5 HMW-GS genes are typically expressed in common wheat cultivars (Payneet al.1982).Moreover,HMW-GSs possess a long central repetitive domain consisting of repeating units surrounded by highly conserved non-repetitive N-and C-terminal domains(Shewry and Halford 2002).The N-terminal domain of HMW-GSs usually has three or five cysteine (Cys)residues,whereas the C-terminal domain has only one Cys and the central repetitive domain either lacks Cys or contains only one (Shewry and Halford 2002).The length of the HMW-GS repetitive domain has crucial effects on wheat processing quality (Shewry and Halford 2002;Shewryet al.2003;Rustgiet al.2019).Because the Cys residues are important for generating intermolecular disulfide bonds,the number and distribution of Cys residues affect the GMP structure and dough strength(Shewryet al.2003).Consequently,HMW-GS alleles vary in terms of their contribution to processing quality.

Previous research indicated that Dx5+Dy10 is the superior HMW-GS subunit pair for wheat processing quality (Anderson and Bekes 2011),and the lack of Dx5+Dy10 adversely affects gluten strength and bread quality (Jianget al.2019).The absence of only Dx5 reduces the sodium dodecyl sulfate sedimentation value(Wuet al.2010).The overexpression ofDx5increases the mixing time and lowers the peak resistance,which may result in overly strong dough that is unsuitable for bread-making (Blechlet al.2007;Leónet al.2009).The expression ofDy10in transgenic wheat increases dough development time and mixing tolerance (Blechlet al.2007;Leónet al.2009).However,the critical effects of Dy10 on wheat processing quality remain uncharacterized.

In this study,we identified aDy10deletion mutant (SM482-Dy10null) by screening an ethyl methanesulfonate (EMS)-induced mutant population of common wheatcv.‘Shumai 482’.The effect of Dy10 on wheat dough properties and processing quality was thoroughly investigated.

2.1.Plant materials and growth conditions

Common wheat (T.aestivum)cv.‘Shumai 482’ produces five HMW-GSs (i.e.,Ax1,Bx7+By9,and Dx5+Dy10).TheDy10deletion mutant (SM482-Dy10null) was isolated from ‘Shumai 482’ seeds treated with 0.8% EMS (Sigma-Aldrich,St.Louis,MO) on the basis of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)analysis (Qiet al.2011).The glutenins analyzed by SDSPAGE were extracted from 10 mg whole-seed powder using 100 μL extraction buffer (62.5 mmol L–1Tris-HCl,pH 6.8,10% (v/v) glycerol,2% (w/v) SDS,0.002% (w/v)bromophenol blue,and 1.5% (w/v) dithiothreitol).

To examine quality-related properties,theDy10deletion mutant was backcrossed two or three times with ‘Shumai 482’ (Appendix A).The wild-type (WT)‘Shumai 482’ andDy10deletion mutant homozygous plants were grown at the experimental farm of Sichuan Agricultural University (30°43´16´´N,103°52´15´´E) for two wheat growing seasons (2019–2020 and 2020–2021).The field trials were performed using a randomized block design,with seven replicates for the WT and mutant lines.Each replicate was grown in a 2 m×2 m area,with 20 cm between rows and 60 plants per row.A compound fertilizer (N:P:K=15:15:15) was applied before sowing at a rate of 450 kg ha–1.The WT and mutant lines were compared in terms of their agronomic performance at maturity.After harvesting,the grains were dried under the sun at about 35°C and stored for 2 mon at room temperature for the subsequent analyses.The harvested seeds for each replicate were analyzed by SDS-PAGE and acid polyacrylamide gel electrophoresis (A-PAGE) as described by Lafiandra and Kasarda (1985).

2.2.Nucleic acid extraction and gene cloning

Genomic DNA was extracted from fresh leaves using the Plant Genomic DNA Kit (Biofit,Chengdu,China).Immature seeds (25 days post-anthesis) were collected and ground to a fine powder in liquid nitrogen.Total RNA was extracted from the ground material using the MiniBEST Universal RNA Extraction Kit with DNase(TaKaRa,Dalian,China).The RNA concentration was determined using a NanoDrop One Spectrophotometer(Thermo Fisher Scientific,Waltham,MA).The RNA samples were reverse transcribed using the Prime Script™First Strand cDNA Synthesis Kit (TaKaRa).A primer pair (F1: 5´-ATGGCTAAGCGGCTGGTCCTCTTTG-3´and R1: 5´-CTATCACTGGCTAGCCGACAATGCG-3´)was designed and used to amplify the fullDy10coding sequence (CDS) (Wanget al.2021a).The 25 μL PCR mixtures consisted of 100 ng genomic DNA or cDNA template,2.5 nmol each dNTP,37.5 nmol Mg2+,4 pmol each primer,0.75 U high-fidelity LATaqpolymerase(TaKaRa),and 2.5 μL of 10× buffer.The PCR amplification was conducted in an Applied Biosystems™Veriti™ PCR instrument (Thermo Fisher Scientific),with the following program: 94°C for 5 min;35 cycles of 94°C for 45 s,61°C for 30 s,and 72°C for 2 min;and 72°C for 12 min.The PCR products were separated on 1.0%agarose gels.The expected PCR fragments were purified and inserted into the pMD19-T vector (TaKaRa) according to the manufacturer’s instructions.The recombinant plasmids in the positive colonies were sequenced by Tsingke Biotechnology (Chengdu,China).All experiments were independently repeated at least three times.

2.3.Determination of the glutenin and gliadin contents

The glutenin and gliadin contents in white flour samples were analyzed by reversed phase high-performance liquid chromatography (RP-HPLC).The total glutenin(including HMW-GSs and LMW-GSs) and gliadin contents were estimated by combining the relevant chromatogram peak areas as described by Zhenget al.(2018).The glutenin:gliadin ratio was then calculated.

2.4.Evaluation of the processing quality parameters

Wheat seeds were milled to produce white flour using the CD1 Laboratory Mill (CHOPIN Technologies,Villeneuvela-Garenne Cedex,France) according to AACC Approved Method 26–70 (AACC International 2010).The processing quality parameters,including grain protein content,Zeleny sedimentation value,wet gluten content,gluten index,and GMP content,were assessed as described by Wanget al.(2021a).Rheological properties were evaluated using a standard farinograph (Brabender GmbH &Co.,KG,Germany) as described by AACC Approved Method 54–21 (AACC International 2010).

Bread-making and biscuit-making qualities were examined as described by Wanget al.(2021a).The loaf volume was determined using the BVM6630 volume meter (Perten,Stockholm,Sweden) following the manufacturer’s instructions.The biscuit surface area (SA),which is an accurate indicator of the biscuit diameter(SA=πd2/4,d=diameter and π=3.14),was measured using the C-Cell Imaging System (Calibre Control International Ltd.,Warrington,UK).Biscuit hardness and thickness were determined using the TA.XTC texture analyzer(BisinTech,Shanghai,China) (Wanget al.2021a).The spread ratio was calculated as the biscuit diameter:biscuit thickness ratio.

2.5.In vitro expression of Dy10 and the Western blot analysis

The ‘Shumai 482’Dy10allele CDS (without the signal peptide-encoding fragment) was amplified by PCR using the F2/R2 primer pair (F2 with theNdeI restriction site:5´-ACCCATATGGAAGGTGAGGCCTCTAGGC-3´ and R2 with theXhoI restriction site: 5´-TTCCTCGAGCT ATCACTGGCTAGCCGAC-3´).The amplified sequence was inserted into the bacterial expression vector pET-30a(Novagen,Merck,Darmstadt,Germany).Escherichia colistrain BL21 (DE3) chemically competent cells (Weidi,Shanghai,China) were transformed with the recombinant plasmid and grown at 37°C until the culture optical density at 600 nm reached 0.6.Bacterial expression was induced by adding 0.8 mmol L–1isopropylβ-Dthiogalactopyranoside (IPTG;Solarbio,Beijing,China)to the culture,which was then incubated for 6 h at 37°C.The Dy10 subunit was purified from theE.colicells as described by Uthayakumaranet al.(2000) and confirmed by SDS-PAGE and the Western blot analyses.Immunoblotting was performed using the mouse anti-HMW-GS polyclonal antibody (1:4 000) and the HRPconjugated anti-mouse secondary antibody (1:5 000;Sangon Biotech,Shanghai,China) as described by Wanget al.(2021a).The mouse anti-HMW-GS polyclonal antibody,which was obtained from Zoonbio Biotechnology(Nanjing,China),was generated using the peptide“GYYPTSPQQPGC” as described by Denery-Papiniet al.(1996).

To express a truncated Dy10 peptide (196 residues)fused to the glutathione S-transferase (GST) tag,theDy10-nullallele CDS (without the signal peptideencoding fragment) was amplified by PCR using the mutant and the F3/R3 primer pair (F3 with theBamHI restriction site: 5´-TTCCAGGGGCCCCTGGGATCCG AAGGTGAGGCCTCTAGG-3´ and R3 with theEcoRI restriction site: 5´-CTCGAGTCGACCCGGGAATTCTT ATTGCCTTTGTCCTGTGTGCTGCA-3´).The amplified sequence was inserted into the pEGX-6p-1 vector(Novagen),after whichE.colicells were transformed with the recombinant plasmid.The bacterial cells were cultured and protein expression was induced as described above.Additionally,an immunoblotting assay using the mouse anti-HMW-GS antibody (1:4 000),the HRPconjugated anti-mouse secondary antibody (1:5 000),the rabbit anti-GST polyclonal antibody (1:5 000;Sangon Biotech),and the HRP-conjugated anti-rabbit secondary antibody (1:5 000;Sangon Biotech) was completed as described above.

2.6.Micro-biscuit processing test

The Dy10 subunit purified fromE.colicells was incorporated into the WT and mutant flour according to a slightly modified reduction–oxidation protocol (Bekeset al.1994).Briefly,10 g wheat flour (14% moisture content)and the purified Dy10 (0,20,40,and 60 mg) were mixed with 1.5 mL dithiothreitol (3 mg mL–1in distilled water) for 2 min and then rested for 5 min.After adding 0.75 mL KIO3oxidant (25 mg mL–1in distilled water),the sample was mixed for 2 min and then rested for 10 min.To prepare micro-biscuits,the following ingredients were added to the dough: 6 g sucrose,300 mg nonfat milk powder,100 mg NaHCO3,50 mg NH4Cl,45 mg NaCl,3 g shortening,and 0.5 mL H2O.The micro-biscuit processing test was performed in triplicate.

2.7.Statistical analysis

Student’st-test was performed using the Data Procession System Software (version 17.10) (Zhejiang University,Hangzhou,China) to determine the significance of any differences in the mean values for the processing parameters and agronomic characteristics.

3.1.ldentification of the Dy10 deletion mutant

ADy10deletion mutant line (SM482-Dy10null),which produces four HMW-GSs (Ax1,Bx7+By9,and Dx5),was identified in an EMS-mutagenized ‘Shumai 482’population that was analyzed by SDS-PAGE (Appendix A).There were no significant differences in the agronomic characteristics between the WT and mutant lines(Appendices B and C).The alignment of the mutantDy10(Dy10-nullallele;GenBank no.OK482716) and the WTDy10(GenBank no.X12929) sequences revealed a nonsense mutation (C to T) in theDy10-nullallele(Appendix D),which resulted in a premature termination of translation at the 217th amino acid residue (Fig.1-A).Reverse transcription (RT)-PCR results indicated that theDy10-nullallele was normally expressed at the transcript level (Appendix E).The anti-HMW-GS antibody crossreacted with the truncated Dy10 peptide (which has a predicted molecular mass of 21.6 kDa) produced by the heterologous expression system (Fig.1-C).In contrast,the anti-HMW-GS antibody did not detect the Dy10 peptide in the mutant seeds (Fig.1-B),thereby confirming the lack of Dy10 in the mutant.

Fig.1 The Dy10-null allele results in the absence of Dy10 in seeds.A,alignment of the deduced amino acid sequences of Dy10 in the wild type (WT) and mutant lines.Cysteine residues are indicated by black asterisks.The thick black arrow indicates the premature stop codon site (red asterisk).The signal peptide,N-terminal domain,repetitive domain,and C-terminal domain are indicated.B,Western blot analysis of the gluten proteins in the mutant (lanes 1 and 3) and WT (lanes 2 and 4) lines using the anti-high-molecular-weight glutenin subunit (HMW-GS) antibody.C,Western blot analysis of the glutathione S-transferase (GST)-tagged truncated peptide (196 residues) using the anti-HMW-GS antibody or the anti-GST antibody.The proteins extracted from Escherichia coli cells expressing the GST-tagged truncated peptide (196 residues) with (lanes 2,4,and 6) and without (lanes 1,3,and 5) the addition of isopropyl β-D-thiogalactopyranoside (IPTG) are presented.Red arrows indicate the target protein band.SDS-PAGE,sodium dodecyl sulfate polyacrylamide gel electrophoresis;WB,Western blot.

3.2.Effects of Dy10 on processing quality

The HMW-GS and gliadin contents were respectively significantly lower and higher in the mutant line than in the WT line (Fig.2).Additionally,the LMW-GS content was slightly higher in the mutant line than in the WT line,but this difference was not significant (Fig.2).The glutenin:gliadin ratio was significantly lower in the mutant than in the WT control.

Fig.2 Glutenin and gliadin contents in the wild type (WT) and mutant lines as determined by reversed phase high-performance liquid chromatography (RP-HPLC).A,RP-UPLC profile of high-molecular-weight glutenin subunits (HMW-GSs) and low-molecularweight glutenin subunits (LMW-GSs);the peaks corresponding to the different HMW-GSs are indicated.B,RP-HPLC profile of gliadins.C,relative contents of HMW-GSs,LMW-GSs,and gliadins.Data are presented as the mean±standard deviation,n=7.＊＊ indicates significance at P<0.01.

The grain protein and wet gluten contents were similar between the mutant and WT lines (Table 1).In contrast,the gluten index,Zeleny sedimentation value,and GMP content were significantly lower in the mutant than in the WT control.Regarding the dough rheological characteristics,the development and stability times of the mutant were significantly shorter than those of the WT control.As expected,the mutant dough was softer than the WT dough (see degree of softening in Table 1).Therefore,the lack of Dy10 resulted in reduced dough strength,but it had no effect on the grain protein content.

Table 1 Comparison of the processing quality parameters of the wild type (WT) and mutant lines

The loaf volume was significantly smaller for the mutant than for the WT (Fig.3-A and B;Table 1).Compared with the WT biscuits,the mutant biscuits were not as thick and had a larger diameter,a higher spread ratio,and a similar hardness (Fig.3-C and D;Table 1).There were no obvious differences in the loaf and biscuit sensory properties between the mutant and WT samples.

Fig.3 Comparison of the wild type (WT) and mutant loaves and biscuits.A,loaf shape.B,loaf slices.C,biscuit shape.D,biscuit thickness.Scale bar=1 cm.

To confirm its effect on the biscuit-making quality,the Dy10 subunit was purified fromE.colicells (Fig.4),after which various amounts (0,20,40,and 60 mg) were incorporated into the WT and mutant flour as described by Bekes and Gras (1999).In all cases,the addition of the purified Dy10 adversely affected the biscuit-making quality.More specifically,the incorporation of purified Dy10 into the wheat flour resulted in a decrease in the biscuit area,an increase in biscuit thickness,and a decrease in the spread ratio (Fig.5).Moreover,the biscuit quality was negatively correlated with the Dy10 content.

Fig.4 Purification of the Dy10 subunit from Escherichia coli cells.The glutenins extracted from the mutant and wild type(WT) lines were used as the controls (lanes 1 and 2).The heterologous expression of the Dy10 subunit was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) (lane 3) and Western blot (lane 4) analyses.

Fig.5 Incorporation of the purified Dy10 subunit negatively affects biscuit quality.A–D,biscuits produced from the mutant flour with the incorporation of 0 mg (A),20 mg (B),40 mg (C),and 60 mg (D) of the Dy10 subunit.E–H,biscuits produced from the wild type (WT) flour with the incorporation of 0 mg (E),20 mg (F),40 mg (G),and 60 mg (H) of the Dy10 subunit.Scale bar=1 cm.I–K,comparisons of the biscuit area (I),biscuit thickness (J),and spread ratio (K) for the mutant and WT flour with and without the addition of Dy10.Data are presented as the mean±standard deviation (n=3).

4.1.Mechanism on the silencing of Dy10-null allele

Deletion mutants are suitable for clarifying the effects of HMW-GSs on wheat processing quality.In this study,aDy10deletion mutant line (SM482-Dy10null)was isolated from an EMS-mutagenized population(Appendix A).Previous research verified the utility of EMS for modifying single nucleotides in order to introduce a stop codon or a missense mutation (a C-to-T mutation is the most common) (Kimet al.2006).TheDy10-nullallele had a single point mutation (C-to-T;Appendix D) that introduced a premature stop codon(TAA).Two major gene-silencing mechanisms in plants have been described,namely transcriptional gene silencing (TGS) and post-TGS (PTGS);and the silenced gene is transcribed normally during PTGS (Sijenet al.2001).In the current study,theDy10-nullallele in the mutant line and theDy10allele in the WT line were similarly transcribed (Appendix E),implying that the silencing of theDy10-nullallele wasviaPTGS (Fig.1).Previous research has suggested that a premature stop codon in HMW-GS genes leads to the production of truncated proteins (De Bustoset al.2000;Chenet al.2021),which is detrimental for investigations regarding the effect of HMW-GSs on processing quality.The Western blot analysis performed in this study revealed the absence of the Dy10 subunit in the mutant with theDy10-nullallele (Fig.1-B and C).Therefore,our analysis of theDy10-nullallele has clearly shown that wheat processing quality is influenced by the Dy10 subunit.

4.2.Mechanism of Dy10-null allele on wheat processing quality

The absence of Dy10 in the mutant likely results in the restructuring of the inherent gluten network,ultimately leading to decreased dough strength.Wheat HMWGSs,especially the Dx5+Dy10 subunit pair,are major determinants of bread-making quality (Shewryet al.2003).Additionally,Dx5,which encodes a protein that contains one more Cys residue in the repetitive domain than the other HMW-GSs,is always expressed as a part of an allelic pair withDy10.Compared with Dx5,the Dy10 subunit contains more Cys residues that form intermolecular disulfide bonds during dough development,enabling the extensive cross-linking of glutenin polymers(Shewry and Halford 2002).There is an evidence that dimers comprising these two subunits are present as“building blocks” in glutenin polymers (Wieser 2007;Wanget al.2021b).In the present study,the lack of Dy10 reduced the gluten index,Zeleny sedimentation value,dough development and stability times,and GMP content,which helps to explain the observed reduction in breadmaking quality (Table 1).

The balance between different gluten protein fractions is an important factor governing the end-use quality of wheat.For different types of food products,diverse dough strength and extensibility values may reflect the optimum balance between gluten fractions (Oliver and Allen 1992).Our results indicated that theDy10-nullallele in the mutant reduced the glutenin:gliadin ratio,but did not alter the grain protein content.TheDy10-nullallele significantly reduced the HMW-GS contents,while significantly increasing the gliadin contents.These findings are in accordance with the reported compensatory interaction between HMW-GSs and gliadins (Diaet al.2022;Liuet al.2022;Scossaet al.2008;Zhanget al.2018).Several studies have revealed that HMW-GSs and gliadins mainly contribute to the viscoelastic properties and extensibility of wheat dough,respectively (Shewryet al.2003;Baraket al.2015).Therefore,it is not surprising that in response to the deletion of Dy10,the biscuit-making quality (Fig.3-C and D;Table 1) and bread-making quality (Fig.3-A and B;Table 1)increased and decreased,respectively.Furthermore,the incorporation of the purified Dy10 subunit had adverse effects on the biscuit-making quality of the wheat dough(Fig.5).In contrast,supplementing wheat flour with purified gliadins reportedly leads to enhanced biscuitmaking quality (Kuraganoet al.1991).Unexpectedly,both positive and negative effects of the silencing of HMWGS genes on biscuit-making quality have been reported.For example,the deletion of a single HMW-GS gene inT.aestivumcv.‘Ningmai 9’ (which normally contains Ax1,Bx7+By8,and Dx2+Dy12) results in improved biscuitmaking quality (Zhanget al.2016),whereas the absence of the Dy12 subunit inT.aestivumcv.‘Kenong 199’ (which normally contains Ax1,Bx7+By9,and Dx2+Dy12) leads to reduced biscuit-making quality (Chenet al.2021).We speculate that the glutenin:gliadin ratio and the HMW-GS composition are essential determinants of biscuit-making quality.

A common wheat mutant carrying theDy10-nullallele was identified by screening an EMS-mutagenized ‘Shumai 482’population.TheDy10-nullallele resulted in the absence of Dy10.Based on an analysis of this mutant,we demonstrated that the Dy10 subunit is essential for maintaining dough strength.Furthermore,theDy10-nullallele positively affects the biscuit-making quality of wheat dough,making it potentially useful for soft wheat breeding programs.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (31971939,32072054,and 31901961),the Science and Technology Department of Sichuan Province,China (2019YFH0066 and 2020YFH0150),and the Designing Future Wheat Strategic Program of the UK (BB/P016855/1).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2022.08.041