High-molecular-weight glutenin subunits (HMW-GS) play a key role in determining wheat processing quality, but their contribution to NCSB quality has not been systematically elucidated. In this study, near-isogenic lines and chromosome substitution lines differing in HMW-GS compositions at the Glu-B1 and Glu-D1 loci were utilized to comprehensively assess the impact of gluten proteins on gluten microstructure, flour functionality, and NCSB quality through multiple analytical methods. Unextractable polymeric protein content (%UPP), disulfide bond content, TGA, rapid viscosity analyzer (RVA) profiles, gluten aggregation behavior, and steamed bread-making trials were used to evaluate gluten structure and functional performance. CLSM and LCM-Raman analyses demonstrated that CB037B and SL/B significantly increased junction density, protein network area, and β-sheet content, while reducing lacunarity and α-helix content. Steamed bread-making trials combined with dough rheological assessments revealed that although CB037B exhibited strong-gluten characteristics, the resulting steamed bread quality was lower than that of CB037A, supporting the concept that superior NCSB quality does not necessarily depend on higher gluten strength. TGA further indicated that the weight loss pattern at 150°C (CB037A>CB037B>SL/B>CS) aligned with steamed bread quality rankings, whereas weight loss at 600°C corresponded more closely with conventional bread quality. Overall, this work provides valuable insights into the structure–function relationships of HMW-GS in wheat and introduces novel indicators for predicting both steamed bread and bread quality, offering guidance for breeding programs aimed at improving wheat processing performance in northern China.

Seed size is regulated by the coordinated growth of the seed coat, embryo, and endosperm, and is modulated by multiple factors. Plant hormones, sugars, and cell cycle-related processes play key roles in this regulation. In this study, we demonstrate that overexpressing the endosperm-specific DOF transcription factor gene TaDOF6 significantly enhances the accumulation of sugars and gibberellin (GA3) in grains during the grain-filling stage. RNA sequencing (RNA-seq), quantitative real-time PCR (RT-qPCR), yeast one-hybrid (Y1H), electrophoresis mobility shift assay (EMSA), and dual-luciferase assays further confirmed that TaSWEET13h is a direct downstream target of TaDOF6. Structural and functional analyses identified TaSWEET13h as a multifunctional cell membrane-localized transporter that transports diverse soluble sugars and GA3. Notably, molecular dynamics (MD) simulations and in vitro assays revealed that hydrophobic interactions among non-polar amino acids primarily drive the transport of sucrose and GA3. Therefore, these findings elucidate the genetic regulatory network involving SWEET sugar transporters in grain size control and highlight promising targets for high-yield wheat breeding.

Wheat (Triticum aestivum L.) is one of the most important food crops globally, and its flour can be processed into a wide variety of foods. The high-molecular-weight glutenin subunits (HMW-GSs) play a crucial role in determining the flour-processing quality. In this study, we used the CRISPR/Cas9 system to generate eight types of wheat mutants with the silencing of one to four HMW-GS-encoding genes simultaneously. These mutations were identified in the T1 generation by PCR-restriction enzyme (PCR-RE) analysis and sequencing. In the T2 generation, mutants were confirmed to express one to four HMW-GSs by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ultra-high-performance liquid chromatography (UPLC). Phenotypic analysis showed that the mutants were comparable to the wild-type (WT) in terms of major agronomic and grain traits. However, glutenin macropolymers (GMP) content in the mutants was significantly lower than in the WT. Transmission electron microscopy (TEM) revealed a flaky GMP structure in the mutant grain endosperms, indicating that the absence of HMW-GSs did not affect GMP formation. SDS-sedimentation volume (SDS-SV) and bread-baking tests revealed that the contribution of HMW-GSs to processing quality was ranked as 1Dx5>1Dy12>1Ax1 in the genetic background of CB037. Interestingly, although bread-baking quality deteriorated, the cookie-making and noodle quality of the mutants improved. The cookie made from the dDx mutant had the thinnest, largest diameter, and the highest spread factor. Mutants with reduced HMW-GS content may provide a new strategy for wheat breeding tailored for cookie and noodle production.

Doubled haploid (DH) technology provides an effective way to generate homozygous genetic and breeding materials over a short period of time. We produced three types of homozygous TtMTL gene-edited mutants (mtl-a, mtl-b, and mtl-ab) by CRISPR/Cas9 in durum wheat. PCR restriction enzymes and sequencing confirmed that the editing efficiency was up to 53.5%. The seed-setting rates of the three types of mutants ranged from 20% to 60%. Abnormal grain phenotypes of kernel, embryo, and both embryo and endosperm abortions were observed in the progenies of the mutants. The average frequency of embryo-less grains was 25.3%. Chromosome counting, guard cell length, and flow cytometry confirmed that the haploid induction rate was in the range of 3%-21% in the cross- and self-pollinated progenies of the mtl mutants (mtl-a and mtl-ab). Furthermore, we co-transformed two vectors, pCRISPR/Cas9-MTL and pBD68-(ZmR + ZmC1), into durum wheat, to pyramide Ttmtl-edited mutations and embryo-specifically expressed anthocyanin markers, and developed a homozygous durum haploid inducer with purple embryo (DHIPE). Using DHIPE as the male parent to be crossed with the wild-type Kronos, the grains with white embryos were identified as haploid, while the grains with purple embryos were diploid. These findings will promote the breeding of new tetraploid wheat varieties.

Selenium (Se) is an essential trace element for humans. Low concentrations of Se can promote plant growth and development. Enhancing grain yield and crop Se content is significant, as major food crops generally have low Se content. Studies have shown that Se biofortification can significantly increase Se content in plant tissues. In this study, the genetic transformation of wheat was conducted to evaluate the agronomic traits of non-transgenic control and transgenic wheat before and after Se application. Se content, speciation, and transfer coefficients in wheat grains were detected. Molecular docking simulations and transcriptome data were utilized to explore the effects of selenium-binding protein-A TaSBP-A on wheat growth and grain Se accumulation and transport. The results showed that TaSBP-A gene overexpression significantly increased plant height (by 18.50%), number of spikelets (by 11.74%), and number of grains in a spike (by 35.66%) in wheat. Under normal growth conditions, Se content in transgenic wheat grains did not change significantly, but after applying sodium selenite, Se content in transgenic wheat grains significantly increased. Analysis of Se speciation revealed that organic forms of selenomethionine (SeMet) and selenocysteine (SeCys) predominated in both W48 and transgenic wheat grains. Moreover, TaSBP-A significantly increased the transfer coefficients of Se from solution to roots and from flag leaves to grains. Additionally, it was found that with the increase in TaSBP-A gene overexpression levels in transgenic wheat, the transfer coefficient of Se from flag leaves to grains also increased.

This study examined the effects of overexpressing the DOF (DNA binding with one finger protein) transcription factor gene, TaDOF6, on wheat grain native starch and III-type resistant starch (RS3). Overexpression of TaDOF6 in the endosperm resulted in a significant increase in amylopectin content, particularly in long-branched chains, as well as larger starch particle size. Additionally, there was an increase in both rapidly and slowly digestible starches, along with an elevated level of native resistant starch. The enhanced crystallinity and orderliness of native starch, along with improved pasting properties, were also observed. However, TaDOF6 overexpression negatively impacted RS3 by reducing its crystal order, thermal stability, and pasting performance. Principal component analysis highlighted the substantial role of amylopectin in determining the crystal structure and physicochemical properties of starch. Moreover, a significant correlation was found between the particle size of RS3 and its physicochemical characteristics. Overall, these findings demonstrate that TaDOF6 overexpression alters the composition of grain starch, leading to improvements in its molecular structure and physicochemical properties.

This study investigated the effects of waxy (Wx) protein on wheat V-type resistant starch (RS5) formation, molecular structure, and physicochemical properties. We discovered that waxy protein deletions led to a rise in B- and C-type starch granules, while reducing A-type starch granules, amylose, and slowly digestible starch contents. Further, dodecyl gallate (DG) addition significantly increased RS5 content, and molecular dynamics simulations indicated that amylose and DG can form stable complexes. Molecular docking indicated that DG could potentially aid in protecting wheat starch from digestion by human α-glucosidase. RS5 content was significantly reduced by waxy protein deletions. X-ray powder diffraction, Fourier-transform infrared spectroscopy, and laser confocal microscopy-Raman analyses revealed that waxy protein deletions decreased long-range crystalline structures and relative crystallinity and increased short-range crystalline structures，and full width at half maximum at 480 cm-1 of RS5. Pearson correlation analysis showed that RS5 content was highly correlated with its crystal structure, functional characteristics, and digestive characteristics. Principal component analysis revealed that five parameters (amylopectin, long-range crystalline structures, amylose, relative crystallinity, and RS5 content) had significant effect on the crystalline structure and functionality of RS5.

Wheat relative species are important for agriculture production, functional genomics study and wheat improvement as useful genetic resources. In this study, a regeneration related wheat gene TaWOX5 was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum, hexaploid triticale, and rye (Secale cereale L.) using their immature embryos. Transgenic plants were efficiently generated. During the transformation process, the Agrobacterium infection efficiency was assessed by histochemical staining for β-glucuronidase (GUS). Finally, the transgenic nature of regenerated plants was verified by polymerase chain reaction (PCR)-based genotyping for presence of the GUS and bialaphos resistance (bar) genes, histochemical staining for GUS protein, and QuickStix strip assay for bar protein. The transformation efficiency of T. monococcum genotype PI428182 was 94.4%; the efficiencies of four hexaploid triticale genotypes Lin456, ZS3297, ZS1257, and ZS3224 were 52.1, 41.2, 19.4, and 16.0%, respectively; the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) analysis indicated that the GUS transgenes were integrated into the distal or near centromere (proximal) region of the chromosomes in transgenic T. monococcum and hexaploid triticale plants; in the transgenic hexaploid triticale plants, the foreign DNA fragment was randomly integrated into the AABB genome and RR genome. Furthermore, the transgene was proved to be almost stably inherited in the next generation by Mendel's law. The findings in this study will promote genetic improvement for grain or forage production of the three plant species and for functional genomics study of cereal species including wheat.

Low-molecular-weight glutenin subunits (LMW-GSs) associated with bread-baking quality and flour nutrient quality accumulate in endosperms of common wheat and related species. However, the mechanism underlying the expression regulation of genes encoding LMW-GSs has not been fully elucidated. In this study, we identified LMW-D2 and LMW-D7, which are highly and weakly expressed, respectively, via the analysis of RNA-sequencing data of Chinese Spring wheat and wheat transgenic lines transformed with 5' deletion promoter fragments and GUS fusion constructs. The 605-bp fragment upstream of the LMW-D2 start codon could drive high levels of GUS expression in the endosperm. The truncated endosperm box located at the -300 site resulted in the loss of LMW-D2 promoter activity, and a single-nucleotide polymorphism on the GCN4 motif was closely related to the expression of LMW-GSs. TCT and TGACG motifs, as well as the others located on the 5' distal end, might also be involved in the transcription regulation of LMW-GSs. In transgenic lines, fusion proteins of LMW-GS and GUS were deposited into protein bodies. Our findings provide new insights into the mechanism underlying the transcription regulation of LMW-GSs and will contribute to the development of wheat endosperm as a bioreactor for the production of nutraceuticals, antibodies, vaccines, and medicinal proteins.

Lysine crotonylation (Kcr) is a novel post-translational modification and its function in plant salt-stress responses remains unclear. In this study, we performed the first comprehensive chloroplast crotonylome analysis of wheat seedling leaves to examine the potential functions of Kcr proteins in salt-stress responses. In a total of 471 chloroplast proteins, 1290 Kcr sites were identified as significantly regulated by salt stress, and the Kcr proteins were mainly involved in photosynthesis, protein folding, and ATP synthesis. The identified Kcr sites that responded to salt stress were concentrated within KcrK and KcrF motifs, with the conserved KcrF motif being identified in the Kcr proteins of wheat chloroplasts for the first time. Notably, 10 Kcr sites were identified in fructose-1,6-bisphosphate aldolase (TaFBA6), a key chloroplast metabolic enzyme involved in the Calvin-Benson cycle. Site-directed mutagenesis of TaFBA6 showed that the Kcr at K367 is critical in maintaining its enzymatic activity and in conferring salt tolerance in yeast. Further molecular dynamic simulations and analyses of surface electrostatic potential indicated that the Kcr at K367 could improve the structural stability of TaFBA6 by decreasing the distribution of positive charges on the protein surface to resist alkaline environments, thereby promoting both the activity of TaFBA6 and salt tolerance. Lysine crotonylation at the K367 site improves the structural stability of FBA6 in wheat chloroplasts under salt stress, thereby promoting its activity and enhancing salt tolerance

Wheat gluten proteins serve as the largest protein molecules in nature and play key roles in breadmaking quality formation. In this study, we used a pair of Glu-A1 allelic variation lines to perform a comprehensive investigation on the effects of Glu-A1a encoded 1Ax1 subunit on gluten physicochemical properties, molecular structures and breadmaking quality. The results showed that the presence of the 1Ax1 subunit significantly increased gluten content, leading to marked improvement of dough rheological properties. Meanwhile, gluten physicochemical properties such as foaming ability and foaming stability, oil/water-holding capacity, emulsifying activity, disulfide 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 secondary 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 physicochemical properties and breadmaking quality. Our results showed that 1Ax1 subunit positively affected gluten molecular structure and quality formation.

Cadmium, one of the toxic heavy metals, robustly impact crop growth and development and food safety. In this study, the mechanisms of wheat (Triticum aestivum L.) selenium-binding protein-A (TaSBP-A) involved in response to Cd stress was fully investigated by overexpression in Arabidopsis and wheat. As a cytoplasm protein, TaSBP-A showed a high expression in plant roots and its expression levels were highly induced by Cd treatment. The overexpression of TaSBP-A enhanced Cd-toleration in yeast, Arabidopsis and wheat. Meanwhile, transgenic Arabidopsis under Cd stress showed a lower H2O2 and malondialdehyde content and a higher photochemical efficiency in the leaf and a reduction of free Cd2+ in the root. Transgenic wheat seedlings of TaSBP exhibited an increment of Cd content in the root, and a reduction Cd content in the leaf under Cd2+ stress. Cd2+ binding assay combined with a thermodynamics survey and secondary structure analysis indicated that the unique CXXC motif in TaSBP was a major Cd-binding site participating in the Cd detoxification. These results suggested that TaSBP-A can enhance the sequestration of free Cd2+ in root and inhibit the Cd transfer from root to leaf, ultimately conferring plant Cd-tolerance via alleviating the oxidative stress and photosynthesis impairment triggered by Cd stress.

The wheat pericarp is the main component of the caryopsis at the early development stage and ultimately converts into a tissue that covers the mature caryopsis. A large number of starch granules are accumulated in the pericarp, but the production of and the role of starch granules in caryopsis development remain- elusive. In the present study, the relationship between accumulated starch granules and starch metabolism-related genes in wheat pericarp was investigated using paraffin section observations, expression analysis, and mutant analysis. Starch synthesis is initiated before anthesis and is dependent on a sucrose uptake and conversion system similar to that in the endosperm. TaPTST2 is required to initiate the production of pericarp starch granules. Pericarp starch granules gradually disappeared at the filling stage with high expression levels of genes encoding β-amylase, sucrose-phosphate synthase, and sucrose-phosphate phosphatase. As a maternal tissue adjacent to the endosperm and embryo, the pericarp plays a temporary reservoir for excess nutrients delivered into the caryopsis during the early development stage and exported at the filling stage. The pericarp contributes to the development of the endosperm and embryo as well as the accumulation of endosperm starch. The metabolism of pericarp starch may affect the weight of the wheat caryopsis.

The content and composition of wheat storage proteins are the major determinants of dough rheological properties and breadmaking quality and are influenced by cultivation conditions. This study aimed to investigate the effects of water deficit and high N-fertilizer application on wheat storage protein synthesis, gluten secondary structure, and breadmaking quality. Reverse-phase ultrahigh-performance liquid chromatography analysis showed that storage protein and gluten macropolymer accumulation was promoted under both independent applications and a combination of water-deficit and high N-fertilizer treatments. Fourier-transform infrared spectroscopy showed that water deficit and high N-fertilizer treatments generally improved protein secondary structure formation and lipid accumulation, and reduced flour moisture. In particular, high N-fertilizer application increased β-sheet content by 10.4% and the combination of water-deficit and high N-fertilizer treatments increased random coil content by 7.6%. These changes in gluten content and secondary structure led to improved dough rheological properties and breadmaking quality, including superior loaf internal structure, volume, and score. Our results demonstrate that moderately high N-fertilizer application under drought conditions can improve gluten accumulation, gluten secondary structure formation, and baking quality.

Activity of BC1 complex kinase (ABC1K) serves as an atypical kinase family involved in plant stress resistance. This study identified 44 ABC1K genes in the wheat genome, which contained three clades (I–III). TaABC1K genes generally had similar structural features, but differences were present in motif and exon compositions from different clade members. More type II functional divergence sites were detected between clade I and clade III and no positive selection site were found in TaABC1K family. The three-dimensional structure prediction by Alphafold2 showed that TaABC1K proteins had more α-helixes with a relatively even distribution, and different clade members had differences in the content of secondary structures. The cis-acting element analysis showed that TaABC1K genes contained abundant cis-acting elements related to plant hormones and environmental stress response in the promoter region, and generally displayed a significantly upregulated expression under drought stress. In particular, both TaABC1K3 and TaABC1K6 genes from clade I was highly induced by drought stress, and their overexpression in yeast and Arabidopsis enhanced drought tolerance by suppressing active oxygen burst and reducing photosynthesis impairment. Meanwhile, TaABC1K3 and TaABC1K6 could, respectively, complement the function of Arabidopsis abc1k3 and abc1k6 mutants and reduce photosynthesis damage caused by drought stress.

GATA transcription factor genes participate in plant growth, development, morphogenesis, and stress response. In this study, we carried out a comprehensive genome-wide analysis of wheat GATA transcription factor genes to reveal their molecular evolutionary characteristics and involvement in salt and drought tolerance. In total, 79 TaGATA genes containing a conserved GATA domain were identified in the wheat genome, which were classified into four subfamilies. Collinear analysis indicated that fragment duplication plays an important role in the amplification of the wheat GATA gene family. Functional disproportionation analysis between subfamilies found that both type I and type II functional divergence simultaneously occurs in wheat GATA genes, which might result in functional differentiation of the TaGATA gene family. Transcriptional expression analysis showed that TaGATA genes generally have a high expression level in leaves and in response to drought and salt stresses. Overexpression of TaGATA62 and TaGATA73 genes significantly enhanced the drought and salt tolerance of yeast and Arabidopsis. Protein-protein docking indicated that TaGATAs can enhance drought and salt tolerance by interacting between the DNA-binding motif of GATA transcription factors and photomorphogenesis-related protein TaCOP9-5A. Our results provided a base for further understanding the molecular evolution and functional characterization of the plant GATA gene family in response to abiotic stresses.

BACKGROUND:Drought stress is the most limiting factor for plant growth and crop production worldwide. As a major cereal crop, wheat is susceptible to drought. Thus, discovering and utilizing drought-tolerant gene resources from related species are highly important for improving wheat drought resistance. In this study, the drought tolerance of wheat Zhongmai 8601-Thinopyrum intermedium 7XL/7DS translocation line YW642 was estimated under drought stress, and then two-dimensional difference gel electrophoresis (2D-DIGE) based proteome analysis of the developing grains was performed to uncover the drought-resistant proteins.RESULTS:The results showed that 7XL/7DS translocation possessed a better drought-tolerance compared to Zhongmai 8601. 2D-DIGE identified 146 differential accumulation protein (DAP) spots corresponding to 113 unique proteins during five grain developmental stages of YW642 under drought stress. Among them, 55 DAP spots corresponding to 48 unique proteins displayed an upregulated expression, which were mainly involved in stress/defense, energy metabolism, starch metabolism, protein metabolism/folding and transport. The cis-acting element analysis revealed that abundant stress-related elements were present in the promoter regions of the drought-responsive protein genes, which could play important roles in drought defense. RNA-seq and RT-qPCR analyses revealed that some regulated DAP genes also showed a high expression level in response to drought stress.CONCLUSIONS:Our results indicated that Wheat-Th. intermedium 7XL/7DS translocation line carried abundant drought-resistant proteins that had potential application values for wheat drought tolerance improvement.

Sucrose, the main synthesized product and transported form of photoassimilates, moves from leaves to support plant growth and storage substance biosynthesis occurring in the heterotrophic sink organs. Enhancing sucrose biosynthesis efficiency is a top priority for crop yield breeding programs. However, the molecular mechanism of sucrose biosynthesis is still elusive, especially in wheat. We performed transcriptome sequencing, subcellular localization, and bioinformatics analysis to identify key isoforms and metabolic branches associated with sucrose biosynthesis in wheat. Our analysis identified 45 genes from 13 families that exhibited high expression in young leaves with an evident diurnal change. The carbon flux from photoassimilates to sucrose was divided into two pathways. In the cytoplasm, assimilates initiating at phosphotrioses (TPs) exported by TaTPT1 from chloroplasts flowed along the TP-Sucrose branch formed by TaALD6, TaFBP5, TacPGI, TacPGM, TaUGP1, TaSPS5, and TaSPP1. Intermediates either from the Calvin cycle or TP-Sucrose branch were converted into ADPGlc to synthesize the simple starch, which was transiently degraded by a series of enzymes, including TaBAM4 and TaSEX4 in the chloroplast. Similar to the starch-biosynthesis branch in endosperms, the TP-Sucrose branch is the most prominent in leaves because each reaction can be catalyzed by at least one highly expressed isoform with expected cytosolic localization. The key isoforms and major branches highlighted in the wheat sucrose biosynthesis pathway expand our molecular understanding of crop sucrose biosynthesis and provide clues to increase wheat yield by enhancing the sucrose synthesis efficiency of leaves.

The Multidrug and toxin efflux (MATE) gene family plays crucial roles in plant growth and development and response to adverse stresses. This work investigated the structural and evolutionary characteristics, expression profiling and potential functions involved in aluminium (Al) tolerance from a genome-wide level. In total, 211 wheat MATE genes were identified, which were classified into four subfamilies and unevenly distributed on chromosomes. Duplication analysis showed that fragments and tandem repeats played the main roles in the amplification of TaMATEs, and Type II functional disproportionation had a leading role in the differentiation of TaMATEs. TaMATEs had abundant Al resistance and environmental stress-related elements, and generally had a high expression level in roots and leaves and in response to Al stress. The 3D structure prediction by AlphaFold and molecular docking showed that six TaMATE proteins localised in the plasmalemma could combine with citrate via amino acids in the citrate exuding motif and other sites, and then transport citrate to soil to form citrate aluminium. Meanwhile, citrate aluminium formed in root cells might be transported to leaves by TaMATEs to deposit in vacuoles, thereby alleviating Al toxicity.

This study investigated the influence of wheat waxy proteins on type III resistant starch (RS3) formation, molecular structure and physicochemical properties. Waxy deletions led to a significant increase in B- and C-type starch granules, particle size of RS3, and slowly digesting starch content, and a decrease in content of amylose and RS3. X-ray powder diffraction and Fourier-transform infrared spectroscopy analyses revealed high relative crystallinity and long-range (1047/1022 cm(-1), IR1) and low short-range (1022/995, IR2) crystalline structures of RS3 in waxy wheat, which suggests that waxy deletions could produce a more ordered crystalline structure and fewer amorphous regions in RS3 crystals. Further laser confocal microscopy Raman spectroscopy analysis found that waxy deletions significantly increased the full width at half maximum and intensity of the bands at 480 cm(-1), as well as leading to more ordered RS3 crystals. These changes in molecular structure resulted in improved physicochemical properties of RS3.