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Proteomic analysis of low-molecular-weight glutenin subunits and relationship with their genes in a common wheat variety.

ABSTRACT: Although many studies on low-molecular-weight glutenin subunit (LMW-GS) function have been reported, a comprehensive comparison between specific genes and their protein product is still lacking. This study aimed to link the 43 genes isolated from the Korean wheat variety "Jokyoung" in the authors' previous study to their protein products. Proteins were separated using two-dimensional gel electrophoresis (2-DGE) and identified by tandem mass spectrometry (MS/MS) at the gene haplotype level. Using MS/MS analysis of 17 protein spots, two spots were identified in the Glu-A3 locus and the corresponding haplotype was GluA3-13(Glu-A3c). Six spots were identified in the Glu-B3 locus and the corresponding haplotypes were GluB3-33 and GluB3-43 (Glu-B3h). Eight spots were identified in the Glu-D3 locus and the corresponding haplotypes were GluD3-11, GluD3-21, GluD3-31, GluD3-5, and GluD3-6 (Glu-D3a), and one spot was contaminated with gamma gliadin. Phylogenetic analysis and alignment of nucleotide and amino acid sequences assigned 35 of the 43 genes to seven haplotypes: GluA3-13, GluB3-43, GluD3-11, GluD3-21, GluD3-31, GluD3-42, and GluD3-5. Taken together, except for GluB3-33 and GluD3-6, which were not isolated, linking of each gene to the corresponding protein products at the gene haplotype level was accomplished using proteomic tools and phylogenetic analysis.

SUBMITTER: Beom HR

PROVIDER: S-EPMC5756151 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Proteomic analysis of low-molecular-weight glutenin subunits and relationship with their genes in a common wheat variety.

Beom Hye-Rang HR Kim Jin Sun JS Jang You-Ran YR Lim Sun-Hyung SH Kim Chang-Kug CK Lee Choung Keun CK Lee Jong-Yeol JY

3 Biotech 20180105 1

Although many studies on low-molecular-weight glutenin subunit (LMW-GS) function have been reported, a comprehensive comparison between specific genes and their protein product is still lacking. This study aimed to link the 43 genes isolated from the Korean wheat variety "Jokyoung" in the authors' previous study to their protein products. Proteins were separated using two-dimensional gel electrophoresis (2-DGE) and identified by tandem mass spectrometry (MS/MS) at the gene haplotype level. Using ...[more]

PMID: 29354367

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Project description: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.

Project description:BackgroundHigh-molecular-weight glutenin subunits (HMW-GS) play important roles in the elasticity of dough made from wheat. The HMW-GS null line is useful for studying the contribution of HMW-GS to the end-use quality of wheat.MethodsIn a previous work, we cloned the Glu-1Ebx gene from Thinopyrum bessarabicum and introduced it into the wheat cultivar, Bobwhite. In addition to lines expressing the Glu-1Ebx gene, we also obtained a transgenic line (LH-11) with all the HMW-GS genes silenced. The HMW-GS deletion was stably inherited as a dominant and conformed to Mendel's laws. Expression levels of HMW-GS were determined by RT-PCR and epigenetic changes in methylation patterns and small RNAs were analyzed. Glutenins and gliadins were separated and quantitated by reversed-phase ultra-performance liquid chromatography. Measurement of glutenin macropolymer, and analysis of agronomic traits and end-use quality were also performed.ResultsDNA methylation and the presence of small double-stranded RNA may be the causes of post-transcriptional gene silencing in LH-11. The accumulation rate and final content of glutenin macropolymer (GMP) in LH-11 were significantly lower than in wild-type (WT) Bobwhite. The total protein content was not significantly affected as the total gliadin content increased in LH-11 compared to WT. Deletion of HMW-GS also changed the content of different gliadin fractions. The ratio of ω-gliadin increased, whereas α/β- and γ-gliadins declined in LH-11. The wet gluten content, sedimentation value, development time and stability time of LH-11 were remarkably lower than that of Bobwhite. Bread cannot be made using the flour of LH-11.ConclusionsPost-transcriptional gene silencing through epigenetic changes and RNA inhibition appear to be the causes for the gene expression deficiency in the transgenic line LH-11. The silencing of HMW-GW in LH-11 significantly reduced the dough properties, GMP content, wet gluten content, sedimentation value, development time and stability time of flour made from this wheat cultivar. The HMW-GS null line may provide a potential material for biscuit-making because of its low dough strength.

Dataset Information

Proteomic analysis of low-molecular-weight glutenin subunits and relationship with their genes in a common wheat variety.

Publications

Proteomic analysis of low-molecular-weight glutenin subunits and relationship with their genes in a common wheat variety.

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