Increased Wheat Protein Content via Introgression of an HMW Glutenin Selectively Reshapes the Grain Proteome.
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ABSTRACT:
SUBMITTER: Cao H
PROVIDER: S-EPMC8214148 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Introgression of a high molecular weight glutenin subunit (HMW-GS) gene, 1Ay21*, into commercial wheat cultivars increased overall grain protein content and bread-making quality by unknown mechanisms. As well as increased abundance of 1Ay HMW-GS, 115 differentially expressed proteins (DEPs) were discovered between three cultivars and corresponding introgressed near-isogenic lines (NILs). Functional category analysis showed that the DEPs were predominantly other storage proteins, and proteins involved in protein synthesis, protein folding, protein degradation, stress response and grain development. Nearly half the genes encoding the DEPs showed strong co-expression patterns during grain development. Promoters of these genes are enriched in elements associated with transcription initiation and light response, indicating a potential connection between these cis-elements and grain protein accumulation. A model of how this HMW-GS enhances the abundance of machinery for protein synthesis and maturation during grain filling is proposed. This analysis not only provides insights into how introgression of the 1Ay21* improves grain protein content, but also directs selection of protein candidates for future wheat quality breeding programmes.
2021-05-30 | PXD021706 | Pride
Project description:The grass species Brachypodium distachyon has emerged as a model system for the study of gene structure and function in temperate cereals. As a first demonstration of the utility of Brachypodium to study wheat gene promoter function, we transformed it with a T-DNA that included the uidA reporter gene under control of a wheat High-Molecular-Weight Glutenin Subunit (HMW-GS) gene promoter and transcription terminator. For comparison, the same expression cassette was introduced into wheat by biolistics. Histochemical staining for β-glucuronidase (GUS) activity showed that the wheat promoter was highly expressed in the endosperms of all the seeds of Brachypodium and wheat homozygous plants. It was not active in any other tissue of transgenic wheat, but showed variable and sporadic activity in a minority of styles of the pistils of four homozygous transgenic Brachypodium lines. The ease of obtaining transgenic Brachypodium plants and the overall faithfulness of expression of the wheat HMW-GS promoter in those plants make it likely that this model system can be used for studies of other promoters from cereal crop species that are difficult to transform.
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Project description:High molecular weight glutenin subunits (HMWGS) are responsible for dough elasticity and bread making quality of bread wheat. Related wild non-progenitor species, Aegilops kotschyi possesses higher molecular weight x and y glutenin subunits than the bread wheat cultivars. A wheat-Aegilops substitution line with 1U chromosome was used for the transfer of (HMWGS) of 1U to wheat by using pollen radiation hybridization approach. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiling showed different patterns of allelic variations with either the presence or absence of HMWGS, Glu-1A (1, null), Glu-1B (7, 7 + 8, 17 + 18) and Glu-1D (5 + 10, 2 + 12, null). The pollen irradiated wheat-Aegilops derivatives, B-56-1-4-2, B-56-1-4-3, B-14-1 and B-14-2 with Glu1Ux and 1Uy and absence or presence of some Glu-1A and Glu-1B HMWGS showed high micro SDS sedimentation test (MST) values while B-16-1 and B-16-2 had moderate MST values and high protein content. However, B-58-3 with transfer of Glu-1Ux + 1Uy for Glu-1D showed very low MST values indicating that Glu-1Ux + 1Uy enhance MST value only in the presence of Glu1D HMWGS. The transfer/substitution of alien HMW-GS for Glu-1A and or Glu-1B loci only can lead to improved bread making quality of wheat.
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Project description:The production of many food items processed from wheat grain relies on the use of high gluten strength flours. As a result, about 80% of the allelic variability in the genes encoding the glutenin proteins has been lost in the shift from landraces to modern cultivars. Here, the allelic variability in the genes encoding the high molecular weight glutenin subunits (HMW-GSs) has been characterized in 152 durum wheat lines developed from a set of landraces. The allelic composition at the two Glu-1 loci (Glu-A1 and -B1) was obtained at both the protein and the DNA level. The former locus was represented by three alleles, of which the null allele Glu-A1c was the most common. The Glu-B1 locus was more variable, with fifteen alleles represented, of which Glu-B1b (HMW-GSs 7 + 8), -B1d (6 + 8) and -B1e (20 + 20) were the most frequently occurring. The composition of HMW-GSs has been used to make inferences regarding the diffusion and diversification of durum wheat. The relationships of these allelic frequencies with their geographical distribution within the Mediterranean basin is discussed in terms of gene-ecology.
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Project description:Bread wheat is the most widely cultivated crop worldwide, used in the production of food products and a feed source for animals. Selection tools that can be applied early in the breeding cycle are needed to accelerate genetic gain for increased wheat production while maintaining or improving grain quality if demand from human population growth is to be fulfilled. Proteomics screening assays of wheat flour can assist breeders to select the best performing breeding lines and discard the worst lines. In this study, we optimised a robust LC-MS shotgun quantitative proteomics method to screen thousands of wheat genotypes. Using 6 cultivars and 4 replicates, we tested 3 resuspension ratios (50, 25, and 17 µL/mg), 2 extraction buffers (with urea or guanidine-hydrochloride), 3 sets of proteases (chymotrypsin, Glu-C, and trypsin/Lys-C), and multiple LC settings. Protein identifications by LC-MS/MS were used to select the best parameters. A total 8738 wheat proteins were identified. The best method was validated on an independent set of 96 cultivars and peptides quantities were normalised using sample weights, an internal standard, and quality controls. Data mining tools found particularly useful to explore the flour proteome are presented (UniProt Retrieve/ID mapping tool, KEGG, AgriGO, REVIGO, and Pathway Tools).
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Project description:High molecular weight glutenin subunits (HMW-GSs), encoded by the genes at Glu-1 loci in wheat and its related species, are significant in the determination of grain processing quality. However, the diversity and variations of HMW-GSs are relatively low in bread wheat. More interests are now focused on wheat wild relatives in Triticeae. The genus Aegilops represents an important germplasm for novel HWM-GSs and other useful genes for wheat genetic improvement.Six novel Glu-1 alleles and HMW-GSs were identified and characterized from three species of Aegilops section Sitopsis (S genome). Both open reading frames (ORFs) and promoter regions of these Glu-1 alleles were sequenced and characterized. The ORFs of Sitopsis Glu-1 genes are approximately 2.9 kb and 2.3 kb for x-type and y-type subunits, respectively. Although the primary structures of Sitopsis HMW-GSs are similar to those of previously reported ones, all six x-type or y-type subunits have the large fragment insertions. Our comparative analyses of the deduced amino acid sequences verified that Aegilops section Sitopsis species encode novel HMW-GSs with their molecular weights larger than almost all other known HMW-GSs. The Glu-1 promoter sequences share the high homology among S genome. Our phylogenetic analyses by both network and NJ tree indicated that there is a close phylogenetic evolutionary relationship of x-type and y-type subunit between S and D genome.The large molecular weight of HMW-GSs from S genome is a unique feature identified in this study. Such large subunits are resulted from the duplications of repetitive domains in Sitopsis HMW-GSs. The unequal crossover events are the most likely mechanism of variations in glutenin subunits. The S genome-encoded subunits, 1Dx2.2 and 1Dx2.2* have independent origins, although they share similar evolutionary mechanism. As HMW-GSs play a key role in wheat baking quality, these large Sitopsis glutenin subunits can be used as special genetic resources for wheat quality improvement.
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Project description:A wheat cultivar "Chinese Spring" chromosome substitution line CS-1S(l)(1B), in which the 1B chromosome was substituted by 1S(l) from Aegilops longissima, was developed and found to possess superior dough and breadmaking quality. The molecular mechanism of its super quality conformation is studied in the aspects of high molecular glutenin genes, protein accumulation patterns, glutenin polymeric proteins, protein bodies, starch granules, and protein disulfide isomerase (PDI) and PDI-like protein expressions. Results showed that the introduced HMW-GS 1S(l)×2.3* and 1S(l)y16* in the substitution line possesses long repetitive domain, making both be larger than any known x- and y-type subunits from B genome. The introduced subunit genes were also found to have a higher level of mRNA expressions during grain development, resulting in more HMW-GS accumulation in the mature grains. A higher abundance of PDI and PDI-like proteins was observed which possess a known function of assisting disulfide bond formation. Larger HMW-GS deposited in protein bodies were also found in the substitution line. The CS substitution line is expected to be highly valuable in wheat quality improvement since the novel HMW-GS are located on chromosome 1S(l), making it possible to combine with the known superior D×5+Dy10 subunits encoded by Glu-D1 for developing high quality bread wheat.
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Project description:Rice (Oryza sativa L.) is a primary global food cereal. However, when compared to wheat, rice has poor food processing qualities. Dough that is made from rice flour has low viscoelasticity because rice seed lacks storage proteins that are comparable to gluten protein from wheat. Thus, current research efforts aim to improve rice flour processing qualities through the transgenic expression of viscoelastic proteins in rice seeds. In this study, we characterized the transgenic expression of wheat glutenin subunits in rice seeds. The two genes 1Dx5_KK and 1Dy10_JK, which both encode wheat high-molecular-weight glutenin subunits that confer high dough elasticity, were cloned from Korean wheat cultivars KeumKang and JoKyung, respectively. These genes were inserted into binary vectors under the control of the rice endosperm-specific Glu-B1 promoter and were expressed in the high-amylose Korean rice cultivar Koami (Oryza sativa L.). Individual expression of both glutenin subunits was confirmed by SDS-PAGE and immunoblot analyses performed using T? generation of transgenic rice seeds. The subcellular localization of 1Dx5_KK and 1Dy10_JK in the rice seed endosperm was confirmed by immunofluorescence analysis, indicating that the wheat glutenin subunits accumulate in protein body-II and novel protein body types in the rice seed. These results contribute to our understanding of engineered seed storage proteins in rice.
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Project description:The processing quality of wheat is affected by seed storage substances, such as protein and starch. High-molecular-weight glutenin subunits (HMW-GSs) are the major components of wheat seed storage proteins (SSPs); they are also key determinators of wheat end-use quality. However, the effects of HMW-GSs absence on the expression of other storage substances and the regulation mechanism of HMW-GSs are still limited. Previously, a wheat transgenic line LH-11 with complete deletions of HMW-GSs was obtained through introducing an exogenous gene Glu-1Ebx to the wild-type cultivar Bobwhite by transgenic approach. In this study, comparative seed transcriptomics and proteomics of transgenic and non-transgenic lines at different seed developmental stages were carried out to explore the changes in genes and proteins and the underlying regulatory mechanism. Results revealed that a number of genes, including genes related to SSPs, carbohydrates metabolism, amino acids metabolism, transcription, translation, and protein process were differentially enriched. Seed storage proteins displayed differential expression patterns between the transgenic and non-transgenic line, a major rise in the expression levels of gliadins were observed at 21 and 28 days post anthesis (DPA) in the transgenic line. Changes in expressions of low-molecular-weight glutenins (LMW-GSs), avenin-like proteins (ALPs), lipid transfer proteins (LTPs), and protease inhibitors (PIs) were also observed. In addition, genes related to carbohydrate metabolism were differentially expressed, which probably leads to a difference in starch component and deposition. A list of gene categories participating in the accumulation of SSPs was proposed according to the transcriptome and proteome data. Six genes from the MYB and eight genes from the NAC transcription families are likely important regulators of HMW-GSs accumulation. This study will provide data support for understanding the regulatory network of wheat storage substances. The screened candidate genes can lay a foundation for further research on the regulation mechanism of HMW-GSs.
| S-EPMC9857648 | biostudies-literature
Project description:High-molecular-weight glutenin subunits (HMW-GSs) have been considered as most important seed storage proteins for wheat flour quality. 1Ay subunits are of great interest because they are always silent in common wheat. The presence of expressed 1Ay subunits in diploid and tetraploid wheat genotypes makes it possible to investigate molecular information of active 1Ay genes.We identified 1Ay subunits with different electrophoretic mobility from 141 accessions of diploid and tetraploid wheats, and obtained the complete ORFs and 5' flanking sequences of 1Ay genes including 6 active and 3 inactive ones. Furthermore, the 5' flanking sequences were characterized from 23 wild diploid species of Triticeae. All 6 active 1Ay possess a typical HMW-GS primary structure and some novel characteristics. The conserved cysteine residue within the repetitive domain of y-type subunits was replaced by phenylalanine residue in subunits of 1Ay (Tu-e1), 1Ay (Tu-e2), 1Ay (Ta-e2) and 1Ay (Td-e). Particularly, 1Ay (Ta-e3) has an unusual large molecular weight of 2202 bp and was one of the known largest y-type HMW-GSs. The translations of 1Ay (Tu-s), 1Ay (Ta-s) and 1Ay (Td-s) were disrupted by premature stop codons in their coding regions. The 5' flanking sequences of active and inactive 1Ay genes differ in a few base substitutions and insertions or deletions. The 85 bp deletions have been found in promoter regions of all 1Ay genes and the corresponding positions of 6 species from Aegilops and Hordeum.The possession of larger molecular weight and fewer conserved cysteine residues are unique structural features of 1Ay genes; it would be interested to express them in bread wheat and further to examine their impact to processing quality of wheat. The 1Ay genes from T. urartu are closer to the genes from T. turgidum dicoccon and T. aestivum, than those from T. monococcum aegilopoides. The 85 bp deletion and some variations in the 5'flanking region, have not interrupted expression of 1Ay genes, whereas the defects in the coding regions could be responsible to the silence of the 1Ay genes. Some mutational events in more distant distal promoter regions are also possible causes for the inactivation of 1Ay genes.
| S-EPMC2667398 | biostudies-literature