International Journal of Biological Macromolecules 225 (2023) 701–714

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Wheat Glu-A1a encoded 1Ax1 subunit enhances gluten physicochemical

properties and molecular structures that confer superior
breadmaking quality
Junwei Zhang 1, Fei Luo 1, Haocheng Sun 1, Jian Wang, Wenjing Duan, Yueming Yan *
College of Life Science, Capital Normal University, 100048 Beijing, China

A R T I C L E I N F O A B S T R A C T

Keywords: Wheat gluten proteins serve as the largest protein molecules in nature and play key roles in breadmaking quality
Bread wheat formation. In this study, we used a pair of Glu-A1 allelic variation lines to perform a comprehensive investigation
1Ax1 subunit on the effects of Glu-A1a encoded 1Ax1 subunit on gluten physicochemical properties, molecular structures and
Gluten proteins
breadmaking quality. The results showed that the presence of the 1Ax1 subunit significantly increased gluten
Physicochemical properties
Molecular structure
content, leading to marked improvement of dough rheological properties. Meanwhile, gluten physicochemical
Breadmaking quality properties such as foaming ability and foaming stability, oil/water-holding capacity, emulsifying activity, di­
sulfide bond content, and gluten degradation temperature were significantly improved. A confocal laser scanning
microscope analysis revealed that the 1Ax1 subunit drastically enhanced gluten microstructure. Gluten sec­
ondary structure analysis by Fourier transform infrared spectroscopy and laser scanning microscope-Raman
spectroscopy indicated that 1Ax1 subunit significantly promoted β-turn and β-sheet content and reduced
α-helix content. Three-dimensional structure analysis by AlphaFold2 revealed a similar structural feature of 1Ax1
with the superior quality subunit 1Ax2*. Correlation and principal component analyses demonstrated that
α-helix and β-sheet content had a significant correlation with dough rheological properties, gluten physico­
chemical properties and breadmaking quality. Our results showed that 1Ax1 subunit positively affected gluten
molecular structure and quality formation.

1. Introduction
Wheat (Triticum aestivum L.) is an agricultural crop of global signif­
icance, and possesses a large percentage of cereal crops. Wheat also is
the main source of proteins and energy needs for a huge amount of the
world's population. Wheat flour can be used to make different kinds of
food products such as various bread, noodles, cookies, and pasta [1].
Storage protein composition and content in wheat endosperm are the
main factors to determine flour quality, which are consist of glutenins
and gliadins that can form gluten proteins linked by intra- and inter­
molecular disulfide bonds. Glutens are considered as the largest protein
molecules in nature [2]. In general, glutenins and gliadins are rapidly
synthesized and accumulated at 10–30 days after flowering [3–5],
which play a role in the formation of dough viscoelasticity and exten­
sibility. Glutenins are divided into high and low molecular weight glu­
tenin subunits (HMW-GS and LMW-GS, respectively). Despite
accounting for only about 10 % of wheat grain proteins, HMW-GS play
critical roles in dough strength and breadmaking quality [6].
HMW-GS have three encoded loci on the long arms of chromosomes
1A (Glu-A1), 1B (Glu-B1) and 1D (Glu-D1), and each locus possesses two
paralogous genes, encoding one larger x-type subunit (80–88 kDa) and
one smaller y-type subunit (67–73 kDa), respectively [1,6]. Although
common hexaploid wheat can express six HMW-GS (1Ax, 1Ay, 1Bx, 1By,
1Dx, and 1Dy) in theory, only 3–5 genes can express due to gene
silencing. The 1Bx, 1Dx and 1Dy genes generally have expression ac­
tivity while the 1Ay gene keeps silent [7]. The allelic variations at Glu-1
are closely related to dough quality, and particularly, Glu-D1a encoding
1Dx5 + 1Dy10 and Glu-A1a encoding 1Ax1 as well as LMW-GS alleles
Glu-A3a and Glu-B3h are highly related to superior breadmaking quality
[8–10].
Molecular characterization showed that HMW-GS have three typical
domains: an N-terminal domain of about 100 amino acids, a repetitive

* Corresponding author at: College of Life Science, Capital Normal University, Xisanhuan Beilu No. 105, 100048 Beijing, China.
E-mail address: yanym@cnu.edu.cn (Y. Yan).
1
Contributed equally to this work.

https://doi.org/10.1016/j.ijbiomac.2022.11.134
Received 14 July 2022; Received in revised form 14 October 2022; Accepted 13 November 2022
Available online 17 November 2022
0141-8130/© 2022 Elsevier B.V. All rights reserved.
J. Zhang et al.
domain of over 400 amino acids, and a C-terminal domain of about 40
residues [6]. The N- and C-terminal domains are non-repetitive and
contain most or all of the cysteine residues whereas the central domain
comprises tandem and interspersed repeats of short peptide motifs:
hexa- (consensus PGQGQQ), nona- (GYYPTSPQQ) and tripeptide (GQQ)
in the x-type subunits, and hexa- (PGQGQQ) and nona-peptides
(GYYPTSLQQ) in the y-type subunits [7]. The N- and C-terminal do­
mains may have globular structures while the middle domain's repeating
sequences may form a loose spiral based on β-turns that are stabilized by
hydrogen bonds between glutamine residues and is inherently elastic
[11]. In general, the x-type subunits contain four cysteine residues
(three in the N-terminal domain and one in the C-terminal domain)
while the y-type subunits have seven cysteine residues (five in the N-
terminal domain, one in the end of the repetitive domain, and one in the
C-terminal domain) [12].
Due to the complexity of their compositions and the difficulty of
crystallization, the molecular structure characteristics of wheat glutenin
subunits and gluten macropolymers are still unknown. To date, various
analysis techniques have been used to reveal the secondary structure
features of HMW-GS and gluten proteins. Circular dichroism (CD)
spectroscopy analysis revealed a similar secondary structure of the Bx7
and Bx20 subunits [13]. Nuclear magnetic resonance (NMR) analysis of
the structure and properties of the intermediate region of the glutenin
subunit showed that the 11-peptide GYYPTSPQQGA was a proline
containing two unrelated prolamines and nine peaks were observed in
the wave spectrum, suggesting the existence of two conformations of the
peptide [14]. Atomic force microscopy (AFM) analysis of the in­
teractions of the HMW glutenin subunits found an interlinked network
of filaments with a width of about 20 nm [15]. Scanning tunneling
microscopy (STM) analysis of 1Bx20 subunit demonstrated a regular
spiral structure with a diameter of about 19.5 Ǻ and a periodicity of
about 14.9 Ǻ [16].
In recent years, various strong approaches for deciphering the mi­
crostructures and secondary structure of wheat gluten proteins, which
are intimately connected to flour processing quality, have been estab­
lished. Confocal laser scanning microscope (CLSM) could visually and
quantitatively characterize the gluten microstructures with different
parameters, which provided insights into the effects of HMW-GS on
gluten structure and function [17–19]. Fourier infrared spectroscopy
(FTIR) analysis can quantitatively reveal the secondary structure fea­
tures of gluten proteins, which were beneficial for investigating the ef­
fects of HMW-GS on gluten structure [20,21]. As a powerful
spectroscopy technique for detecting the conformation of protein sec­
ondary structures, laser scanning microscope (LCM)-Raman spectros­
copy can also characterize the backbones and side chains [22]. The
variations in the amide I bands in gluten proteins from HMW-GS near-
isogenic lines by LCM-Raman spectroscopy indicated significant differ­
ences in the secondary structure. At the same time, the side chain vi­
brations of gluten also exhibited differences in the content of disulfide
bonds [23]. LCM-Raman spectroscopy was recently used to dissect the
molecular structure and function of wheat resistant starch in different
waxy protein deletion lines, demonstrating that waxy protein deletions
significantly increased the half-width and intensity at 480 cm− 1 and
produced a more ordered crystal structure of resistant starch [24].
At present, revealing the three-dimensional (3D) structures of wheat
glutenin subunits and gluten protein is still a great challenge. Early ef­
forts tried to decipher the 3D structure features of HMW-GS through
homology modelling and fold identification algorithm. The N-terminal
structural fold of the 1Dy10 subunit was found by using a fold identi­
fication algorithm [25]. In addition, a molecular model of 42 k LMW-GS
was established by gene-derived amino acid sequences, secondary
structure predictions, and potential intramolecular and intermolecular
disulfide linkages [26]. However, the effective structural information in
these studies is very limited. The recent work has reported the 3D
structure of gluten in wheat dough at millimeter and sub-micron reso­
lution by combined two-photon microscopy [27], but the internal
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International Journal of Biological Macromolecules 225 (2023) 701–714
structure still remains unclear. The recently developed protein predict­
ing tool Alphafold has provided the possibility to dissect the 3D structure
of glutenin subunits, which is the first computational method that can
regularly predict protein structures with atomic accuracy even in cases
in which no similar structure is known [28]. AlphaFold2's prediction
results generally possess low alignment errors and a higher per-residue
confidence score for more accurate predictions [28]. The AlphaFold2
protein 3D structure prediction model by using the transformer design
displayed a high accuracy and easy-to-use for wheat MATE proteins
[29]. Recently, AlphaFold2 has been used to fully predict the structure
of 350,000 proteins in the whole proteome of 21 organisms, including
humans, E. coli, fruit flies, and rice [30].
In this work, we used a pair of Glu-A1a allelic variation lines to
perform a comprehensive study on the effects of the 1Ax1 subunit on
gluten physicochemical properties, molecular structures and bread­
making quality by combining different spectroscopic techniques and
AlphaFold 3D prediction. We aim to further reveal the relationships
between structures and functions of glutenin subunits, which would
improve our understanding for the molecular mechanisms of wheat
processing quality formation.
2. Materials and methods
2.1. Plant materials and field trial
The wheat materials used in this study included common wheat
Chinese Spring (CS) substitution line CS-1Sl/1B (the 1B chromosome of
CS substituted by 1Sl from Aegilops longissima) with HMW-GS composi­
tions (Null, 2.3* + 16*, 2 + 12) [31] and its Glu-A1a allelic variation line
AVL-1Ax1 (1, 2.3* + 16*, 2 + 12) recently developed in our group
through crossing between CS-1Sl/1B and common wheat variety
CB037A (1, 17 + 18, 2 + 12) and consecutive self-crossing for seven
times combined with selection and identification. CB037A is a wheat-
Haynaldia villosa 6VS/6AL translocation line developed at the Capital
Normal University and Institute of Crop Science, Chinese Academy of
Agricultural Science, China, which carries Pm21 gene with a HMW-GS
composition of 1Ax1, 1Bx17 + 1By18 and 1Dx2 + 1Dy12, and has
high resistance to powdery mildew and superior gluten quality [32].
Both lines were planted in the experimental field of Chinese Academy of
Agricultural Science, Beijing during 2021 growing season under field
cultivation conditions. Three biological replications were used for each
material and each replication had 30 m2. Mature grain samples were
collected for later analysis.
2.2. HMW-GS extraction and SDS-PAGE
HMW-GS were extracted referred to the previous method [33]. The
albumins, globulins and gliadins were firstly extracted and excluded
from 15 mg seeds by using 75 μL distilled water, 75 μL dilute salt so­
lutions, and 120 μL 30 % ethanol, respectively. Then glutenins were
extracted by using commonly used glutenin extraction buffer (50 %
isopropanol, 80 μL Tris-HCl, pH 8.0) with 64.83 μL dithiothreitol (DTT)
(Beijing Solarbio Science & Technology Co. Ltd., Beijing, China) and 1.4
% 4-vinylpyridine (v/v) (Sigma-Aldrich Trading Co, Ltd., Shanghai,
China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed with Bio-Rad PRO-TEAN II XL equipment
(Bio-Rad Laboratories Co., Ltd., Shanghai, China) in 12 % gel and
electrophoresed at 15 mA for 2 h based on the previously described
method [34].
2.3. Quality parameter testing
Different quality parameters were determined based on the recent
report [20], including basic quality parameters and Farinograph pa­
rameters. Grain hardness was measured using Perten Single Kernel
Characterization System (SKCS) 4100 (Perten Instrument North
J. Zhang et al.
American Inc., Spring-field, IL, USA) according to the AACC approved
method 55-31. Flour moisture and ash contents (% dry basis) were
measured by using the AACC, 2000 44-15A and 08-02, respectively. The
flour protein content was tested using NIR Product Analyzer (Instalab
610, NIR Product Analyzer, Dickey-john Co. Ltd., USA) based on the
method of AACC 39-10A. Farinograph parameters were obtained by
Farinograph-AT (Brabender GmbH & Co. KG, Duisburg, Germany)
equipped with a 50 g mixing bowl and MetaBridge software. All dough
samples were prepared at optimal water absorption, and sufficient water
was added to 50 g of flour to attain a consistency of 500 ± 20 Farino­
graph Units (FU).
2.4. Preparation of gluten samples
Gluten proteins were extracted by using a hand washing method
(AACC Method 38e10.01, 2000), and then lyophilized for 48 h in a
freeze dryer. After milling to fine powder with a pestle and mortar, the
fine gluten powder was stored in a refrigerator (4 ◦ C) for later use.
2.5. Determination of gluten foaming properties
The gluten suspension (1 % w/v, 30 mL) at pH 7.0 in a measuring
cylinder (100 mL) was homogenized by using a digital homogenizer
(Ultra-Turrax T 25, IKA Co., Germany) at 15,000 rpm for 2 min.
Foaming ability (FA) was calculated as the percent increase in the vol­
ume of the suspensions upon mixing while foaming stability (FS) was
estimated as the percentage of foam remaining after 30 min [35].
2.6. Determination of gluten oil/water absorption capacity and
emulsifying activity index
Oil absorption capacity (OAC) and water absorption capacity (WAC)
of gluten proteins were tested [36]. Gluten samples (2.5 g) were mixed
with 20 mL distilled water and mustard oil in a centrifuge tube, and then
vortexed for 30 min at 25 ◦ C, respectively. The slurry was then centri­
fuged at 5000 ×g for 15 min (5810R, Eppendorf, Hamburg, Germany)
and the supernatant was decanted. The weight of the residue was tested,
expressing as weight of oil/water absorbed per gram of the sample.
Emulsifying activity index (EAI) of gluten proteins was measured by
using the method of Pearce et al. [37].
2.7. Determination of gluten surface hydrophobicity and S-S content
Surface hydrophobicity (H0) of wheat gluten was tested using the
reported method [38]. Gluten hydrophobicity was measured by bro­
mophenol blue sodium salt (Sigma-Aldrich Trading Co, Ltd., Shanghai,
China), and 200 μL of 1 mg/mL BPB (in distilled water) was added to 1
mL of gluten solution. The weak acid BPB (Beijing Solarbio Science &
Technology Co. Ltd., Beijing, China) could slightly reduce the pH of the
solution from 6 to 5.8. The control without gluten consisted of the
addition of 200 μL of 1 mg/mL BPB (in distilled water) to 1 mL of 20 mM
phosphate buffer at pH 6. Samples and control were kept under agitation
at room temperature for 10 min, and then centrifuged at 2000 ×g for 15
min. The absorbance of the supernatant (diluted 1/10) was tested at 595
nm against a blank of phosphate buffer. The amount of BPB bound was
determined using the formula: BPB bound (μg) = 200 μg × (A control −
A sample) / A control, A = absorbance at 595 nm.
The number of free sulfhydryl groups was tested using the reported
method [39]. Briefly, the Ellman's reagent DTNB (5,5′ -dithiobis-(2-
nitrobenzoic acid)) (Shanghai Macklin Biochemical Co, Ltd., Shanghai,
China) and gluten samples were dissolved in Tris-HCl buffer (86 mM
Tris, 90 mM glycine and 4 mM ethylenediamine tetraacetic acid, EDTA,
pH 8.0) to get 0.4 % (w/w) DTNB solution and 0.5 % (w/w) gluten
solution, respectively. Then, 50 μL DTNB was added to 5 mL gluten
solution. The solution was mixed and reacted for 15 min at 25 ◦ C, and
then the absorbance of solution was measured at 412 nm. The buffer was
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International Journal of Biological Macromolecules 225 (2023) 701–714
used as the reagent blank. An extinction coefficient of sulfhydryl groups
of 13,600 M− 1 cm− 1 was used to calculate the micromoles of SH/g of
protein. The total sulfhydryl groups in the proteins were measured using
the reported method [40]. The content of S-S was calculated by (Q1 −
Q2) / 2, in which Q1 and Q2 represented the content of total SH and free
SH, respectively.
2.8. Thermogravimetric analysis
Thermogravimetric analysis (TGA) of the gluten proteins was con­
ducted on a Mettler Toledo Star TGA unit (Mettler Toledo Corp., Zurich,
Switzerland). Argon was used as a purge gas (mL/min), and freeze dried
gluten samples (2–5 mg) were put in oxide aluminum pans and heated
from 20 ◦ C to 900 ◦ C at a heating rate of 10 ◦ C/min. The degradation
temperature (Td) and weight loss at 600 ◦ C were determined by ORIGIN
(version 9.0 PRO, OriginLab Corp., USA).
2.9. Gluten microstructure observation
Gluten microstructures were observed based on Gao et al. [21].
Gluten samples (10 g) and rhodamine B solution (6 mL, 0.01 mg/mL)
were kneaded together to form dough, and a tiny dough sample was put
on the glass slide and pressed by the coverslip. The gluten samples were
observed by using FV1000 confocal laser scanning microscope (CLSM,
Olympus, Tokyo, Japan), and images (512 × 512 pixels and 317 × 317
μm) were captured for each sample. The gluten microstructures were
quantitatively characterized by AngioTool software with the parameters
of protein area, protein percentage area, protein junctions, junction
density, protein length, protein end-points, end-point rate, lacunarity
and branching rate.
2.10. Fourier transform infrared spectroscopy (FTIR)
The gluten FTIR spectra of seven dietary fibres were recorded with a
Nicolet 6700 FTIR spectrometer (Thermo Scientific, Madison, WI, USA)
equipped with a diamond attenuated total reflectance attachment ac­
cording to Liu et al. [20]. The FTIR spectra were obtained between 4000
and 400 cm− 1 at 4 cm− 1 intervals. Each spectrum resulted from 128
scans to obtain an optimal signal-to-noise ratio. Gluten secondary
structures were deconvoluted from the amide I region (1600–1700
cm− 1) via the secondary derivation. Gaussian curve fitting was used
OMNIC software (v. 8.2, Thermo Fischer Scientific Inc., Madison, WI,
USA).
2.11. Laser scanning microscope (LCM)-Raman spectroscopy
Gluten LCM-Raman spectra were recorded by using a Raman Spec­
trometer with a 50 × lens LCM-Raman spectra (HORIBA XPLORA PLUS,
Horiba Jobin-Yvon, Villeneuve d'Ascq, France) according to Chang et al.
[24] and Wang et al. [41], with minor modifications. Gluten samples
were excited at 785 nm by an argon-ion laser with the laser power 100
mW. The laser wavelength was calibrated at 520.7 nm with mono­
crystalline silicon for improving sample testing accuracy. Three different
spots of the Raman spectra were tested in the range of 400–2000 cm− 1.
Each spectrum was collected for 60 s exposure time, with 2 cm− 1 reso­
lutions. According to the previous report [42], the amide I bands were
assigned as follows: intermolecular β-sheets due to protein aggregation
(1612–1614 cm− 1), β-sheets (1618–1640 cm− 1), α-helix (1650–1660
cm− 1), β-turns (1660–1670 cm− 1) and antiparallel β-sheets (1675–1695
cm− 1). As the major vibrational motions of the side chains, inter-chain
disulfide bands (497 cm− 1), tryptophan bands (760 cm− 1) and tyro­
sine bands (I850/830) were analyzed by comparing with Raman data
reported [43,44]. The samples in three replicates were collected and the
mean values are reported.
J. Zhang et al.
2.12. Three-dimensional (3D) structure prediction by AlphaFold2
The 3D structures of HMW-GS were predicted by AlphaFold2
(DeepMind, London, UK) recently developed by Jumper et al. [28]. The
amino acid sequences of HMW-GS were used for sequence alignments/
templates by MMseqs2 and HHsearch, and protein structures and com­
plexes were predicted by AlphaFold2 and Alphafold2-multimer. Simu­
lations were carried out for 5 cycles. In this study, the 3D protein
prediction model was evaluated in terms of three parameters: confi­
dence score per residue, number of matched template sequences and
prediction alignment error. Then, editing was conducted by Pymol
software (version 1.7.4 Schrödinger, Warren Lyford DeLano, New York
City, NY, USA).
2.13. Breadmaking quality testing
Bread baking and evaluation were carried out following Sun et al.
[45], including loaf volume and appearance score and C-Cell parameters
related to bread inner structures. Image analysis of crumb bread was
performed using C-Cell image analysis software and equipment (Calibre
Control International Ltd.; Warrington, UK).
2.14. Statistical analysis
Data collected from three replicate experiments were used for sta­
tistical analyses with SPSS (version 26.0, SPSS Inc., Chicago, IL, USA).
Student's t-test was used to determine significant differences among
different samples. Pearson's correlation coefficients were calculated to
determine the relationship among basic quality parameters, dough
rheological properties, gluten physicochemical properties, gluten mo­
lecular structure and breadmaking quality. Principal component anal­
ysis (PCA) of different parameters was performed on Tutools platform
(http://www.cloudtutu.com), a free online data analysis website ac­
cording to Zheng et al. [46].
3. Results and discussion
3.1. Grain protein identification of CS-1Sl/1B and AVL-Ax1
SDS-PAGE analysis of grain glutenin proteins showed that both CS-
1Sl/1B and AVL-Ax1 had same HMW glutenin subunit compositions at
Glu-B1 (1Bx2.3* + 1By16*) and Glu-D1 (1Dx2 + 1Dy12), but differed in
Glu-A1 locus. AVL-Ax1 expressed 1Ax1 subunit encoded by GluA1a at
Glu-A1, which was absent in CS-1Sl/1B substitution line. In addition,
both CS-1Sl/1B and AVL-Ax1 had similar LMW-GS compositions at Glu-3
loci (Fig. S1). These results indicated that CS-1Sl/1B and AVL-Ax1 were
a pair of Glu-A1a allelic variation lines and suited for investigating the
effects of Glu-A1a allele on gluten structures and functional properties.
Table 1
Comparison of main quality parameters between CS-1Sl/1B and AVL-1Ax1.
Materials Basic quality parameters
Protein content (%) Moisture content (%) Ash content (%)
l
CS-1S /1B 13.15 ± 0.02 14.44 ± 0.25 0.49 ± 0.005
NIL-Ax1 14.12 ± 0.09* 14.97 ± 0.01 0.65 ± 0.01**
Materials Farinograph parameters
Water absorption (500BU) Development time (min) (500BU)
CS-1Sl/1B 55.55 ± 0.05 3.1 ± 0.4
NIL-Ax1 57.05 ± 0.05* 4.25 ± 0.25**
The data were expressed as the mean values ± standard deviations.
*
Significant at the 0.05 probability level.
**
Significant at the 0.01 probability level.
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International Journal of Biological Macromolecules 225 (2023) 701–714
3.2. Effects of 1Ax1 subunit on basic quality parameters and dough
rheological properties
The basic quality parameters of CS-1Sl/1B and AVL-Ax1 included
protein content, moisture content, ash content, gluten over sieves, total
gluten content, gluten index, and grain hardness. The results showed
that AVL-Ax1 containing the 1Ax1 subunit had a significant increase in
protein and ash content, grain hardness, gluten over sieves, total gluten,
and gluten index compared to CS-1Sl/1B lacking the 1Ax1 subunit
(Table 1). The protein content is a measure of flour gluten, and higher
protein content generally leads to stronger gluten and superior bread­
making quality [47]. Ash refers to the mass ratio of inorganic impurities
after burning wheat grains to pre-combustion, and studies demonstrated
that the ash content of wheat grain and flour was positively related to
protein and gluten content [48]. The gluten index indicates the ratio of
gluten over sieves to total gluten content, and gluten strength grows
stronger as the gluten index rises [49]. Therefore, our results indicated
that the presence of the 1Ax1 subunit had a positive effect on grain
quality.
The main parameters reflecting dough rheological properties were
tested by Farinograph-E, including consistency, water absorption,
development time, stabilization time, and degree of softening. The re­
sults showed that all Farinograph parameters of AVL-Ax1 were signifi­
cantly promoted, including increased dough consistency, water
absorption, development time and stabilization time, and decreased
degree of softening (Table 1). The consistency refers to the degree to
which the dough surface resists deformation caused by external forces
[50], and the greater consistency generally causes better dough exten­
sibility. The water absorption rate is associated with the elasticity of the
dough, and higher water absorption rate of flour generally has better
elasticity and stronger dough [51]. The development time of the dough
represents the time to reach the consistency of 500 Brabender Units
(BU), reflecting a positive indication of elasticity and gluten content
[52]. The stability time of the dough, referring to the time difference
from the first arrival at 500 BU of the farinograph curves to departure
the 500 BU, reflects the resistance of the dough [53]. The degree of
softening related to the difference between the consistency of the peak
and the peak of 12 min later in the Farinograph curves, negatively re­
lates to gluten strength [54]. Our results confirmed that 1Ax1 subunit
significantly promoted grain protein and gluten content and dough
rheological properties (Table 1).
3.3. Effects of 1Ax1 subunit on gluten physicochemical properties
The gluten physicochemical properties of CS-1Sl/1B and AVL-Ax1
were detected, including foaming ability, foaming stability, oil-holding
capacity, water-holding capacity, emulsifying activity index, surface
hydrophobicity, S-S content, weight loss, and degradation temperature
Gluten over sieves (%) Total gluten (%) Gluten index Grain hardness
2.27 ± 0.03 4.71 ± 0.01 48.22 ± 0.49 68 ± 18
3.34 ± 0.04** 3.77 ± 0.01** 88.75 ± 0.89** 99 ± 16**
Stability time (min) (500BU) Softening degree (BU)
3.85 ± 0.05 146 ± 2
9.35 ± 0.35** 51 ± 4**
J. Zhang et al.
(Td). The foaming properties of wheat gluten proteins can reflect that
gluten content and the protein molecular structures are rearranged to
form bubbles during mixing [55]. The results showed that the gluten
proteins from AVL-1Ax1 have more pores and a tight and continuous
internal structure, creating more air bubbles, and significantly promoted
foaming ability and foaming stability (Fig. 1A–B). The oil/water-holding
capacity of AVL-1Ax1 glutens was also higher than those of CS-1Sl/1B
(Fig. 1C–D), probably resulting from the increased pores and structural
stability that facilitate the penetration of water and oil and produced the
improved gluten 3D network structures more effective for water
adsorption and water/oil-holding capacity during storage [56]. Oil-
holding capacity represents the ability of proteins to retain lipids
through binding action with free fatty acids, which is associated with the
flavor of processed such as bread [57].
Surface hydrophobicity (H0) analysis indicated that the presence of
the Ax1 subunit caused a significant decrease of gluten H0 (Fig. 1E),
which might be resulted from the changes in gluten molecular structures
[58]. When the Ax1 subunit was present, the gluten structures become
tightly ordered and fewer hydrophobic residues within the gluten pro­
tein were exposed to the surface of the protein molecule, thereby
reducing the surface hydrophobicity of the gluten. The surface hydro­
phobicity is related to the protein conformational changes, and the
gluten proteins with low free sulfhydryl content generally have weak
surface hydrophobicity [59]. At the same time, the 1Ax1 subunit
significantly increased emulsifying activity index (EAI) of gluten pro­
teins (Fig. 1F). Protein emulsification reflects the protein solubility, and
higher protein solubility generally causes higher emulsifying activity.
Fig. 1. Physicochemical properties of the gluten proteins from wheat CS-1Sl/1B and AVL-1Ax1.
(A) FA: foaming ability; (B) FS: foaming stability; (C) OBC: oil-binding capacity; (D) WHC: water-holding capacity; (E) H0: surface hydrophobicity; (F) EAI:
emulsifying activity index; (G) SH: sulfhydryl group; (H) Total SH: total sulfhydryl group; (I) -S-S: disulfide bond; (J) thermogravimetric profile; (K): Weight loss at
600 ◦ C; (L) Degradation temperature (Td).
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International Journal of Biological Macromolecules 225 (2023) 701–714
The Ax1 subunit might improve the formation of a more regular gluten
structure, resulting in higher solubility, greater surface area of gluten
proteins, and improved emulsifying activity [60].
Sulfhydryl groups have important roles in maintaining protein sec­
ondary structure [61], and the content of disulfide bonds shows a pos­
itive correlation with the quality of the dough products [62]. The results
showed that the Ax1 subunit significantly reduced free sulfhydryl con­
tent and promoted total sulfhydryl content and disulfide bond content
(Fig. 1G–I). A disulfide bond is a covalent bond created by the oxidation
of two cysteine sulfhydryl groups and functions in maintaining the
three-dimensional structure and biological activity of proteins [56].
Disulfide bonds can be formed within the same peptide chain or between
different peptide chains, and the number and dispersion of sulfhydryl
groups affect the state of gluten proteins, which is closely related to the
quality of the dough [63,64]. The gluten proteins from CS-1Sl/1B
without 1Ax1 subunit had a higher level of free sulfhydryl groups and
higher surface hydrophobicity, which may be related to the fact that
protein molecules at higher levels of free sulfhydryl groups were
unfolded to a greater extent and more hydrophobic residues were
exposed on the surface of the protein molecules, which in turn caused an
increase in surface hydrophobicity.
Further thermogravimetric analysis showed that the 1Ax1 subunit
significantly reduced the weight loss and promoted gluten degradation
temperature (Td) (Fig. 1J–L). Initial weight loss in the gluten samples at
60–150 ◦ C is connected with the loss of free and bound water with
increasing temperature. Further increase in the temperature caused
breakage of the covalent peptide bonds, disulfide bridges, and O–N and
J. Zhang et al. International Journal of Biological Macromolecules 225 (2023) 701–714

O–O linkages, leading to decomposition of the gluten proteins. The
gluten thermal properties can reflect the extent of proteins' tertiary
conformation [65]. Changes in the weight loss provided information
about the structure of the gluten network based on Khatkar et al. [66].
An increase in the parameter value suggested the formation of the more
open and weak gluten structure whereas a decrease is resulted from the
more compact and stronger gluten structure. Thus, the gluten protein
structures in AVL-1Ax1 became continuous and more compact, which
could be hardly damaged at high temperatures. This finding was in
general agreement with the previous report: changes in the secondary
structure and microstructure of gluten could affect the thermal stability
of gluten [24].
3.4. Effects of 1Ax1 subunit on gluten microstructure
The freshly-washed gluten samples from CS-1Sl/1B and AVL-Ax1
were detected with CLSM. The gluten proteins from both lines showed
sponge-like structures with significant differences in pore size.
Compared with CS-1Sl/1B, the gluten proteins from AVL-1Ax1 had a
smaller pore size and a significantly tighter structure, and the “fibrous
rope” protein strands in the gluten were more tightly cross-linked
(Fig. 2).
The quantitative analysis of the gluten microstructure showed that
1Ax1 subunit significantly increased the protein area, protein area
percentage, number of junctions, junction density, total protein length,
and lacunarity of the gluten (Table 2). Higher protein area indicates less
aperture area, reflecting that 1Ax1 subunit produced a more compact
gluten network, consistent with the CLSM observation (Fig. 2). The
junction density and branching rate were calculated from protein
junctions according to the previous report [19]. Fewer protein junctions
formed in the gluten network reflect fewer cross linkages formed in the
gluten network, and protein end-points represent the open-ended

Fig. 2. Protein network analysis of the gluten proteins from CS-1Sl/1B and AVL1-Ax1 detected by confocal laser scanning microscopy (CLSM).
(A) Original CLSM pictures of gluten samples from CS-1Sl/1B and AVL1-Ax1,with the scale bar represents 50 μm; (B) the row pictures analyzed by Angio-Tool; (C) the
partial enlargement with junctions shown in white, protein skeleton shown in green and protein area shown in yellow.

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Table 2
Network parameters of gluten proteins from CS-1Sl/1B and AVL-1Ax1 determined by AngioTool.
Materials Protein area Protein Number of Junction Total protein End-points End-points Branching rate Lacunarity
(×104 μm2) percentage area junctions (×102) density length (×103μm) (×102) rate (×10− 3) (×10− 3)
(×10− 3)

CS-1Sl/ 33.16 ± 2.13 0.32 ± 0.02 9.17 ± 0.57 0.88 ± 0.05 35.24 ± 0.49 8.10 ± 2.19 ± 0.07** 3.08 ± 0.35* 0.10 ±
1B 0.45** 0.01
NIL-Ax1 36.95 ± 1.94* 0.35 ± 0.02* 11.34 ± 0.78** 1.09 ± 0.08** 38.25 ± 1.65** 6.6367 ± 2.00 ± 0.06 2.77 ± 0.01 0.12 ±
0.25 0.01*

The data were expressed as the mean values ± standard deviations.

*
Significant at the 0.05 probability level.
**
Significant at the 0.01 probability level.

protein threads. At this point, 1Ax1 subunit could improve the gluten
microstructure and dough quality, consistent with the results of the
rheological properties (Table 1).
3.5. Effects of 1Ax1 subunit on gluten secondary structure and side-chain
structure
Gluten secondary structure changes between CS-1Sl/1B and AVL-
1Ax1 were detected by FTIR (Fig. S2A). The wave number of the
amide I band was shifted, indicating some variations were present in the
secondary structure content of gluten proteins between CS-1Sl/1B and
AVL-1Ax1 (Fig. S2B). Five components related to gluten secondary
structures were obtained by deconvoluting the amide I band, and the
relative area sizes of the five spectra peaks differed between CS-1Sl/1B
and AVL-1Ax1 glutens (Fig. 3A–B). The secondary structures were
expressed as percentages of the corresponding area relative to the total
amide I band area (Fig. 3C). The β-sheets were found to be dominant in
both gluten samples, but AVL-1Ax1 glutens had more intermolecular
β-sheets, β-sheets, β-turns, and antiparallel β-sheets, and less α-helix
content and α-helix/β-sheet ratio. These results indicated that the 1Ax1
subunit could produce significant changes in gluten secondary structure.
The β-sheets were closely associated with protein networks and the
β-sheet content was positively correlated with the viscoelasticity of
dough [67]. Thus, the 1Ax1 subunit enhanced gluten secondary struc­
ture by increasing intermolecular β-sheets and antiparallel β-sheets and
decreasing α-helix content and α-helix/β-sheet ratio, which provided a
structural backbone in the gluten network of the dough.
To quantify the secondary and amino acids side-chain structures
reflecting the protein backbone conformation, we further used LCM-
Raman spectroscopy to detect gluten secondary structure changes
caused by 1Ax1 subunit. Amide I bands in 400–2000 cm− 1 of LCM-
Raman spectroscopy showed the intensity changes of each wave num­
ber between CS-1Sl/1B and AVL-1Ax1 (Fig. 4A). The results from amide
I bands (1600–1700 cm− 1) showed that the glutens from AVL-Ax1
showed a slight shift towards higher wave numbers at 1659 cm− 1
compared to CS-1Sl/1B glutens at 1657 cm− 1 (Fig. 4B), which could
reflect the secondary structure changes of the gluten proteins. Further
quantitative analysis of gluten secondary structures at this region
showed similar results with FTIR (Fig. 4C–D). The presence of 1Ax1
subunit significantly increased the content of intermolecular β-sheets,
β-sheets, β-turns and antiparallel β-sheets and reduced α-helix content
(Table 3). It is known that the gluten secondary structure is closely
associated with gluten physicochemical properties and dough rheolog­
ical characteristics. In particular, the content of β-sheets and α-helices is
positively and negatively related to dough viscoelasticity, respectively
[68].
The amino acids side-chain structure changes including inter-chain
disulfide bands, tryptophan bands and tyrosine bands in the gluten
proteins demonstrated that Ax1 subunit significantly increased the in­
tensities of disulfide and tryptophan bands and intensity ratio I850/830

Fig. 3. FTIR spectroscopy analysis of the secondary structure percentages of gluten proteins in CS-1Sl/1B and AVL-1Ax1.
(A) Amide I band fitting peak of gluten IR spectroscopy from CS-1Sl/1B; (B) amide I band fitting peak of gluten IR spectroscopy from AVL-1Ax1; (C) secondary
structure percentages of gluten proteins from CS-1Sl/1B and AVL-1Ax1.

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Fig. 4. CLSM-Raman spectra of the secondary structure and side chain vibrations of gluten proteins from CS-1Sl/1B and AVL-1Ax1.
(A) Profile of LCM-Raman spectrogram band patterns at 400–2000 cm− 1; (B) amide I band of the glutens extracted from LCM-Raman spectroscopy; (C) second
derivative fitted peaks of amide I bands of CLSM-Raman spectroscopy from CS-1Sl/1B glutens; (D) second derivative fitted peaks of amide I bands of LCM-Raman
spectroscopy from AVL-Ax1 glutens; (E) normalized intensity of inter-chain disulfide band appeared at 497 cm− 1; (F) normalized intensity of tryptophan band
appeared at 760 cm− 1; (G) intensity ratios I850/830 indicating tyrosine residues. Normalized intensity values correspond to the mean values of three independent
experiments. Vertical bars represent confidence intervals at p < 0.05.

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Table 3
Gluten secondary structure percentages determined by CLSM-Raman spectroscopy.
Materials Secondary structure

α-Helixes Intermolecular β-sheets β-Sheets β-Turns Antiparallel β-sheets

CS-1Sl/1B 59.84 ± 1.97228** 3.0533 ± 0.66531 13.5133 ± 0.64501 12.53 ± 0.57689 7.9767 ± 0.53529
AVL-Ax1 48.8167 ± 0.40501** 4.5433 ± 0.32192* 17.44 ± 1.24531** 16.5733 ± 1.13094* 11.6733 ± 0.67337**

The data were expressed as the mean values ± standard deviations.

*
Significant at the 0.05 probability level.
**
Significant at the 0.01 probability level.

(Fig. 4E–G). The inter-chain disulfide bands at 497 cm− 1 can reflect the
strength of intermolecular disulfide cross-links [42]. The tryptophan
bands at 760 cm− 1 can be used as an indicator of the strength of
hydrogen bonding, and I850/830 ratio is a monitor of the hydrogen
bonding of the phenolic hydroxyl group [69]. An increase in I850/830
ratio was reported to reflect a decrease in buriedness of tyrosyl residues
in inter- or intra-molecular interactions [70]. Thus, the significantly
promoted intensities of disulfide and tryptophan bands and intensity
ratio I850/830 by Ax1 subunit could enhance dough mixing properties,
consistent with the previous study [44].
3.6. 3D structure analysis of 1Ax1 subunit by AlphaFold2
The 3D structures of 1Ax1 subunit with 830 amino acid residues
were predicted by AlphaFold2 and 1Ax2* subunit encoded by the same
Glu-A1 locus with 815 amino acid residues was used as control, which
was considered as a superior quality subunit [3,71]. The results showed
that Alphafold2 predictions with five cycles for 1Ax1 and 1Ax2* sub­
units were almost identical, including three indicators alignment errors,
per-residue confidence score, and matching template (Fig. S3A–C). The
1Ax1 and 1Ax2* subunits had different confidence scores in 3D structure
models after five cycles of prediction, which were divided into five

Fig. 5. Comparative analysis of the predicted three-dimensional structure of HMW-GS 1Ax1 and 1Ax2* by AlphaFold2.
(A) Top rank of the predictive model for the 3D structure of 1Ax1 and 1Ax2 subunits; (B) localized view of the 3D structure of 1Ax1 and 1Ax2 subunits.

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different ranks according to their scores (Fig. S4A–B). The highest level
for both subunits is shown in Fig. 5A, and the confidence score for 1Ax1
was 34.9, slightly larger than 1Ax2* (34.4). Similarly, both 1Ax1 and
1Ax2* had a typical 3D structure: two non-repetitive structural domains
(N-terminus and C-terminus) with a repetitive fragment in the middle
and a large number of α-helices at the N- and C-terminus (Fig. 5B).
Interestingly, both 1Ax1 and 1Ax2* subunits usually contained four
(three in the N-terminal domain and one in the C-terminal domain)
cysteine residues. The cysteines in both subunits did not form intra­
molecular disulfide bonds, and they might form disulfide bonds with
other glutenins or gliadins to further form gluten macropolymers [72].
This result is consistent with the recently reported x-type subunit
1Ax2.1* [73]. Combined with the results of disulfide bonds and free
sulfhydryl groups (Fig. 1), 1Ax1 subunit could form more disulfide
bonds in the gluten, resulting in a decreased free sulfhydryl groups and
improved gluten strength.
3.7. Effects of 1Ax1 subunit on breadmaking quality
The breadmaking quality of AVL-Ax1 and CS-1Sl/1B was detected
and AVL-Ax1 showed significantly promoted loaf volume (Fig. 6A) and
improved bread texture (Fig. 6B). In particular, the loaf volume of AVL-
Ax1 was up to 933 mL3, increasing by 43.54 % compared to CS-1Sl/1B
with only 650 mL3. The inner structure score of AVL-Ax1 was 33, also
much higher than CS-1Sl/1B (9). Ultimately, the loaf score of AVL-Ax1
was 94, almost twice more than CS-1Sl/1B (Table 4).
C-Cell image and parameter analyses were further performed to
evaluate the inner structural features of the bread slices in AVL-Ax1 and
CS-1Sl/1B, including slice brightness, slice area, wrapper length,
attenuation ratio, cell contrast, volume of course cell, average cell
elongation, cell diameter, number of cells, and cell density (Table 4).
Some parameters such as slice brightness, slice area, wrapper length, cell
contrast, number of cells and average cell elongation are positively
related to bread texture while the others such as cell diameter, wall
thickness, and volume of course cell have negative effects on bread
Fig. 6. Loaf performance of CS-1Sl/1B and AVL-1Ax1.
(A) The loaf baking images; (B) C-Cell structures.
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International Journal of Biological Macromolecules 225 (2023) 701–714
quality [74]. The results indicated that the existence of 1Ax1 subunit
significantly improved most of the C-Cell parameters, including slice
brightness, slice area, wrapper length, attenuation ratio, cell contrast,
number of cells, and average cell elongation related to inner structures.
The recent study has shown that lacking of 1Ax1 subunit displays
significantly lower values in protein junctions, junction density and
branching rate, indicating less protein junctions formed in the gluten
network [75]. Thus, 1Ax1 subunit improved breadmaking quality by
enhancing gluten microstructures and molecular structures that confer
superior dough rheological properties, gluten physicochemical proper­
ties, loaf volume and inner structures.
3.8. Pearson correlation and principal component analysis of quality
related parameters
The Pearson correlation matrix of 51 wheat quality and gluten
structure related parameters is shown in Fig. 7 and Table S1. The results
demonstrated that most of the parameters related to basic quality pa­
rameters, dough rheological properties, gluten physicochemical prop­
erties and molecular structures, and breadmaking quality had significant
correlations. Some key quality parameters were significantly positively
correlated with loaf volume, inner structure and loaf score, such as
gluten content, dough development time and stability time, oil-binding
capacity and foaming stability. In particular, the gluten secondary
structure parameters were significantly correlated with the parameters
related to dough rheological properties, gluten physicochemical prop­
erties and breadmaking quality. For example, the content of intermo­
lecular β-sheets, β-sheets, β-turns and antiparallel β-sheets was
significantly and positively correlated with loaf volume, inner structure
and loaf score, and most of the other quality related parameters.
Contrarily, the content of α-helixes was negatively related to most
quality parameters. These results confirmed that the α-helix and β-sheets
had important effects on dough rheological properties, gluten physico­
chemical properties and breadmaking quality and can be used as key
quality predictors.
The PCA-based chemometric multivariate analysis of 51 wheat
quality and gluten related parameters was carried out (Fig. 8). The re­
sults showed that the sum of principal components (PCA1 and PCA2)
was >90 %, indicating a high correlation between these parameters as
shown in Pearson correlation analysis above. PCA1 and PCA2 repre­
sented most of the information on quality parameters, gluten molecular
structure and physicochemical properties. As shown in Table S2, the
contribution of PCA1 was 58.99 %, which mainly integrated the infor­
mation related to wheat quality and gluten molecular structures,
including gluten content, dough development time and stability time,
softening degree, loaf volume, inner structures, loaf score, gluten
microstructure and secondary structure. The contribution of PCA2 was
36.40 %, which mainly integrated the information of gluten physico­
chemical properties such as oil- and water-binding capacity, foaming
stability, total sulfhydryl group, disulfide bond, and grain hardness.
These PCA results indicated that the presence of 1Ax1 subunit could lead
to significant alterations of main gluten molecular structure and wheat
quality.
4. Conclusions
Glu-A1a encoded 1Ax1 subunit enhanced grain protein and gluten
content and dough rheological properties, including significantly pro­
moted dough consistency, water absorption, development time and
stabilization time, and reduced softening degree. Meanwhile, the pres­
ence of 1Ax1 subunit positively promoted gluten physicochemical
properties such as significantly increased foaming ability and foaming
stability, oil/water-holding capacity, emulsifying activity, disulfide
bond content, and gluten degradation temperature, and decreased
gluten surface hydrophobicity and weight loss. Gluten microstructure
analysis showed that 1Ax1 subunit significantly enhanced the protein
J. Zhang et al. International Journal of Biological Macromolecules 225 (2023) 701–714

Table 4
Comparison of main breadmaking quality parameters between CS-1Sl/1B and AVL 1Ax1.
Materials Loaf parameters C-Cell parameters
3
Loaf volume (mL ) Inner structures (35) Loaf score Slice brightness Slice area/px Wrapper length/px Attenuation ratio

CS-1Sl/1B 650 ± 7.4 9±1 43 ± 2 141.85 ± 0.55 231,100 ± 1287 1793 ± 8.2 66.86 ± 0.33
NIL-Ax1 933 ± 3.8** 33 ± 3** 94 ± 3** 151.05 ± 0.25** 305,161 ± 2471** 2094.5 ± 2.5** 69.37 ± 0.08*

Materials C-Cell parameters

Cell contrast Volume of course cell Average cell elongation Cell diameter/px Number of cells Cell density Wall thickness/px
l
CS-1S /1B 0.725 ± 0.001 10.88 ± 0.34 1.54 ± 0.01 15.45 ± 0.44 2504 ± 16 0.011 ± 0.0001 3.34 ± 0.02
NIL-Ax1 0.765 ± 0.001* 10.19 ± 0.36 1.73 ± 0.02** 15.81 ± 0.67 3637 ± 31** 0.012 ± 0.0001 3.18 ± 0.01*

The data were expressed as the mean values ± standard deviations.

*
Significant at the 0.05 probability level.
**
Significant at the 0.01 probability level.

Fig. 7. Pearson correlation coefficients among 51 parameters related to gluten quality from CS-1Sl/1B and AVL-1Ax1.
*Red indicates a positive correlation, green indicates a negative correlation, the shade of the colour indicates the magnitude of the correlation coefficient. *Sig­
nificance at the 0.05 probability level; **Significance at the 0.01 probability level. ***Significance at the 0.001 probability level.
PC, protein content; MC, moisture content; AC, ash content; GOS, gluten over sieves; TG, total gluten; GI, gluten index; GH, grain hardness; WA, water absorption;
DDT, dough development time; DST dough stability time; SD, softening degree; SB, slice brightness; SA, slice area; WL, wrapper length; AR, attenuation ratio; CC, cell
contrast; CCV, volume of course cell; ACE, average cell elongation; CD, cell diameter; CN, number of cells; CDE (CD1), cell density; WT, wall thickness; LV, loaf
volume; IS, inner structures; LS, loaf score; PA, protein area; PPA, protein percentage area; JN, number of junctions; JD, junction density; TPL, total protein length;
EP, end-points; EPR, end-points rate; BR, branching rate; LA, lacunarity; α-H, α-helixes; β-I, intermolecular β-sheets; β-S, β-sheets; β-T, β-turns; β-A, antiparallel
β-sheets; S-S (497 cm− 1), inter-chain disulfide band appeared at 497 cm− 1; Trp, tryptophan band; Tyr, tyrosine residues; OBC, oil-binding capacity; WHC, water-
holding capacity; FA, foaming ability; FS, foaming stability; H0, surface hydrophobicity; EAI, emulsifying activity index; SH, sulfhydryl group; TSH, total sulfhy­
dryl group; DB (-S-S), disulfide bond.

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CRediT authorship contribution statement

JZ, FL and HS, performed the experiments, data analysis, and wrote
the paper. JW and WD performed the field trial and protein identifica­
tion. YY designed and supervised the experiments.

Declaration of competing interest

This manuscript has no financial or non-financial competing

interests.

Acknowledgments

We sincerely thank Dr. Hui Sun and Liu Chang in the laboratory of
National Food and Strategic Reserves Administration of the people's
Republic of China for the help of wheat quality analysis.

Funding

This research was financially supported by the grant from the Na­
tional Natural Science Foundation of China (31971931).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.ijbiomac.2022.11.134.
Fig. 8. Principal component analysis (PCA) loading describing relationships
among 51 parameters related to gluten quality from CS-1Sl/1B and AVL-1Ax1.
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