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Biotechnology and Biological Transformations

Effects of additional cysteine residue of Avenin-like b protein by sitedirected mutagenesis on dough properties in wheat (Triticum aestivum L.) Yaqiog Wang, Miao Li, Yanbin Guan, Li Li, Fusheng Sun, Jiapeng Han, Junli Chang, Mingjie Chen, Guangxiao Yang, Yuesheng Wang, and Guangyuan He J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02814 • Publication Date (Web): 12 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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Journal of Agricultural and Food Chemistry

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Effects of additional cysteine residue of Avenin-like b protein by site-directed

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mutagenesis on dough properties in wheat (Triticum aestivum L.)

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Yaqiong Wang,† Miao Li,‡ Yanbin Guan,† Li Li,† Fusheng Sun,† Jiapeng Han,† Junli

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Chang,† Mingjie Chen,† Guangxiao Yang,† Yuesheng Wang,†,* Guangyuan He†,*

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†The

Genetic Engineering International Cooperation Base of Chinese Ministry of Science

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and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of

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Education, College of Life Science and Technology, Huazhong University of Science &

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Technology, Wuhan 430074, China

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‡College

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450052, China

of Grain Oil and Food Science, Henan University of Technology, Zhengzhou

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Corresponding authors

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*(Y.W.) E-mail: [email protected]. Tel: 0086-27-87792271. Fax: 0086-27-87792272

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*(G.H.) E-mail: [email protected]. Tel: 0086-27-87792271. Fax: 0086-27-87792272

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ACS Paragon Plus Environment


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ABSTRACT: Avenin-like b protein is rich in cysteine residues, providing the

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possibility to form inter-molecular disulfide bonds and then facilitate glutenin

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polymerization. Site-directed mutagenesis was adopted to produce mutant avenin-like b

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gene encoding mutant Avenin-like b protein, in which one tyrosine codon at the

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C-terminal substituted by cysteine codon. Compared with control lines, both transgenic

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lines with wild-type and mutant avenin-like b genes demonstrated superior dough

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properties. While compared within the transgenic lines, the mutant lines showed relative

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weaker dough strength and decreased sodium-dodecyl-sulfate sedimentation volumes

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(from 69.7 ml in line WT alb-1 to 41.0 ml in line Mut alb-4). These inferior dough

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properties were accompanied with the lower contents of large-sized glutenin polymers,

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the decreased particle diameters of glutenin macropolymer (GMP), due to the lower

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contents of inter-molecular β-sheets (from 39.48% for line WT alb-2 to 30.21% for line

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Mut alb-3) and the varied contents of disulfide bonds (from 137.37 µmol/g for line WT

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alb-1 to 105.49 µmol/g for line Mut alb-4) in wheat dough. The extra cysteine might alter

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the original disulfide bond structure, allowed cysteine residue usually involved in

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inter-molecular disulfide bond becoming available for intra-chain disulfide bond.

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Avenin-like b proteins were detected in glutenin macropolymers, providng further

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evidence for this protein to participate in the polymerization of glutenin. This is the first

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time to investigate the effect of specific cysteine residue in the Avenin-like b protein on

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flour quality.

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KEYWORDS: Site-directed mutagenesis, wild-type and mutant avenin-like b,

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non-conserved cysteine residue, disulfide bond, glutenin polymers, mixing properties,

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transgenic wheat lines

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INTRODUCTION

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Wheat is one of the gramineous plants widely cultivated all over the world and is

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considered as the world’s ‘big three’ cereal crops together with corn and rice.1,2 Despite

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the high adaptability to environmental conditions and high yield potential, the important

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characteristic which has given wheat an advantage over other temperate crops is the

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gluten protein in its grains.3 Gluten protein is the major contribution to determine the

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unique baking quality of wheat flour due to its characteristics to confer dough water

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absorption capacity, cohesivity, viscosity and elasticity.4-6 The alcohol-insoluble glutenins

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and alcohol-soluble gliadins are the main components of gluten. Glutenins confer dough

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viscoelasticity are polymeric proteins, which are formed by the cross-linking of several

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subunits through inter-molecular disulfide bonds.7 Gliadins are described as monomeric

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proteins imparting dough extensibility, based on the different molecular weights they can

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be further divided into three types: α/β- (MW ~ 31 kDa), γ- (~ 35 kDa) and ω-gliadin

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(44-80 kDa). The conserved structural regions of α/β-gliadin and γ-gliadin are similar and

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contained six and eight cysteine residues respectively, corresponding to take part in

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forming three pairs and four pairs of intra-molecular disulfide bonds. These polymeric

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glutenins and monomeric gliadins are cross-linked by strong covalent and non-covalent

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forces, conferring the diversified dough viscoelasticity and resulting in the difference in

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the quality of gluten.8 A widely accepted view on the structure of gluten network is that

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the HMW-GS form an “elastic backbone” consisting largely of head-to-tail polymers

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through inter-molecular disulfide bonds and the LMW-GS as “branches” connecting to

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the “backbone”, while monomeric gliadins adhering to the gaps of the gluten matrix.

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In addition to the typical gluten proteins, Avenin-like proteins (ALPs) which are

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considered as atypical gluten proteins due to the lack of repetitive domains contain only a

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short sequence of glutamine and proline residues in the mature proteins, also have an

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important impact on wheat flour quality. The discovery of ALPs can be traced back to

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nearly twenty years ago. Anderson et al. used the expressed-sequence-tag (EST) project

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to reveal four new classes proteins that related to wheat gliadin proteins, but lacking the

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prominent repeat domain.9 Clark et al. identified a new class of functional genes

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associated with wheat end-use, these genes were related to the gliadins and the LMW-GS. 3



Vensel et al. and DuPont et al. also found the presence of this type of protein by mass

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10

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spectrometry and proteome analysis, respectively.11,12 In particular, Kan et al. named the

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discovered genes which have ‘weakly similar’ to the genes of avenin storage proteins of

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oats as avenin-like for the first time and they cloned several full-length sequences of

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avenin-like genes in Aegilops species and wheat. According to the structural differences

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of deduced ALPs, they were divided into Avenin-like a and Avenin-like b protein.13 It

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was predicted that seven pairs of intra-molecular disulfide bonds can be formed in

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Avenin-like a protein, which mainly presents in the storage proteins in the form of

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monomer. However, Avenin-like b protein contains 18 or 19 cys residues, sixteen

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cysteine residues located in the central repetitive region were considered conserved.

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However, the two cysteine residues located in the N-terminal and one in the C-terminal

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are not conserved. It was speculated that the non-conserved cysteine residues affect the

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polymerization of glutenin via forming inter-molecular disulfide bonds and thus affect the

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processing quality of wheat. Later, De Caro et al. used proteomic techniques to reveal the

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protein characterization of Avenin-like b and they detected the presence of this protein in

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the glutenin fraction, predicting that it might participate in glutenin polymers by forming

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inter-molecular disulfide bonds.14 Chen and Ma had proved Avenin-like b protein could

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significantly improve dough mixing properties in vitro and vivo.15-17 Later, Chen et al.

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mapped ALP coding genes to the chromosomes 7AS, 7DS and 4AL. Alleles on each

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chromosome were analyzed and the allele effect of each locus was quantified.18 Recently,

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Zhang et al. analyzed the molecular characteristics of the avenin-like b gene in the wild

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emmer wheat (Triticum dicoccoides) populations and emphasized that natural selection

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through the diversity of climate and soil factors was the main driving force of ALP allele

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diversity.19

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The proportion of cysteine residues in the total amino acids of gluten proteins (about

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2%) is very small, they are most critical to the structure and function of gluten protein due

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to their ability to form intra- and inter-molecular disulfide bonds.20,21 It is widely accepted

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that inter-molecular disulfide bonds are essential for glutenin viscoelasticity because they

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are involved in the formation of the glutenin polymers within gluten matrix.22 Since the

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non-conserved cysteine residues of Avenin-like b protein are most likely supposed to 4


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form inter-molecular disulfide bonds, in the present study, the effect of specific cysteine

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residue in the Avenin-like b protein on flour quality was investigated that is important for

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further understanding the effect of Avenin-like b protein on dough properties.

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MATERIALS AND METHODS

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Plasmid constructs. According to the avenin-like b gene on chromosome 7D of the

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common wheat Chinese Spring (CS), the wild-type avenin-like b was artificially

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synthesized. The target gene was then cloned into the eukaryotic expression vector named

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as pLRPT-WT avel.23,24 The artificially synthesized site-directed mutagenesis gene mut

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avenin-like b with the adenine at the 836 bp position replaced by guanine, encoding a

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subunit (mutant Avenin-like b) in which one specific tyrosine codon in the C-terminal

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was replaced by a cysteine codon. To facilitate the subsequent detection of genetically

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modified materials, the 48 bp sequence of myc epitope tag was inserted after the signal

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peptide sequence in N-terminal of mutant Avenin-like b. The recombinant vector carrying

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mut avenin-like b gene was constructed in a similar way with pLRPT-WT avel and named

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as pLRPT-Mut avel (Supporting Information Figure. S1). Although it has been reported

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that the use of myc tag did not affect the transgenic protein trafficking and its

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incorporation into glutenin polymers, to ensure the rigor of the experiment, we still used

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the plasmid without myc tag for genetic transformation.23

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Wheat transformation and plant regeneration. The common wheat (T. aestivum)

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cultivar ZM 9023 was used for genetic transformation. The immature scutella isolated

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from young seeds (12 d-18 d after pollination) were used as the target explants and then

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used for genetic transformation by gene gun (PDS-1000/He System, Bio-Rad, USA).25

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The T0 regenerated putative transgenic plants obtained after a series of tissue culture

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processes were transferred to the soil under the greenhouse conditions.16

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Identification of integration and expression of foreign genes in transgenic plants.

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To detect the integration of foreign genes, two pairs of specific primers with different

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lengths of PCR products were designed using Primer 6.0 software. The amplified

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fragment size was 585 bp for one pair of primers (SF1:AACCATTCCATTGGTGGCT,

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SR4:ACAAGCATTCCCTTAGCG) and 2926 bp for the other pair of primers (ORF

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alb-F2:GCATGCAAATATGCAACATAATTTCC, 5


ORF

alb-R2:


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TACCCATCGCATGCGATCC). The genomic DNA of putative transgenic plants that

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were simultaneously identified as positive for two pairs of primers and the confirmed

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plants were reconsidered as candidates for propagation. In order to further confirm the

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expression of foreign genes, Western blotting was carried out with the anti-Avenin-like b

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polyclonal antibody and anti-myc-tag monoclonal antibody (Medical & Biological

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Laboratories, Japan) for all candidate plants.

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The total protein was extracted from the single seeds using the endosperm part, the

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other part containing embryo was used for plant generation.26 The proteins were separated

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on the 12% SDS-PAGE gel and transferred onto the polyvinylidene fluoride (PVDF)

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membrane. The using dilution concentration of primary anti-myc monoclonal antibody

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was 1:5000 in TBST/5% BSA and the polyclonal antibody for Avenin-like b protein was

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1:200000. The dilution concentration of the corresponding secondary antibody was both

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1:5000. The bands can be detected using UVP ChemiDoc-It HR 410 Imaging System

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(American). The β-actin was used to quantify the loading volume of each sample and to

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calculate the relative expression amounts of avenin-like b for each sample.

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Field trials of transgenic lines. The materials of transgenic and negative control

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lines were planted in the experimental field of Huazhong University of Science and

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Technology (Wuhan, Hubei Province, China) with 2016-2017 and 2017-2018 two cycles,

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these plants were planted in a randomized complete block design in triplicate.27

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Agronomic traits including spike length, number of seeds per spike, number of spikes,

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grain weight per spike, 1,000-seed weight and plant height, were measured from 30

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individual plants collected from three central rows of each plot. Wheat seeds were then

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sun-dried and stored for 60 d before further tests.

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Quantitative PCR (qPCR) analysis. For RNA extraction, the developmental seeds

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were collected at 5, 11, 13, 15, 18, 20, 22, 25, 28 days post anthesis (DPA) from

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field-grown plants. The total RNA was extracted using a Plant Total RNA kit (Zoman Bio,

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Beijing, China). RNA was reversed transcribed using a FastQuant RT kit (Tiangen,

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Beijing, China). Amplification reactions were performed on the CFX96 Real-Time

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System (Bio-Rad, Hercules, CA, USA) using the AceQ qPCR SYBR Green Master Mix

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(Vazyme, Nanjing, China). The specific primer sequence for the target gene was used for 6


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qPCR analysis (ORF F3:5’-ATGAAGGTCTTCATCCTGGCTCTCCTT-3’ and ORF

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R3:5’GGCAGCATTGGTATTGCACGATCT). The reaction was conducted as the

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following cycling parameters: 95°C for 15 s, 62.3°C for 15 s and 72°C for 20 s with 40

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cycles. The amplification efficiency and the specificity of primer were tested before using

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for qPCR. The β-actin gene was used for calculating and standardizing. Each PCR

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reaction was performed independently at least three times.

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Seed storage protein characterization. About 500-gram grains of each line were

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tempered at 15% moisture level for 24h under 25°C and milled into flour on a Chopin

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CD1 laboratory mill (Chopin technologies, Villeneuve-la-Garenne Cedex, France). The

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method of Dumas was used to determine the flour protein content. Gliadins and glutenins

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from 10mg flours were extracted according to the report by León et al..28 Using a

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Bradford assay kit (Beyotime Biotechnology, Shanghai, China) the contents of extracted

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glutenins and gliadins were determined.29 For densitometry analysis, the glutenins

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extracted from flour samples were alkylated for 30 min and then mixed separately with

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running buffer solution [62.5 mM Tris–HCl buffer (pH 6.8), 2% (w/v) sodium dodecyl

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sulfate, 1.5% (w/v) dithiothreitol, 40% (v/v) glycerol, 0.02% (w/v) Bromophenol blue].

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The extracted glutenins were separated by SDS-PAGE using 12% polyacrylamide gel and

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analyzed with the software of Quantity One (Bio-Rad, Hercules, CA, USA).26

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Dough mixograph study. The specific operation steps were carried out following

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the approved AACCI Method 54-40A. In the present study, 11 representative parameters

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highly related to dough mixing characteristic were selected for statistical analysis.30

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Repeat four times of this analysis for each sample. In the fourth repeat, dough samples

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mixed to peak were collected from the mixing bowl and divided into two parts and then

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freeze-dried. One part was used for determining the contents of free sulfhydryl and

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disulfide bond and the other for SEM analysis.

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Determination of the gluten content and sodium dodecyl sulfate sedimentation

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volume. Wet gluten content (%) and dry gluten content (%) were determined following

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the AACCI approved Method 38-12A using the Glutomatic 2200 (Perten Instruments Ltd.,

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Hägersten, Sweden). SDSS volume was determined following the AACCI Method

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56-70.01. Each sample was performed independently at least three times for gluten 7



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content and SDSS volume analyses.

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Glutenin macropolymer content test and the particle size distribution analysis.

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The GMP gel was extracted from wheat flour following the method described by Mueller

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et al..31 The determination of GMP content was conducted according to the protocol of

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Yan et al..

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extracted from flour should be fully dispersed in 1.5% (w/v) SDS (10 ml).33 Particle size

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distributions of GMP dispersion suspensions were determined using the Mastersizer 3000

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(Malvern Instrument Ltd., Worcestershire, UK).

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In order to carry out the particle size distribution analysis, 1 g fresh GMP

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Size Exclusion-HPLC analysis. Determination the molecular weight distribution

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and solubility of protein which are important indicators to measure the degree of protein

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cross-linking by SE-HPLC.34 The analyses were conducted both in wheat flour and GMP

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gel samples of all wheat lines. The details of analysis were accorded to the previous

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reports.35,36

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Determination of free sulfhydryl (SHfree) and disulfide bond (SS) contents. The

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contents of SHfree and thiol equivalent groups (SHeq) in dough samples mixed to peak

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were determined using the Ellman’s reagent.37,38 The protein standard curve was obtained

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by the reaction of gradient diluted L-cysteine (1mM) with Ellman’s reagent under the

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same conditions. According to the absorbance at 412 nm and the standard curve, the

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corresponding concentration of SHfree and SHeq would be calculated. The SS content was

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calculated as follows: SHeq = 2SS + SHfree. Three technical and biological repeats were

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conducted for each sample.

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Scanning electron microscopy. Scanning electron microscopy (SEM) was carried

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out to observe the microstructure of dough samples. Freeze-dried samples were cut into

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small pieces and attached to a silicon wafer using double-sided tape in order. After coated

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with gold particles the images were taken in a scanning electronic microscope (Hitachi

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High-Technologies Corp., Tokyo, Japan) with a 15 kV acceleration voltage.

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Fourier transform infrared spectroscopy. The secondary structure of dough was

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studied by attenuated total reflectance-Fourier transform infrared (ATR-FTIR)

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spectroscopy. FT-IR spectrometer (Model VERTEX 70, Bruker, German) record spectra

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in the 4000-400 cm-1 at room temperature. Acquisition of 128 scan interferograms with a 8


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spectral resolution of 4 cm-1. After subtracting the water spectra, the protein spectra were

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obtained. Baseline correction and smoothing of the infrared spectrum was performed

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using OPUS software (version 6.5). Fourier self-deconvolution identified the position of

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the band. Second-order derivative spectrum supported spectral deconvolution. The

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‘goodness of fit’ fitting curve was obtained.

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Statistical analyses. All analyses in the present study were repeated at least three

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times. The software SPSS 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical

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analysis via one-way analysis of variance (ANOVA) followed by Tukey HSD and

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Scheffe. The diagrams of statistical analyses were generated by Origin 8.5. The criterion

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for statistical significance used was P ˂ 0.05.

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RESULTS

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Over-expressions of mut avenin-like b and wt avenin-like b genes in wheat

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transgenic plants. PCR amplification with two different pairs of primers had been used

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to confirm the positive putative transgenic wheat plants in the T0 generation (Figure 1).

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The products were sequenced to confirm the validity, the results showed that target mut

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avenin-like b gene with site-mutation had been successfully introduced into wheat plants

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genomes (Supporting Information Figure S2). Two homozygous transgenic wheat lines

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over-expressing avenin-like b gene (WT alb-1 and WT alb-2) and four lines

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over-expressing mut avenin-like b gene (Mut alb-1, 2, 3 and 4) were obtained in the T5

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generation, among which two contained the myc tag (Mut alb-1 and Mut alb-2). The flour

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of these six transgenic lines and the two negative control line (N-4 and ZM 9023) were

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also analyzed by SDS-PAGE and Western blotting to verify the homozygosity and

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stability of transgene (Figure 2).

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The Western blotting results (Figure 2B) showed that compared with the

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non-transformed line ZM 9023, the relative expression levels of avenin-like b in six

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transgenic lines (Mut alb-1, 2, 3, 4 and WT alb-1, 2) were increased 2.45-, 2.43-, 2.47-,

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2.35-, 2.20- and 2.10-fold, respectively (Figure 2C). There was almost no difference in

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protein expression between the two negative control lines, although the expression levels

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of the mutant lines were slightly higher than that of the wild-type lines it did not reach a

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significant level. The anti-myc-tag monoclonal antibody was used for Western blotting to 9



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further confirm the expression of mut avenin-like b as shown in Figure 2D. It could find

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that the target bands only existed in lines Mut alb-1 and 2 while no signal was obtained

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from other lines.

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The qPCR analysis (Supporting Information Figure S3) indicated the negative and

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transgenic lines had a similar expression trend with a much lower expression at 5 DPA

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but rapidly up-regulated at 11 DPA, and then down-regulated from 18 to 28 DPA. The

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maximum expression level of avenin-like b gene occurred at 11 to 15 DPA. These results

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were consistent with the previous research.39 The six transgenic lines have a much higher

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level expression than negative lines ZM 9023 and N-4 throughout the whole endosperm

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developmental period.

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Agronomic performance. The agronomic characteristics of transgenic wheat lines

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may be poorer than their control lines.40-42 The result (Supporting Information Table S1)

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revealed that no significant difference existed in the agronomic traits such as plant height,

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number of spikelets per spike, growth period and seeds weight per spike among all lines.

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Except for the transgenic lines Mut alb-1 and Mut alb-3, no significant difference at the

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0.01 probability level was observed in spike length between transgenic and negative

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control lines. With the exception of Mut alb-1, the trait of number of seeds per spike was

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similar among all lines. In addition, 1,000-seed weight of transgenic line Mut alb-3 was

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significantly higher than other lines. Altogether, no significant difference was observed

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among the lines in most of the measured traits in this study.

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Characterization of storage proteins. As shown in Table 1, the flour protein

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contents ranged from 9.52% to 12.02% for the non-transgenic line ZM 9023 and line WT

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alb-1. All transgenic lines over-expression avenin-like b had relative higher flour protein

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contents compared with the negative control lines. The amount of total proteins in

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wild-type transgenic lines was higher than that in mutant transgenic lines. In addition, the

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amounts of glutenin proteins in flour increased from 7.40 µg/mg in N-4 to 11.46 µg/mg in

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WT alb-1, indicating that the over-expression of avenin-like b could increase the amounts

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of glutenin proteins. The highest glutenin quantities were detected in lines WT alb-1

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(11.46 µg/mg) and WT alb-2 (9.51µg/mg). All transgenic wheat lines showed relative

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higher glutenin/gliadin ratio than the two negative controls, especially for the two 10


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wild-type lines the ratios were higher than all other lines. But no significant difference

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was observed among in the contents of gliadin proteins between these lines.

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In addition, no difference was observed in the expression patterns of major storage

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proteins between the transgenic and control lines, indicating that the expression patterns

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did not appear to be affected by over-expression of wt avenin- like b gene or mut

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avenin-like b (Figure 2A). Glutenin proteins play an important role in determining the

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quality characteristics of flour. SDS-PAGE combined with densitometry was conducted

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to analyze whether the foreign gene wt avenin-like b/mut-avenin like b affected the

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expressions of endogenous glutenin components. As shown in Figure 3, the relative

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contents of endogenous HMW-GS (1Dx2, 1Bx7, 1By8 and 1Dy12) subunits was similar

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between the transgenic and negative control wheat lines. No significant difference was

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observed in the ratios of HMW/glutenin and LMW/glutenin among wheat lines. Overall,

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the over-expression of foreign genes including wt avenin-like b and mut-avenin like b had

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little effect on the proportions of glutenin components.

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Effect of over-expression of foreign genes on wheat flour quality parameters.

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SDSS volume and gluten content had been used as important index to predict and

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evaluate wheat quality. As shown in Table 1, almost similar SDSS volume values, wet

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and dry gluten contents were observed among the two negative control lines (ZM 9023

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and N-4). Compared with the negative control lines, the six transgenic wheat lines had

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significantly higher (P < 0.01) SDSS volume values with Mut alb-1 (50.7), Mut alb-2

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(51.1), Mut alb-3 (48.8), WT alb-1 (69.7) and WT alb-2 (67.1), while no significant

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difference of Mut alb-4 (41.0). Wet gluten contents of all transgenic lines were

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significantly higher than the control lines. However, when compared with WT alb-1 and 2,

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significant decreases (P < 0.01) of the four transgenic lines over-expressing mut-avenin

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like b were observed in the SDSS volumes. The four mutant lines had relative lower

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gluten contents than the WT alb-1 and 2 lines except for the wet gluten content for the

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Mut alb-2 and the dry gluten contents for the line Mut alb-2 and 3.

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The contents of GMP and the GMP wet weight were also significantly different

328

among lines. The over-expression of foreign avenin-like b genes significantly increased

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the concentration and wet weight of GMP (Table 1). Meanwhile, similar to the SDSS 11



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volume, all mutant transgenic lines had remarkably lower GMP wet weight than the line

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WT alb-1 and 2. The line WT alb-1 had the highest GMP concentration followed by the

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line Mut alb-3 and the other three mutant transgenic lines were all lower than the

333

wild-type lines.

334

Dough mixing property analysis. From the results, (Figure 4 and Supporting

335

Information Figure S4 and Supporting Information Table S2) we found the two negative

336

control lines (N-4 and ZM 9023) had no statistically significant difference in the mixing

337

parameters, suggesting that the genetic transformation had little effect on dough mixing

338

quality under the same genetic background. However, significant differences were

339

observed between transgenic lines and control lines in mixing characteristics. The

340

negative control lines showed narrower and lower Mixogram curves than transgenic lines

341

(Supporting Information Figure S4), it could be preliminarily judged the dough stability

342

and its resistance to extension had been improved to some extent because of the

343

incorporation of subunit WT Avenin-like b or Mut Avenin-like b. To more clearly display

344

the effects of WT Avenin-like b or Mut Avenin-like b on dough property, AVONA and

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multiple comparisons were performed on the 11 key mixing parameters. The complete

346

results of mixing parameters were provided in Figure 4 and Supporting Information Table

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S2, over-expression of avenin-like b in transgenic lines showed differences in the dough

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mixing properties compared with the negative control lines. The parameters describing

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the height of mixograph curve including MLV, MPV, MRV and MTXV were

350

significantly increased in transgenic lines and these parameters were positively correlated

351

with dough elasticity. The MPW and MRW were considered had positively correlated

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with dough resistance to extension, and the MTxW had positively correlated with dough

353

stability. In addition, the MTxI value which is positively related to the dough strength was

354

raised in the transgenic lines compared with the negative lines. Unlike the mutant

355

transgenic lines, MPT from lines WT alb-1 and 2 were similar to the control lines. Only

356

the lines Mut alb-1 and 4 showed lower weakening slope compared with the

357

non-transformed control line ZM 9023.

358

However, when compared with the wild-type transgenic lines WT alb-1 and 2, the

359

mutant transgenic lines presented different mixing behaviors, as shown in Figure 4, the 12


Page 12 of 42

Page 13 of 42


360

wild-type transgenic line WT alb-1 and 2 had significantly higher values in the four

361

parameters describing the curve height and four describing the width of the curve as

362

compared with those of mutant transgenic lines, with the exception of the MTxW which

363

showed almost similar level between mutant lines and wild-type lines. Besides that, the

364

MPT in the mutant lines was significantly increased with respect to the wild-type lines

365

and the weakening slope was the opposite. Significant reduction in MTxI was observed in

366

mutant lines compared with that of lines WT alb-1 and 2.

367

Protein analysis of flour and GMP by SE-HPLC. As shown in Figure 5, the

368

SE-HPLC curve fitting profiles of the total protein extracted from flour/GMP gel could be

369

divided into four peak areas, the peak areas 1 and 2 correspond to the large-sized

370

polymers and the medium-sized polymers, respectively, and the peak area 3 corresponds

371

to small oligomers, the peak area 4 corresponds to non-gluten proteins and monomeric

372

gliadins that were similar to the previous results.43,44 In the present study, the parameters

373

F1%, F1%/F2% and (F3%+F4%)/F1% which were considered to have closely related to

374

the gluten strength were used to determine the effect of over-expression of avenin-like b

375

gene on the formation of gluten aggregate. The peak height of negative control lines

376

unmistakably decreased compared to the transgenic lines (Figure 5A and C). The six

377

transgenic wheat lines had obviously higher values of %F1 and %F1%F2 compared with

378

the negative control lines ZM 9023 and N-4, but the ratios of (%F3+%F4)/%F1 were

379

lower (Figure 5B and D). The result indicated that the amounts of large-sized polymers in

380

transgenic lines were increased.

381

In addition, the parameters were different between the transgenic lines

382

over-expressing mut avenin-like b and wt avenin-like b. The relative amounts of

383

large-sized polymers (%F1) of the mutant line Mut alb-1 and 2 were significantly lower

384

than the two lines over-expressing wt avenin like b in flour/GMP gel and a different

385

amplitude reductions were also present in line Mut alb-3 and 4. However, the %F1/F2%

386

ratio in mutant transgenic lines was slightly lower than the wild-type transgenic lines but

387

did not reach a significant level in flour/GMP gel samples, while the four mutant lines

388

had a little higher (%F3+%F4)/%F1 ratio in the flour and GMP gel samples but this

389

difference is very small. 13



390

Effects of Avenin-like b on the GMP particle size distribution. As shown in

391

Figure 6A, the GMP particle number distribution presented a single peak curve, and the

392

particle sizes (diameter) ranged from 1 µm to 300 µm. With the exception of Mut alb-1,

393

the particle diameters of GMP in six transgenic lines were slightly shifted to the right

394

compared with the negative control ZM 9023 and N-4, indicating that the size of GMP

395

was increased. These results were in agreement with the variation of weighted average

396

surface area D[3,2] and weighted average volume particle size D[4,3] (Figure 5B).

397

Relative to the negative control lines ZM 9023 and N-4, the D[3,2] and D[4,3] of Mut

398

alb-1, 2, 3, -4 and WT alb1, -2 all increased significantly (P < 0.01). However, the values

399

of D[3,2] in lines over-expressing mut-avenin like b were 59.37, 63.00, 66.87 and 67.47

400

µm respectively, and the D[4,3] were 66.67, 70.53, 74.20, 75.63 µm. These values in

401

mutant lines were significantly reduced when compared with the line WT alb-1 and this

402

obvious reduce was also observed in Mut alb-1 and 2 when compared with WT alb-2.

403

Effects of over-expression of avenin-like b on the amount of disulfide bonds. The

404

transgenic lines had significantly higher SS contents when compared with the negative

405

control lines (ZM 9023 and N-4) (Figure 7). Compared with line WT alb-1, the four

406

wheat lines over-expressing mut avenin-like b had significant lower SS content (P < 0.01)

407

in dough samples. The SS contents of Mut alb-1 and 2 were similar to that in line WT

408

alb-2. Furthermore, no significant difference was observed for the contents of SHfree

409

among each line.

410

Effects of over-expression of avenin-like b on the microstructure of dough. As

411

shown in Figure 8, obvious distinctions in the structure of the gluten network among

412

transgenic lines and control lines were discovered. Although starch granules were closely

413

adjacent and were surrounded by gluten protein matrix in the two negative control lines

414

ZM 9023 and N-4, these protein matrix is relatively discontinuous and unapparent.

415

Conversely, the starch granules were almost completely embedded in the gluten matrix

416

and the more continuous and obvious gluten structures were discovered in all transgenic

417

wheat lines.

418

Effects of over-expression of avenin-like b on protein secondary structure. The

419

deconvolution spectra of dough mixed to peak from all wheat lines were shown in 14


Page 14 of 42

Page 15 of 42


420

Supporting Information Figure S5. At least seven peaks have been found at 1600, 1615,

421

1630, 1645, 1655, 1670 and 1685 cm-1. Due to the changes in the protein secondary

422

structures, the bands of the six transgenic lines were slightly shifted in the amide I

423

measurements compared with the control lines ZM 9023 and N-4. Generally, the

424

secondary structure included the intra-molecular β-sheet, inter-molecular β-sheet, β-turn,

425

α-helix and random coil. The percentage of each secondary structure relative area

426

compared with the total area was evaluated from the models, which estimated from

427

relative band areas in the spectral sections 1600-1625 cm-1/ 1680-1700 cm-1, 1630-1640

428

cm-1 ,1650-1660 cm-1, 1660-1680 cm-1, and ~1645 cm-1, respectively.45 Form the result

429

shown in Figure 9, we discovered the β-sheet and α-helix structures occupied the vast

430

majority of the total area which was similar to previously reported results.36,46 The

431

curve-fitting analysis of Amide I spectra in the dough sample of ZM 9023 control line

432

revealed 23.95% inter-molecular β-sheet, 19.74% intra-molecular β-sheet, 20.4% α-helix,

433

12.79% β-turn and 23.12% random coils. The N-4 is similar to ZM 9023 with 24.56%

434

inter-molecular β-sheet, 19.54% intra-molecular β-sheet, 19.82% α-helix, 13.43% β-turn

435

and 22.65% random coils. Significant increases of the inter-molecular β-sheet content

436

(from 30.21% to 39.48%) were observed in the transgenic lines compared with the

437

control lines. Meanwhile, the transgenic lines had a slight decrease in contents of

438

intra-molecular β-sheet, random coil and α-helix. However, there was no remarkable

439

difference in the content of β-turn structure between lines.

440

The differences in the contents of protein conformations also existed between lines

441

expressing mut avenin-like b and wt avenin-like b. The line WT alb-1 and WT alb-2 had

442

the highest contents of inter-molecular β-sheet, 37.45% and 39.48%, respectively, higher

443

than the four mutant lines. However, the intra-molecular β-sheet, β-turn and random coils

444

contents were 15.63%, 12.09%, 15.99% (WT alb-1) and 16.24%, 11.9%, 15.39% (WT

445

alb-2) which were slightly lower than the four mutant lines. No significant difference was

446

observed in the contents of α-helix.

447

DISCUSSION

448

To a certain extent, the location and number of cysteine residues of wheat glutenin

449

proteins are the possible reasons for the differences in the composition and functionality 15



450

of glutenin polymers.44,47 It has been thought that wheat glutenin subunits participated in

451

forming GMP through inter-molecular disulfide bonds, which in turn determines the

452

elasticity of gluten. Although for specific gliadin subunits with an odd number of cysteine

453

residues due to mutations, the pattern of common intra-molecular disulfide bonds may be

454

altered, causing cysteine residues that typically form intra-molecular disulfide bonds

455

being available for inter-molecular disulfide bonds, but this assumption has not been

456

further supported by experiments.48 Seed storage protein ALPs were considered as

457

atypical gluten constituents capable of influence the elasticity of dough. The solubility

458

and amino acid sequence characteristics of wheat Avenin-like b protein are different from

459

glutenins, which has aroused our research interest.

460

Site-directed mutagenesis of avenin-like b and over-expression of foreign genes.

461

Site-directed mutagenesis provides technical support for in-depth understanding of gene

462

function, and it has been successfully applied in genetic engineering of seed storage

463

proteins of wheat.36,49,50 This method was also adopted in our study and the transgenic

464

materials with the expected mutation site were obtained. In the current study, more

465

avenin-like b genes were transcribed in the transgenic lines, and in these lines the

466

avenin-like b gene was still transcribed at the late stage of grain development. This may

467

be due to large glutenin polymer formations required for more Avenin-like b subunits

468

accumulation.

469

Due to the critical role of glutenin in dough function, the glutenin profiles were

470

determined among each line to ensure whether any difference existed. The result (Figure

471

3) showed that in the present study the over-expression of target genes did not alter any

472

related expressions of endogenous glutenin components. In addition, the four transgenic

473

mutant lines with over-expression of mut avenin-like b gene and the two transgenic

474

wild-type lines with over-expression of wt avenin-like b gene had approximate transgene

475

expression levels. Based on the above-mentioned results, it was considered that the

476

comparative analyses of the subsequent experimental results are reasonable between the

477

wild-type and mutant lines.

478

Effect of over-expression of avenin-like b on mixing properties of dough. The

479

higher gluten content and SDSS volume values are generally associated with strong 16


Page 16 of 42

Page 17 of 42


480

gluten and superior bread-making quality. Compared with the control, the wet gluten

481

content and SDSS volume value of the transgenic lines increased significantly, indicating

482

that the over-expression of avenin-like b improved the gluten quality. Consistent with the

483

above-mentioned results, the result of mixograph indicated that the over-expression of

484

avenin-like b played a positive role in improving the flour mixing characteristics, which

485

significantly enhanced the dough elasticity, dough resistance to extension and the stability.

486

These results were consistent with the results reported previously that over-expression of

487

avenin-like b both in vivo and in vitro will significantly improved dough

488

characteristics.15-17

489

The rheological properties of flour are mainly determined by the composition,

490

structure, content and molecular size distribution of flour protein. Importantly, the content

491

and molecular structure of GMP are closely related to the processing quality of wheat,

492

which determines the strength of gluten and the characteristics of dough. Consistent with

493

expectations, the amounts of large- and medium-sized polymers in all transgenic lines

494

were significantly increased and the contents of small monomer proteins were decreased

495

compared with the control lines (Figure 5). It indicated that over-expression of

496

Avenin-like b changed the distribution and interaction of protein components in flour and

497

GMP gel, which not only promote the formation of more large-sized polymers but also

498

assist the recruitment of small molecular proteins to participate in the formation of

499

glutenin polymers and thus affecting the molecular weight distribution of storage protein

500

in dough. Furthermore, the result of GMP particle size distribution indicating an increase

501

in the volume percentage of large particle GMP in the transgenic lines (Figure 6). The

502

relative higher amounts of large- and medium-sized polymers and the increase of GMP

503

particle size in transgenic lines may be one of the proofs for Avenin-like b protein

504

participate in the formation of glutenin polymers. In addition, we also detected the

505

presence of the mutant Avenin-like b subunit in the extracted GMP by Western blotting,

506

which provided further evidence for the participation of Avenin-like b subunit in the

507

formation of glutenin aggregates.

508

The changes in the secondary conformation of gluten protein in dough are based on

509

the inter-molecular interactions between protein molecules and are closely related to the 17



510

rheological properties of wheat dough.34,45,51 The content of β-sheets are positively related

511

to viscoelasticity of dough, while the contents of α-helices are negatively correlated to

512

these characteristics.52,53 In our results, the significantly increased content of the

513

inter-molecular β-sheet at the cost of α-helix and random coil in the above-mentioned

514

transgenic lines, indicating more formation of the ordered structure and an increase in

515

glutenin aggregation content. The result of SEM demonstrated that the dough of all

516

transgenic lines had more sufficient and obvious gluten network provided intuitive

517

evidence for the above-mentioned results.

518

In the process of wheat grain development, the contents of SHfree and SS and the

519

mutual transformation between them are main guarantees for the correct folding of grain

520

storage proteins, so it is closely related to the formation of glutenin polymer. In our study,

521

disulfide bonds were significantly increased in all transgenic lines in freeze-dried dough

522

samples compared to control lines. So we can reasonably associate the increase of

523

glutenin macropolymer with the increase of SS content.

524

Different effects of wild-type Avenin-like b and mutant Avenin-like b on wheat

525

dough properties. Results indicated that lines over-expressing the mut avenin-like b gene

526

were not as good as the lines over-expressing avenin-like b gene in improving flour

527

quality in general. Consistent with that the amounts of large- and medium-sized polymers

528

were decreased in the mutant lines relative to the wild-type lines. The results of the GMP

529

particle size distribution and the analysis of protein secondary structure also supported the

530

SE-HPLC results.

531

Due to the same genetic background and similar protein expression patterns between

532

wild-type and mutant transgenic lines, it is reasonable to attribute the difference in the

533

accumulation of glutenin polymers and properties of wheat gluten to the

534

tyrosine-to-cysteine mutation in the mutant lines which led to an extra cysteine residue in

535

the C-terminal of Avenin-like b protein. Although it has been reported that tyrosine

536

linkages existed in wheat flour, dough and bread in addition to disulfide bonds, less than

537

0.1% of tyrosine residues being cross-linked and they only play a minor role in gluten

538

structure. The loss of tyrosine is unlikely to be the main factor affecting glutenin

539

polymerization.54 So, the effect of an additional cysteine would be more significant. 18


Page 18 of 42

Page 19 of 42


540

Intuitively, it might be expected that the presence of additional cysteine residue would

541

increase the opportunity for inter-molecular cross-linkage and then enhance

542

polymerization, yet the effects observed here appear to be more consistent with the

543

reduced polymerization. One explanation is that the extra cysteine altered the previous

544

disulfide bond structure, allowed cysteine residue usually involved in inter-molecular

545

disulfide bond becoming available for intra-chain disulfide bond. This is confirmed by the

546

prediction online of the distribution of disulfide bonds in mutant and wild-type

547

Avenin-like b protein (http://www.ics.uci.edu/~baldig/scratch/explanation.html). As

548

shown in Figure 10, in the wild-type Avenin-like b protein, fourteen cysteine residues

549

were devoted to forming seven intra-molecular disulfide bonds and the four remaining

550

cysteine residues at the position of 57, 174, 200 and 257, respectively, may be involved in

551

forming inter-molecular disulfide bonds. However, in the mutant avenin like b protein

552

which contains nineteen cysteine residues, the disulfide bond pattern was changed with

553

forming eight pairs of intra-molecular disulfide bonds, resulting in the decrease of

554

cysteine residues available to form inter-molecular disulfide bonds. The cysteine residue

555

at the position of 257 in the wild-type Avenin-like b protein was devoted to forming

556

inter-molecular disulfide bond, however, the additional cysteine residue changed the

557

conformation of the protein resulting in the cysteine residue at position of 257 cross-links

558

with the adjacent cysteine residue to form intra-molecular disulfide bonds. The decreased

559

number of inter-molecular disulfide bonds may be the reason for mutant lines are inferior

560

to wild-type lines in flour quality. Our results regarding the determination of disulfide

561

bond contents showed the SS contents of mutant lines were lower than that in wild-type

562

lines, which supported the above explanation.

563

In addition, our results showed that the myc tag at the N-terminal of the mutant

564

Avenin-like b subunit did not appear to affect the correct folding and trafficking of the

565

transgenic subunits and their incorporation into the glutenin polymers. Because no

566

significant difference was observed between lines Mut alb-1 and 2 with myc tag and Mut

567

alb-3 and 4 without myc tag in the analyzed results mentioned above. Therefore, the myc

568

tag can be used to detect the exogenous transgenic subunits in the complex glutenin

569

complexes. 19



570

Possible mechanisms of the effect of wild-type Avenin-like b and mutant

571

Avenin-like b on dough properties. Based on the above-mentioned results, the

572

mechanism of the effect of Avenin-like b protein on the functional properties of wheat

573

flour was deduced as follow. Some cysteine residues in the Avenin-like b protein

574

especially for the cysteine residue at the position of 257 participated in the formation of

575

inter-molecular disulfide bonds and then enhanced the degree of cross-linking with other

576

protein subunits, which facilitated the polymerization of GMP, the size of GMP particles

577

and the proportion of large-sized glutenin polymers. It is beneficial for the formation of

578

denser protein gluten network structure, which enhanced the interaction among the

579

various components of the flour, thereby improving the dough quality.

580

However, for the non-conserved cysteine residue of Avenin-like b protein in the

581

C-terminal, its presence may not form inter-molecular disulfide bond as expected to

582

improve the processing quality of flour. Contrary to this, it may interfere with glutenin

583

polymerization by changing the original disulfide bond mode. This indicated that the

584

number and location of disulfide bonds in Avenin-like b protein can affect the pattern of

585

disulfide bond formation and the more number of cysteine residues did not always mean

586

superior flour quality. However, additional experiments, for example, the determination

587

of contents of inter-molecular and intra-molecular disulfide bonds separately in transgenic

588

lines and the further study of more Avenin-like b mutants with different number of

589

cysteine residues on dough properties, would be necessary to support this view.

590

In the present study, from the perspective of the effect of the specific non-conserved

591

cysteine residue at the C-terminal of Avenin-like b protein on gluten elasticity, our work

592

confirms that the number and position of cysteine residues of Avenin-like b protein affect

593

the content of inter-molecular disulfide bonds, which affect the gluten strength. This

594

provided an explanation for the function of this atypical gluten constituent in promoting

595

the formation of gluten elasticity. Our study confirmed the existence of inter-molecular

596

disulfide bonds in Avenin-like b protein and identified the sites most likely to form

597

inter-molecular disulfide bonds. This also indicated that the presence of special

598

components other than glutenin in wheat storage proteins participated in the formation of

599

gluten elasticity through inter-molecular disulfide bonds. This provided research evidence 20


Page 20 of 42

Page 21 of 42

600


for correcting or improving the molecular mechanism of wheat gluten elasticity.

601 602

ASSOCIATION CONTENT

603

Supporting Information

604

Agronomic performance of transgenic and control wheat lines; comparisons of mixograph

605

parameters of the transgenic and control wheat lines; structure of the transformation

606

plasmid pLRPT-WT avel/ pLRPT-Mut avel used for this study; sequencing analysis for

607

the PCR products of mutant and wild transgenic lines and the comparison of derived

608

amino acid sequence of mutant Avenin-like b and wild Avenin-like b; expression levels

609

of the endogenous avenin-like b genes in developing endosperms from 5 to 28 DAP of

610

the transgenic and control wheat lines; dough sample mixing curves of the transgenic and

611

control wheat lines; amide I band for dough samples of transgenic and control wheat lines

612 613

AUTHOR INFORMATION

614

Corresponding authors

615

*(Y.W.) E-mail: [email protected]. Tel: 0086-27-87792271. Fax: 0086-27-87792272

616

*(G.H.) E-mail: [email protected]. Tel: 0086-27-87792271. Fax: 0086-27-87792272

617

Author Contributions

618

G.H. and Y.W. conceived, designed and led the research. Y.W., M.L and Y.G.

619

participated in all experiments and analyzed the data. F.S., J.C. performed genetic

620

transformation. L.L. participated in part of the selection and propagation of transgenic

621

offspring. J.H. performed part of the sequencing and qRT-PCR experiments. M.C. and

622

G.Y. put forward valuable suggestions to this research. Y.W. and M.L. wrote the

623

manuscript. G.H. and Y.W. revised and finalized the manuscript. All authors read and

624

approved the final manuscript. Y.W., M.L. and Y.G. contributed equally to this work.

625

Funding

626

This work was financially supported by the National Natural Science Foundation of

627

China (31071403, 31371614) to Y.W., the National Natural Science Foundation of Hubei,

628

China (2016CFB549) to G.H., and National Genetically Modified New Varieties of 21



629

Major Projects of China (2016ZX08010004-004) to G.H.. The cost of all experiment

630

expenses in this research was supported by the four projects.

631

Notes

632

The authors declare no competing financial interests.

633 634

ABBREVIATIONS USED

635

AACCI, American association of cereal chemists international; ATR, attenuated total

636

reflectance; AVONA, one-way analysis of variance; DPA, days post anthesis; FT-IR,

637

fourier transform infrared spectroscopy; GMP, glutenin macropolymer; HMW-GS, high

638

molecular weight glutein subunits; ICC, international association for cereal science and

639

technology; LMW-GS, low molecular weight glutein subunits; mut avenin-like b, mutant

640

avenin-like b; MLV, midline left value; MLW, midline left width; MPT, midline peak

641

time; MPV, midline peak value; MPW, midline peak width; MRV, midline right value;

642

MRW, midline right width; WS, weakening slope; MTxI, area under midline for 8 min;

643

MTxV, midline value at 8 min; MTxW, midline width at 8 min; qPCR, quantitative real

644

time polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel

645

electrophoresis; SDSS, sodium dodecyl sulfate sedimentation; SE-HPLC, size exclusion

646

high performance liquid phase chromatography; SEM, scanning electron microscope; SH,

647

sulfhydryl; SS, disulfide bond; wt avenin-like b, wild-type avenin-like b; PVDF,

648

polyvinylidene fluoride

649 650

ACKNOWLEDGMENTS

651

We thank the Analytical and Testing Center of Huazhong University of Science and

652

Technology (HUST) for technical assistance in the measurements of nitrogen content and

653

FTIR. We thank the Research Core Facilities for Life Science (HUST) for the assistance

654

in the extract of GMP gel and determination the amount of disulfide bonds.

655 656 657 658 22


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improves dough mixing properties by their incorporation into glutenin polymers. PLoS One 2013, 8, e66758. (18) Chen, X.; Cao, X.; Zhang, Y.; Islam, S.; Zhang, J.; Yang, R.; Liu, J.; Li, G.; Appels, R.; Keeble-Gagnere G.; Ji, W.; He, Z.; Ma, W. Genetic characterization of cysteine-rich type-b avenin-like protein coding genes in common wheat. Sci. Rep. 2016, 6, 30692. (19) Zhang, Y.; Hua, X.; Islam, S.; She, M.; Peng, Y.; Yu, Z.; Wylie, S.; Juhasza, A.; Dowla, M.; Yang, R.; Zhang, J.; Wang, X.; Dell, B.; Chen, X.; Nevo, E.; Sun, D.; Ma, W. New insights into the evolution of wheat avenin-like proteins in wild emmer wheat (Triticum dicoccoides). Proc. Natl. Acad. Sci. U. S. A. 2018, 115, 13312-13317. (20) Shewry, P.; Tatham, A. Disulphide bonds in wheat gluten proteins. J. Cereal Sci. 1997, 25, 207-227. (21) Grosch, W., Wieser, H. Redox reactions in wheat dough as affected by ascorbic acid. J. Cereal Sci. 1999, 29, 1-16. (22) Keck, B.; Kiihler, P.; Wieser, H. Disulphide bonds in wheat gluten: cysteine peptides derived from gluten proteins following peptic and thermolytic digestion. Z. Lebensm. Unters. Forsch. 1995, 200, 432-439. (23) Tosi, P.; D’Ovidio, R.; Napier, J.; Bekes, F.; Shewry, P. Expression of epitope-tagged LMW glutenin subunits in the starchy endosperm of transgenic wheat and their incorporation into glutenin polymers. Theor. Appl. Genet. 2004, 108, 468-476. (24) He, G.; Jones, H.; D'Ovidio, R.; Masci, S.; Chen, M.; West, J.; Butow, B.; Anderson, O.; Lazzeri, P.; Fido, R.; Shewry, P. Expression of an extended HMW subunit in transgenic wheat and the effect on dough mixing properties. J. Cereal Sci. 2005, 42, 225-231. (25) Sparks, C.; Jones, H. Transformation of wheat by biolistics. In Transgenic Crops of the World; Springer: New York, 2004; pp 19−34. (26) He, G.; Rooke, L.; Steele, S.; Békés, F.; Gras, P.; Tatham, A.; Fido, R.; Barcelo, P.; Shewry, P.; Lazzeri, P. Transformation of pasta wheat (Triticum turgidum L. var. durum) with high-molecular-weight glutenin subunit genes and modification of dough functionality. Mol. Breed. 1999, 5, 377−386. (27) Barro, F.; Barceló, P.; Lazzeri, P.; Shewry, P.; Martín, A.; Ballesteros, J. Functional properties and agronomic performance of transgenic tritordeum expressing high molecular weight glutenin subunit genes 1Ax1 and 1Dx5. J. Cereal Sci. 2003, 37, 65-70. (28) León, E.; Marín, S.; María J.; Giménez; Piston, F.; Rodríguez-Quijano, M.; Shewry, P.; Barro, F. Mixing properties and dough functionality of transgenic lines of a commercial wheat cultivar expressing the 1Ax1, 1Dx5 and 1Dy10 HMW glutenin subunit genes. J. Cereal Sci. 2009, 49, 148-156. (29) Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. (30) Li, Y.; Wang, Q.; Li, X.; Sun, F.; Wang, C.; Hu, W.; Feng, Z.; Chang, J.; Chen, M.; Wang, Y.; Li, K.; Yang, G.; He, G. Coexpression of the high molecular weight

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glutenin subunit 1Ax1 and puroindoline improves dough mixing properties in durum wheat (Triticum turgidum L. ssp. durum). PLoS One 2012, 7, e50057. (31) Mueller, E.; Wieser, H.; Koehle, P. Preparation and chemical characterisation of glutenin macropolymer (GMP) gel. J. Cereal Sci. 2016, 70, 79-84. (32) Wang, S.; Yu, Z.; Cao, M.; Shen, X.; Li, N.; Li, X.; Ma, W.; Zeller, F., Hasm, S.; Yan Y. Molecular mechanisms of HMW glutenin subunits from 1S1 genome of Aegilops longissima positively affecting wheat breadmaking quality. PLoS One 2013, 8, e58947. (33) Herpen, T.; Cordewener, J.; Klok, H.; Freeman, J.; America, A.; Bosch, D.; Smulders, M.; Gilissen, L.; Shewry, P.; Hamer, R. The origin and early development of wheat glutenin particles. J. Cereal Sci. 2008, 48, 870-877. (34) Luo, Y.; Li, M.; Zhu, K.; Guo, X.; Peng, W.; Zhou, H. Heat-induced interaction between egg white protein and wheat gluten. Food Chem. 2016, 197, 699-708. (35) Wang, Q., Li, Y.; Sun, F.; Li, X.; Wang, P.; Sun, J.; Zeng, J.; Wang, C.; Hu, W.; Chang, J.; Chen, M.; Wang, Y.; Li, K.; Yang, G.; He, G. Tannins improve dough mixing properties through affecting physicochemical and structural properties of wheat gluten proteins. Food Res. Int. 2015, 69, 64-71. (36) Li, M.; Wang, Y.; Ma, F.; Zeng, J.; Chang, J.; Chen, M.; Li, K.; Yang, G.; Wang, Y.; He, G. Effect of extra cysteine residue of new mutant 1Ax1 subunit on the functional properties of common wheat. Scientific Rep. 2017, 7, 7510. (37) Zhou, Y.; Zhao, D.; Foster, T.; Liu, Y.; Wang, Y.; Nirasawa, S.; Tatsumi, E.; Cheng, Y. Konjac glucomannan-induced changes in thiol/disulphide exchange and gluten conformation upon dough mixing. Food Chem. 2014, 143, 163-169. (38) Morel, M.; Redl, A.; Guilbert, S. Mechanism of heat and shear mediated aggregation of wheat gluten protein upon mixing. Biomacromolecules 2002, 3, 488-497. (39) Chen, P.; Wang, C.; Li, K.; Chang, J.; Wang, Y.; Yang, G.; Shewry, P.; He, G. Cloning, expression and characterization of novel avenin-like genes in wheat and related species. J. Cereal Sci. 2008, 48, 734-740. (40) Barro, F.; Barceló, P.; Lazzeri, P.; Shewry, P.; Martín, A.; Ballesteros, J. Field evaluation and agronomic performance of transgenic wheat. Theor. Appl. Genet. 2002, 105, 980-984. (41) Bregitzer, P.; Halbert, S.; Lemaux, P. Somaclonal variation in the progeny of transgenic barley. Theor. Appl. Genet. 1998, 96, 421-425. (42) Hernändez, P.; Barceló, P.; Lazzeri, P.; Flores, F.; Lörz, H.; Martín, A. Morphological and Agronomic Variation in Transgenic Tritordeum Lines Grown in the Field. J. Plant Physiol. 2000, 156, 223-229. (43) Tosi, P.; Masci, S.; Giovangrossi, A.; D’Ovidio, R.; Bekes, F.; Larroque, O.; Napier, J.; Shewry, P. Modification of the low molecular weight (LMW) glutenin composition of transgenic durum wheat: Effects on glutenin polymer size and gluten functionality. Mol. Breeding 2005, 16, 113-126. (44) Pirozi, M.; Margiotta, B.; Lafiandra, D.; MacRitchie, F. Composition of polymeric proteins and bread-making quality of wheat lines with allelic HMW-GS differing in number of cysteines. J. Cereal Sci. 2008, 48, 117-122. (45) Nawrocka, A.; Krekora, M.; Niewiadomski, Z.; Mis, A. A FTIR studies of gluten matrix dehydration after fibre polysaccharide addition. Food Chem. 2018, 252, 198-206. 25



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(46) Seabourn, B.; Chung, O.; Seib, P.; Mathewson, P. Determination of secondary structural changes in gluten proteins during mixing using fourier transform horizontal attenuated total reflectance spectroscopy. J. Agric. Food Chem. 2008, 56, 4236-4243. (47) Gao, X.; Zhang, Q.; Newberry, M.; Chalmers K.; Mather, D. A cysteine in the repetitive domain of a high-molecular-weight glutenin subunit interferes with the mixing properties of wheat dough. Amino Acids 2012, 44, 1061-1071. (48) Lutz, E.; Wieser, H.; Koehler, P. Identification of disulfide bonds in wheat gluten proteins by means of mass spectrometry/electron transfer dissociation. J. Agric. Food Chem. 2012, 60, 3708-3716. (49) Washida, H.; Wu, C.; Suzuki, A.; Yamanouchi, U.; Akihama, T.; Harada, K.; Takaiwa, F. Identification of cis-regulatory elements required for endosperm expression of the rice storage protein glutelin gene GluB-1. Plant Mol. Biol. 1999, 40, 1. (50) Saumonneau, A.; Rottier, K.; Conrad, U.; Popineau, Y.; Guéguen, J.; Francin-Allami, M. Expression of a new chimeric protein with a highly repeated sequence in tobacco cells. Plant Cell Rep. 2011, 30, 1289-1302. (51) Pourfarzad, A.; Ahmadian, Z.; Habibi-Najafi, M. Interactions between polyols and wheat biopolymers in a bread model system fortified with inulin: A Fourier transform infrared study. Heliyon 2018, 4, e01017. (52) Li, X.; Liu, T.; Song, L.; Zhang, H.; Li, L.; Gao, X. Influence of high-molecular-weight glutenin subunit composition at Glu-A1 and Glu-D1 loci on secondary and microstructures of gluten in wheat (Triticum aestivum L.) Food Chem. 2016, 213, 728-734. (53) Gao, X.; Liu, T.; Yu, J.; Li, L.; Feng, Y.; Li, X. Influence of high-molecular-weight glutenin subunit composition at Glu-B1 locus on secondary and micro structures of gluten in wheat (Triticum aestivum L.). Food Chem. 2016, 197, 1184-1190. (54) Hanft, F.; Koehler, P. Quantitation of dityrosine in wheat flour and dough by liquid chromatography-tandem mass spectrometry. J. Agric. Food Chem. 2005, 53, 2418-2423.

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828

FIGURE LEGENDS

829

Figure 1. PCR analysis of the transgenic wheat plants (A) PCR amplification of the

830

CaMV35S terminator and partial target gene sequence. (B) PCR amplification of the

831

vector partial sequence and full-length sequence of the target gene. Lane M: DNA marker

832

III (A) or DNA marker DL5000 (B); lanes 1-20: genomic DNA of regenerated wheat

833

plants. Among them lanes 1-6 were genomic DNA of mut avenin-like b with myc tag,

834

lanes 7-15 were genomic DNA of mut avenin-like b without the myc tag and lanes 16-20

835

were genomic DNA of wt avenin-like b. lane 21: water for negative control; lane 22:

836

genomic DNA of ZM 9023 for negative control; lane 23: pLRPT-WT avel/ pLRPT-Mut

837

avel for positive control.

838

Figure 2. SDS-PAGE and Western blotting analyses of Avenin like b protein in

839

transgenic and control lines (A) Total proteins extracted from flours of transgenic and

840

control wheat lines were visualized on stained SDS-PAGE gel. (B) Western blotting of

841

flour from the transgenic and negative lines using anti-avenin like b polyclonal antibody.

842

The target protein was indicated on Western blotting. The housekeeping protein β-actin

843

was used as control to calibrate for equal amounts of proteins and to calculate the relative

844

loading volume for each sample. (C) Densitometry quantified for the Western blotting

845

result. Data are given as means ± SEM. * and ** indicated the significant differences with

846

the relative levels of control cultivar ZM 9023 at 0.05 and 0.01 probability level,

847

respectively. (D) Western blotting analysis of flour from the transgenic and negative

848

wheat lines using anti-myc monoclonal antibody. (E) Western blotting analysis of GMP

849

protein extracted from the transgenic and negative lines with anti-myc monoclonal

850

antibody.

851

Figure 3. Characterizations of glutenin proteins in transgenic and control lines (A)

852

SDS-PAGE of glutenin proteins extracted from transgenic and negative control lines. The

853

endogenous HMW-GS 1Dx2, 1Bx7, 1By8 and 1Dy12 were indicated by short lines on

854

the left side of the gel. (B) The relative amounts and proportions of endogenous

855

HMW-GS 1Dx2, 1Bx7, 1By8 and 1Dy12, the proportions of total HMW-GS and

856

LMW-GS were determined by densitometry analyses for all lines. Data were given as the

857

means ± SEM. Three independent analyses were conducted for all wheat lines. 27



858

Figure 4. Effects of mutant Avenin-like b and wild-type Avenin-like b on the dough

859

mixing properties Comparations of the eleven mixograph parameters among dough

860

samples of all wheat lines. */** show the comparisons between all transgenic lines and

861

negative controls (ZM 9023 and N-4) on the mixing parameters, while */** and */**

862

represent comparisons between mutant lines and line WT alb-1 and WT alb-2,

863

respectively (*P < 0.05, **P < 0.01 by Student’s t-test). Bars are mean (± SD).

864

Figure 5. Effects of mutant Avenin-like b and wild-type Avenin-like b on molecular

865

size distribution of gluten proteins in flour and GMP gel SE-HPLC analyses of flour

866

and GMP gel samples of transgenic and control lines are shown in (A) and (C), the

867

corresponding comparisons of three parameters (%F1, %F1/%F2, and (%F3+%F4)/%F1)

868

analyzed statistically in (B) and (D). */** show the comparisons between all transgenic

869

lines and negative controls (ZM 9023 and N-4) on the three parameters above-mentioned,

870

while */** and */** represent comparisons between mutant lines and line WT alb-1 and

871

WT alb-2, respectively (*P < 0.05, **P < 0.01 by Student’s t-test). Bars are mean (± SD),

872

all wheat lines were performed three times.

873

Figure 6. Effects of mutant Avenin-like b and wild-type Avenin-like b on GMP

874

particles size distribution (A) The size distribution patterns of GMP particles extracted

875

from flour samples of all wheat lines. (B) Statistical analyses of weighted average

876

diameters D[3,2] and D[4,3] of GMP particles. */** show the comparisons between all

877

transgenic lines and negative controls (ZM 9023 and N-4) on the two parameters above,

878

while */** and */** represent comparisons between mutant lines and line WT alb-1 and

879

WT alb-2, respectively (*P < 0.05, **P < 0.01 by Student’s t-test). Bars are mean (± SD),

880

all wheat lines were performed in triplicate.

881

Figure 7. Effects of mutant Avenin-like b and wild-type Avenin-like b on the

882

contents of free sulfhydryl (SHfree) and disulfide bonds (SS) in dough samples

883

Relative content of SHfree and SS in freeze-dried dough mixed to peak samples from all

884

wheat lines. Three biological experiments were performed. */** show the comparisons

885

between all transgenic lines and negative controls (ZM 9023 and N-4) on the contents of

886

SHfree and SS, while */** and */** represent comparisons between mutant lines and line 28


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Page 29 of 42


887

WT alb-1 and WT alb-2, respectively (*P < 0.05, **P < 0.01 by Student’s t-test). Bars are

888

mean (± SEM).

889


890

microstructure of dough mixed to peak by SEM The letter S in the figure refers to

891

starch granules and the G refers to the gluten network structures. Scale bar = 30 um.

892


893

secondary structures of dough mixed to peak Proportions of the five secondary

894

structures of dough determined by FT-IR. Values are given as the means of the

895

percentage of each secondary structure with three replicates.

896

Figure 10. The online prediction of disulphide bond of wild-type Avenin-like b and

897

mutant Avenin-like b The numbers indicated positions of the cysteine residues. S-S

898

indicated the intra-molecular disulfide bonds. * indicated the cysteine residues forming

899

inter-molecular disulfide bonds.

29



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Table 1. Comparisons of flour quality-related parameters of the transgenic and control wheat lines Lines Parameters ZM 9023

N-4

Flour protein content (%)a

9.52±0.01gG

9.57±0.01gG

9.98±0.01dD

10.55±0.01cC

9.86±0.00fF

9.92±0.01eE

12.02±0.01aA

11.68±0.01bB

Glutenin (µg mg-1 flour)b

7.67±0.25deCD

7.40±0.09eD

9.16±0.05bcB

8.61±0.32bcdBCD

8.41±0.16cdeBCD

8.76±0.32bcBC

11.46±0.05aA

9.51±0.27bB

Gliadin (µg mg-1 flour) b

11.71±0.59

11.56±0.02

11.99±0.16

11.25±0.42

11.75±0.17

12.44±0.19

12.73±0.13

12.16±0.21

Glutenin/gliadin

0.65±0.02cCD

0.64±0.02cD

0.71±0.03bcBCD

0.77±0.05bBC

0.72±0.02bcBCD

0.70±0.04bcBCD

0.90±0.08aA

0.78±0.03bB

Wet gluten (%)c

24.96±0.18dD

24.07±0.47dD

28.77±0.12bcC

33.82±0.30aA

30.10±0.20bB

27.65±0.08cC

34.38±0.19aA

33.82±0.03aA

Dry gluten (%) c

8.21±0.09cdD

8.05±0.26dD

9.29±0.16bCD

11.38±0.22aAB

10.16±0.16bBC

9.19±0.04bcCD

11.27±0.12aAB

11.95±0.20aA

SDS sedimentation (ml) d

38.5±0.3cD

37.9±0.1cD

50.7±0.8bB

51.1±0.6bB

48.8±1.0bBC

41.0±0.5cCD

69.7±1.4aA

67.1±1.5aA

GMP wet weight (g)

1.65±0.04cC

1.77±0.03cC

2.61±0.03bB

2.47±0.04bB

2.68±0.02bB

2.57±0.01bB

3.30±0.05aA

3.12±0.06aA

GMP concentration (mg g-1) e

7.53±0.02dD

8.61±0.38dD

11.69±0.12cC

11.64±0.26cC

14.37±0.30abAB

12.79±0.37bcBC

15.92±0.21aA

13.38±0.36bcBC

a

Mut alb-1

Mut alb-2

Mut alb-3

Mut alb-4

WT alb-1

WT alb-2

Results are expressed based on 14% moisture. b Protein contents were determined by the Dumas method with an average of three replications.

Glutenins and gliadins were determined using the Bradford assay with an average of 4 replications.

d

c

Wet gluten and dry gluten content were

determined using AACCI Method 38-12A with an average of 3 replications. e SDSS volume was determined according to AACCI Method 56-70.01 with an average of 4 replications. f GMP concentration was measured with biuret reagent with an average of 3 replications. All data are presented as


Page 31 of 42


mean ± SD. Values within the same parameter followed by the same letter are not significantly different at the 0.05 (small letter) and 0.01 (capital letter) probability levels. Significant differences between lines for each parameters were calculated using the Scheffe test by SPSS software.




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Figure 1. PCR analysis of the transgenic wheat plants 173x74mm (600 x 600 DPI)



Figure 2. SDS-PAGE and Western blotting analyses of Avenin like b protein in transgenic and control lines 173x173mm (600 x 600 DPI)


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Figure 3. Characterizations of glutenin proteins in transgenic and control lines 173x90mm (600 x 600 DPI)



Figure 4. Effects of mutant Avenin-like b and wild-type Avenin-like b on the dough mixing properties 173x115mm (600 x 600 DPI)


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Figure 5. Effects of mutant Avenin-like b and wild-type Avenin-like b on molecular size distribution of gluten proteins in flour and GMP gel 173x139mm (600 x 600 DPI)



Figure 6. Effects of mutant Avenin-like b and wild-type Avenin-like b on GMP particles size distribution 173x63mm (600 x 600 DPI)


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Figure 7. Effects of mutant Avenin-like b and wild-type Avenin-like b on the contents of free sulfhydryl (SHfree) and disulfide bonds (SS) in dough samples 83x64mm (600 x 600 DPI)



Figure 8. Effects of mutant Avenin-like b and wild-type Avenin-like b on the microstructure of dough mixed to peak by SEM 173x74mm (600 x 600 DPI)


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Figure 9. Effects of mutant Avenin-like b and wild-type Avenin-like b on the secondary structures of dough mixed to peak 173x106mm (600 x 600 DPI)



Figure 10. The online prediction of disulphide bond of wild-type Avenin-like b and mutant Avenin-like b 173x62mm (600 x 600 DPI)


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