Wheat Endosperm Research Articles

Molecular cloning, phylogenetic analysis, and expression profiling of endoplasmic reticulum molecular chaperone BiP genes from bread wheat (Triticum aestivum L.).

BackgroundThe endoplasmic reticulum chaperone binding protein (BiP) is an important functional protein, which is involved in protein synthesis, folding assembly, and secretion. In order to study the role of BiP in the process of wheat seed development, we cloned three BiP homologous cDNA sequences in bread wheat (Triticum aestivum), completed by rapid amplification of cDNA ends (RACE), and examined the expression of wheat BiP in wheat tissues, particularly the relationship between BiP expression and the subunit types of HMW-GS using near-isogenic lines (NILs) of HMW-GS silencing, and under abiotic stress.ResultsSequence analysis demonstrated that all BiPs contained three highly conserved domains present in plants, animals, and microorganisms, indicating their evolutionary conservation among different biological species. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that TaBiP (Triticum aestivum BiP) expression was not organ-specific, but was predominantly localized to seed endosperm. Furthermore, immunolocalization confirmed that TaBiP was primarily located within the protein bodies (PBs) in wheat endosperm. Three TaBiP genes exhibited significantly down-regulated expression following high molecular weight-glutenin subunit (HMW-GS) silencing. Drought stress induced significantly up-regulated expression of TaBiPs in wheat roots, leaves, and developing grains.ConclusionsThe high conservation of BiP sequences suggests that BiP plays the same role, or has common mechanisms, in the folding and assembly of nascent polypeptides and protein synthesis across species. The expression of TaBiPs in different wheat tissue and under abiotic stress indicated that TaBiP is most abundant in tissues with high secretory activity and with high proportions of cells undergoing division, and that the expression level of BiP is associated with the subunit types of HMW-GS and synthesis. The expression of TaBiPs is developmentally regulated during seed development and early seedling growth, and under various abiotic stresses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0260-0) contains supplementary material, which is available to authorized users.

Tandem Mass Spectrometric Determination of Glycolipids in Wheat Endosperm: A New Tool for Breeders to Rank and Select Early Seed Generations

AbstractWheat breeding for end‐use quality is enhanced by development and application of a selective and sensitive method for ranking early generation cultivars as part of the effort of generating desirable wheat lipids. A desirable wheat lipid, the increased concentration of which in hard wheat flour correlates positively with bread loaf volume, is digalactosyldiglyceride (DGDG). A mass spectrometric test for DGDG was applied to 100‐mg samples of flour isolated from the F6 generation of hard winter wheats. The test enables prediction of desirable lipids in early generations so that decisions about the F6 progeny improve wheat breeding efficiency. Previous attempts to screen wheat cultivars for desirable glycolipids were constrained by sample size, lipid extraction and purification, and time. Here, we present flour glycolipid data obtained from individual wheat plants grown in short rows in two nurseries. The endosperm (100 mg) representing several kernels was exhaustively extracted with a total lipid solvent. Following isolation of the lipids and addition of an internal standard, the lipids were subjected to tandem mass spectrometry (MS–MS). The concentration of DGDG in the flour from five to seven kernels was found to vary by a factor of two within single nurseries. Thus, early generation lipid screening enables informed selection for this hard wheat quality indicator.

Development of endosperm cells and starch granules in common wheat

The objective of the present study was to understand the developmental regularity of wheat endosperm cells at different Days After Pollination (DAP) using microscopic and histochemical methods. Resin semi-thin sections of the endosperm and the enzymatically dissociated Starchy Endosperm Cells (SECs) were observed under a light microscope. The results showed that: (1) SECs were irregular-shaped and had two types of starch granules: large oval-shaped A-type starch granules and small spherical B-type starch granules. (2) The growth shape of SECs was referred to as S-curve and the fastest cell growth period was at 16–24 DAP. (3) The largest increase and growth of A-type starch granules were mainly at 4–16 DAP. B-type starch granules increased rapidly after 16 DAP and made up over 90% of the total starch granules in SEC during the late stage of endosperm development. (4) The nuclei of SEC deformed and degenerated during the middle and late stages of endosperm development and eventually disappeared. However, starch granules still increased and grew after the cell nuclei had degenerated. The investigations showed the development regularity of starch endosperm cells and starch granules, thereby improving the understanding of wheat endosperm development.

Gluten quality of bread wheat is associated with activity of RabD GTPases.

In the developing endosperm of bread wheat (Triticum aestivum), seed storage proteins are produced on the rough endoplasmic reticulum (ER) and transported to protein bodies, specialized vacuoles for the storage of protein. The functionally important gluten proteins of wheat are transported by two distinct routes to the protein bodies where they are stored: vesicles that bud directly off the ER and transport through the Golgi. However, little is known about the processing of glutenin and gliadin proteins during these steps or the possible impact on their properties. In plants, the RabD GTPases mediate ER-to-Golgi vesicle transport. Available sequence information for Rab GTPases in Arabidopsis, rice, Brachypodium and bread wheat was compiled and compared to identify wheat RabD orthologs. Partial genetic sequences were assembled using the first draft of the Chinese Spring wheat genome. A suitable candidate gene from the RabD clade (TaRabD2a) was chosen for down-regulation by RNA interference (RNAi), and an RNAi construct was used to transform wheat plants. All four available RabD genes were shown by qRT-PCR to be down-regulated in the transgenic developing endosperm. The transgenic grain was found to produce flour with significantly altered processing properties when measured by farinograph and extensograph. SE-HPLC found that a smaller proportion of HMW-GS and large proportion of LMW-GS are incorporated into the glutenin macropolymer in the transgenic dough. Lower protein content but a similar protein profile on SDS-PAGE was seen in the transgenic grain.

Structure of clusters and building blocks in amylopectin from developing wheat endosperm

Changes in internal structure of amylopectin (AP) during wheat endosperm development were studied by isolating clusters and building blocks of AP from both large A-type and small B-type starch granules at different maturity stages up to harvest time at 49 days after anthesis (DAA). Clusters isolated from B-type granules had a degree of branching (DB) of 16.5–16.8% and were more tightly branched than those isolated from A-type granules (DB 15.7–16.2%). The degree of polymerization (DP) of the clusters increased in both types of granules during the pre-physiological maturity stage up to 28 DAA. Clusters at maturity were smaller with less branches and building blocks than at the end of the pre-maturity stage. It is suggested that this was due to a continuous trimming of the cluster structure after the active period of starch synthesis. Differences were evident between A- and B-type granules with regards to glucan trimming and the type of new chains produced.

Evolution of amylopectin structure in developing wheat endosperm starch

In this study, starches extracted from wheat grains harvested at 7, 14, 28, and 35 days after anthesis (DAA) were used as a means of examining the molecular structure of amylopectin (AP) from developing wheat grain. Scanning electron microscopy of wheat grain cross-sections revealed the presence of endosperm at 7 DAA and contained lenticular-shaped developing large (A-type) granules. From 14 DAA onward, spherical-shaped small (B-type) granules coexisted with large granules in the endosperm. During granule development, the fine structure of AP varied with maturity in both large and small granules. Towards the end of the pre-physiological maturity stage (28 DAA), AP in small and large granules had shortest external chain length (ECL), longest internal chain length (ICL) and lowest amount of A-chains. At physiological maturity (35 DAA), these changes in ECL, ICL and amount of A-chains were reversed when compared to 28 DAA. In both large and small granules, the external AP structure was apparently more organized at physiological maturity than at pre-physiological maturity.

A novel approach to the generation of seamless constructs for plant transformation.

BackgroundWhen creating plant transformation vectors, full control of nucleotides flanking the insert in the final construct may be desirable. Modern ligase-independent methods for DNA-recombination are based on linearization by classical type II restriction endonucleases (REs) alone or in combination with nicking enzymes leaving residual nucleotides behind in the final construct. We here explore the use of type IIS and type IIB REs for vector linearization that combined with sequence and ligase-independent cloning (SLIC) overcomes this problem and promotes seamless gene-insertion in vectors. Providing the basis for a collection of biolistic plant transformation vectors ready to be cloned with different genes-of-interest, we present two vectors, where promoter and terminator are joined by a spacer. During spacer-removal linearization (SRL), type IIS and type IIB REs remove their own recognition sequences from the vector leaving no undesired, short sequences behind.ResultsWe designed two plant transformation vectors prepared for SRL in combination with SLIC, pAUrumII and pAUrumIII, harboring a spacer with recognition sites for a type IIS and IIB RE, respectively. The gene for a green fluorescent protein, gfp, was successfully cloned into both vectors; traces of pAUrumIII, however, contaminated the transformation due to incomplete linearization, an issue not encountered with the type IIS linearized pAUrumII. Both constructs, pAUrumII-gfp and pAUrumIII-gfp, were functional, when tested in vitro on wheat and barley endosperm cells for transient gfp expression.ConclusionsAll nucleotides flanking an insert in a biolistic plant transformation vector can be customized by means of SRL in combination with SLIC. Especially type IIS REs promote an efficient cloning result. Based on our findings, we believe that the SRL system can be useful in a series of plant transformation vectors, favoring the presence of functional sequences for optimal expression over redundant cloning-site remnants.

Enzymatic modification and particle size reduction of wheat bran improves the mechanical properties and structure of bran-enriched expanded extrudates

The aim of this study was to examine enzymatic modification of wheat bran, performed in a low-moisture process, and the reduction of bran particle size as means of improving the technological performance of wheat bran in expanded extrudates. Modification of bran by hydrolytic enzymes increased the crispiness and decreased the hardness and piece density of extrudates containing wheat bran and endosperm rye flour in 20:80 ratio. These improvements correlated (P < 0.01 or 0.05) with an increased content of water extractable arabinoxylan and decreased water holding capacity of the bran, as well as with increased longitudinal expansion of the extrudates. Furthermore, bran with a fine average particle size (84 μm) produced extrudates with improved mechanical properties and higher radial expansion than coarse bran (particle size 702 μm). The impact of bran particle size was also observed in the cellular structure of the extrudates as differences in cell size and homogeneity. The bran drying method, oven or freeze drying after enzymatic modification, did not have a major impact on the properties of the extrudates. The study showed that the functionality of wheat bran in extrusion can be improved by enzymatic modification using a low-water process and by reduction of bran particle size.

Characterisation of soluble and insoluble cell wall fractions from rye, wheat and hull-less barley endosperm flours

Within cereal endosperm flours, arabinoxylan and β-glucan molecules exist in either a soluble or an insoluble form. From a nutritional functionality viewpoint, soluble and insoluble forms offer different potential health advantages, so it is important to define both the features controlling solubilisation and the properties of each of the soluble and insoluble fractions. Factors known to affect the stability of arabinoxylan (AX) and β-glucan (BG) solutions include AX branching extent and type, and the ratio of cellotriose to cellotetraose units (DP3/DP4) in BG. Through studying the solubilisation of AX and BG from wheat, rye, and hull less barley endosperm under conditions that avoid the use of alkali or ethanol during the solubilisation process, we report (a) similar A/X ratios and fine structures for extracted soluble arabinoxylan and the corresponding insoluble AX within the cell walls for rye and wheat endosperm flours, (b) comparable DP3/DP4 ratios for soluble β-glucan, flour and insoluble β-glucan within the endosperm cell wall of hull less barley, and (c) evidence for enrichment of β-glucan at the exterior of residual insoluble cell walls. Therefore, the factors determining solubilisation of AX and BG from endosperm cell walls are different to those that determine the stability of aqueous solutions of the same polymers, and β-glucan may show limited solubilisation by being trapped within restraining cross-linked arabinoxylans in the cell wall.

Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic genes CrtB and CrtI.

Carotenoid content is a primary determinant of wheat nutritional value and affects its end-use quality. Wheat grains contain very low carotenoid levels and trace amounts of provitamin A content. In order to enrich the carotenoid content in wheat grains, the bacterial phytoene synthase gene (CrtB) and carotene desaturase gene (CrtI) were transformed into the common wheat cultivar Bobwhite. Expression of CrtB or CrtI alone slightly increased the carotenoid content in the grains of transgenic wheat, while co-expression of both genes resulted in a darker red/yellow grain phenotype, accompanied by a total carotenoid content increase of approximately 8-fold achieving 4.76 μg g(-1) of seed dry weight, a β-carotene increase of 65-fold to 3.21 μg g(-1) of seed dry weight, and a provitamin A content (sum of α-carotene, β-carotene, and β-cryptoxanthin) increase of 76-fold to 3.82 μg g(-1) of seed dry weight. The high provitamin A content in the transgenic wheat was stably inherited over four generations. Quantitative PCR analysis revealed that enhancement of provitamin A content in transgenic wheat was also a result of the highly coordinated regulation of endogenous carotenoid biosynthetic genes, suggesting a metabolic feedback regulation in the wheat carotenoid biosynthetic pathway. These transgenic wheat lines are not only valuable for breeding wheat varieties with nutritional benefits for human health but also for understanding the mechanism regulating carotenoid biosynthesis in wheat endosperm.

Suppression of starch synthase I expression affects the granule morphology and granule size and fine structure of starch in wheat endosperm.

Studies in Arabidopsis and rice suggest that manipulation of starch synthase I (SSI) expression in wheat may lead to the production of wheat grains with novel starch structure and properties. This work describes the suppression of SSI expression in wheat grains using RNAi technology, which leads to a low level of enzymatic activity for SSI in the developing endosperm, and a low abundance of SSI protein inside the starch granules of mature grains. The amylopectin fraction of starch from the SSI suppressed lines showed an increased frequency of very short chains (degree of polymerization, dp 6 and 7), a lower proportion of short chains (dp 8-12), and more intermediate chains (dp 13-20) than in the grain from their negative segregant lines. In the most severely affected line, amylose content was significantly increased, the morphology of starch granules was changed, and the proportion of B starch granules was significantly reduced. The change of the fine structure of the starch in the SSI-RNAi suppression lines alters the gelatinization temperature, swelling power, and viscosity of the starch. This work demonstrates that the roles of SSI in the determination of starch structure and properties are similar among different cereals and Arabidopsis.

An asparagine residue at the N-terminus affects the maturation process of low molecular weight glutenin subunits of wheat endosperm

BackgroundWheat glutenin polymers are made up of two main subunit types, the high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. These latter are represented by heterogeneous proteins. The most common, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. The mature sequences differ as a consequence of three extra amino acids (MET-) at the N-terminus of LMW-m types. The nucleotide sequences of their encoding genes are, however, nearly identical, so that the relationship between gene and protein sequences is difficult to ascertain.It has been hypothesized that the presence of an asparagine residue in position 23 of the complete coding sequence for the LMW-s type might account for the observed three-residue shortened sequence, as a consequence of cleavage at the asparagine by an asparaginyl endopeptidase.ResultsWe performed site-directed mutagenesis of a LMW-s gene to replace asparagine at position 23 with threonine and thus convert it to a candidate LMW-m type gene. Similarly, a candidate LMW-m type gene was mutated at position 23 to replace threonine with asparagine. Next, we produced transgenic durum wheat (cultivar Svevo) lines by introducing the mutated versions of the LMW-m and LMW-s genes, along with the wild type counterpart of the LMW-m gene.Proteomic comparisons between the transgenic and null segregant plants enabled identification of transgenic proteins by mass spectrometry analyses and Edman N-terminal sequencing.ConclusionsOur results show that the formation of LMW-s type relies on the presence of an asparagine residue close to the N-terminus generated by signal peptide cleavage, and that LMW-GS can be quantitatively processed most likely by vacuolar asparaginyl endoproteases, suggesting that those accumulated in the vacuole are not sequestered into stable aggregates that would hinder the action of proteolytic enzymes. Rather, whatever is the mechanism of glutenin polymer transport to the vacuole, the proteins remain available for proteolytic processing, and can be converted to the mature form by the removal of a short N-terminal sequence.

Enantioselectivity of the Bioconversion of Chiral Citronellal during the Inhibition of Wheat Seeds Germination

Citronellal is one of the most prominent monoterpenes present in many essential oils. Low persistence of essential oils as bioherbicides has often been addressed because of the high volatility of these compounds. Bioconversion of citronellal by wheat seeds releases less aggressive and injurious compounds as demonstrated by their diminished germination. We demonstrated that optically pure citronellal enantiomers were reduced to optically pure citronellol enantiomers with retention of the configuration both in isolated wheat embryos and endosperms. Our findings reveal the potential of essential oils as allelopathic agents providing an insight into their mechanism of action and persistence.

Production of gliadin-poly(ethyl cyanoacrylate) nanoparticles for hydrophilic coating

Cyanoacrylate nanoparticles have been usually prepared by anionic polymerization initiated by hydroxyl ions derived from dissociation of water. In the current research, amine groups on the surface of gliadin aggregates were utilized as initiator for the polymerization of ethyl cyanoacrylate (ECA). Gliadin, a protein found in the endosperms of wheat (Triticum aestivum L.), is not soluble in water, but dissolves in aqueous ethanol in the form of aggregates. As a result of the reaction with ECA monomers, gliadin molecules are chemically bound to poly(ethyl cyanoacrylate) (PECA) chains. The nanoparticles thus produced are made up of block copolymers that are consisted of hydrophilic moiety (gliadin) and hydrophobic moiety (PECA). The suspension containing these nanoparticles showed an excellent coating capability on the surface of hydrophobic materials such as glass or plastics. A simple spray coating changed the wetting property of the material instantly and dramatically. Since both protein and poly(alkyl cyanoacrylate) are degradable polymers, the developed nanoparticles are degradable.

The colours of durum wheat: a review

Pigments are essential to the life of all living organisms. Animals and plants have been the subjects of basic and applied research with the aim of determining the basis of the accumulation and physiological roles of pigments. In crop species, the edible organs show large variations in colour. In durum wheat grain, which is a staple food for humans, the colour is mainly due to two natural classes of pigment: carotenoids and anthocyanins. The carotenoids provide the yellow pigmentation of the durum wheat endosperm, and consequently of the semolina, which has important implications for the marketing of end products based on durum wheat. Anthocyanins accumulate in the aleurone or pericarp of durum wheat and provide the blue, purple and red colours of the grain. Both the carotenoids and the anthocyanins are known to provide benefits for human health, in terms of decreased risks of certain diseases. Therefore, accumulation of these pigments in the grain represents an important trait in breeding programs aimed at improving the nutritional value of durum wheat grain and its end products. This review focuses on the biochemical and genetic bases of pigment accumulation in durum wheat grain, and on the breeding strategies aimed at modifying grain colour.

In situ Digestion of Wheat Cell-wall Polysaccharides

Cell walls of the wheat endosperm are mostly composed of arabinoxylans (AX) and mixed (1→3), (1→4)-β-glucans (BG) (Saulnier et al., 2012). Here, we present an optimized protocol to degrade enzymatically these cell-wall polysaccharides into oligosaccharides, directly from wheat grain cross sections. The main difficulty is to provide a sufficient amount of humidity for the enzyme to be active, while the amount of liquid at the surface of the tissue should stay low to prevent any delocalization of the released products. With this protocol, enzymatic degradation was shown to be efficient and delocalization of released oligosaccharides was estimated below 50 &#181;m (Veličković et al., 2014). Although it can be employed for other purposes, this in situ enzymatic digestion was primarily developed to obtain molecular images of the cross-sections of wheat endosperm by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Veličković et al., 2014). The cell wall polysaccharides are heterogeneous in structure, exhibit high masses and are entangled into complex networks. Thus, they are not amenable to direct analysis by mass spectrometry and they need to be degraded into smaller compounds as a first step. In this protocol, additional steps corresponding to the deposition of the MALDI matrix are also described.

The reducing end sequence of wheat endosperm cell wall arabinoxylans

Walls from wheat (Triticum aestivum L.) endosperm are composed primarily of hetero-(arabino)xylans (AXs) (70%) and (1→3)(1→4)-β-d-glucans (20%) with minor amounts of cellulose and heteromannans (2% each). To understand the differential solubility properties of the AXs, as well as aspects of their biosynthesis, we are sequencing the xylan backbone and examining the reducing end (RE) sequence(s) of wheat (monocot) AXs. A previous study of grass AXs (switchgrass, rice, Brachypodium, Miscanthus and foxtail millet) concluded that grasses lacked the comparable RE glycosyl sequence (4-β-d-Xylp-(1→4)-β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp) found in dicots and gymnosperms but the actual RE sequence was not determined.Here we report the isolation and structural characterisation of the RE oligosaccharide sequence(s) of wheat endosperm cell wall AXs. Walls were isolated as an alcohol-insoluble residue (AIR) and sequentially extracted with hot water (W-sol Fr) and 1M KOH containing 1% NaBH4 (KOH-sol Fr). Detailed structural analysis of the RE oligosaccharides was performed using a combination of methylation analysis, MALDI-TOF-MS, ESI-QTOF-MS, ESI-MSn and enzymic analysis. Analysis of RE oligosaccharides, both 2AB labelled (from W-sol Fr) and glycosyl-alditol (from KOH-sol Fr), revealed that the RE glycosyl sequence of wheat endosperm AX comprises a linear (1→4)-β-d-Xylp backbone which may be mono-substituted with either an α-l-Araf residue at the reducing end β-d-Xylp residue and/or penultimate RE β-d-Xyl residue; β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp and/or an α-d-GlcpA residue at the reducing end β-d-Xylp residue; β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp-(1→4)-[α-d-GlcAp-(1→2)]-β-d-Xylp. Thus, wheat endosperm AX backbones lacks the RE sequence found in dicot and gymnosperm xylans; a finding consistent with previous reports from other grass species.

The influence of nitrogen on the development and accumulation of protein bodies in the developing endosperm of wheat caryopses

The aim of the present work was to reveal the histological changes of protein bodies (PBs) in the developing wheat endosperm under nitrogen (N) treatment. For this purpose, the development and accumulation of PBs in the dorsal and ventral regions of wheat endosperm affected by N application at booting stage were investigated using light microscopy and Image-Pro Plus 6.0 software. The endosperm without N treatment contained many smaller PBs that were scattered in endosperm cells in an unordered pattern, whereas the endosperm with N treatment contained many larger PBs or aggregations that were concentrated in a certain region of endosperm cells. The amount and relative areas of PBs in wheat varieties cvs. Xumai 30 and Yangmai 13 were significantly increased by N application. However, the cultivars differed with the degree of response to N being cv. Xumai 30>cv. Yangmai 13. These differences also varied with position in the endosperm in the order ventral>dorsal region. The initiation of PBs occurred 3days earlier in N-treated endosperm than the control.

Effect of nitrogen fertilizer on distribution of starch granules in different regions of wheat endosperm

This study provided visual evidence of a nitrogen effect on starch granules (SGs) in wheat endosperm. Winter wheat (Titicum aestivum L.) cultivar Xumai 30 was cultured under no nitrogen (control) and 240kgha−1 of nitrogen applied at the booting stage. The number, morphology, and size of A- and B-type SGs in subaleurone of dorsal endosperm (SDE), center of dorsal endosperm (CDE), modified aleurone (MA), subaleurone of ventral endosperm (SVE), and center of ventral endosperm (CVE) were observed under light and electron microscopes. (1) The distribution of SGs in SDE was similar to that in SVE, the distributions of SGs in CDE and CVE were similar, but the distribution of SGs in MA was different from those in the other four endosperm regions. The number of SGs in the five endosperm regions was in the order SDE>CDE>SVE>CVE>MA. (2) Nitrogen increased the number of A- and B-type SGs in SDE and SVE. Nitrogen also increased the number of B-type SGs but decreased the number of A-type SGs in CDE and CVE. Nitrogen decreased the numbers of A-type and B-type SGs in MA. The results suggest that increased N fertilizer application mainly increased the numbers of small SGs and decreased the numbers of large SGs, but that the results varied in different regions of the wheat endosperm.

The Interactive Effects of Transgenically Overexpressed 1Ax1 with Various HMW-GS Combinations on Dough Quality by Introgression of Exogenous Subunits into an Elite Chinese Wheat Variety

Seed storage proteins in wheat endosperm, particularly high-molecular-weight glutenin subunits (HMW-GS), are primary determinants of dough properties, and affect both end-use quality and grain utilization of wheat (Triticum aestivum L). In order to investigate the interactive effects between the transgenically overexpressed 1Ax1 subunit with different HMW-GS on dough quality traits, we developed a set of 8 introgression lines (ILs) overexpressing the transgenic HMW-glutenin subunit 1Ax1 by introgression of this transgene from transgenic line B102-1-2/1 into an elite Chinese wheat variety Chuanmai107 (C107), using conventional crossing and backcrossing breeding technique. The donor C107 strain lacks 1Ax1 but contains the HMW-GS pairs 1Dx2+1Dy12 and 1Bx7+1By9. The resultant ILs showed robust and stable expression of 1Ax1 even after five generations of self-pollination, and crossing/backcrossing three times. In addition, overexpression of 1Ax1 was compensated by the endogenous gluten proteins. All ILs exhibited superior agronomic performance when compared to the transgenic parent line, B102-1-2/1. Mixograph results demonstrated that overexpressed 1Ax1 significantly improved dough strength, resistance to extension and over-mixing tolerance, in the targeted wheat cultivar C107. Further, comparisons among the ILs showed the interactive effects of endogenous subunits on dough properties when 1Ax1 was overexpressed: subunit pair 17+18 contributed to increased over-mixing tolerance of the dough; expression of the Glu-D1 allele maintained an appropriate balance between x-type and y-type subunits and thereby improved dough quality. It is consistent with ILs C4 (HMW-GS are 1, 17+18, 2+12) had the highest gluten index and Zeleny sedimentation value. This study demonstrates that wheat quality could be improved by using transgenic wheat overexpressing HMW-GS and the feasibility of using such transgenic lines in wheat quality breeding programs.