Zitong Yu

Molecular Mechanisms of HMW Glutenin Subunits from 1Sl Genome of Aegilops longissima Positively Affecting Wheat Breadmaking Quality

A wheat cultivar “Chinese Spring” chromosome substitution line CS-1Sl(1B), in which the 1B chromosome was substituted by 1Sl 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 1Sl×2.3* and 1Sly16* 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 1Sl, making it possible to combine with the known superior D×5+Dy10 subunits encoded by Glu-D1 for developing high quality bread wheat.

Rapid Characterization of Wheat Low Molecular Weight Glutenin Subunits by Ultraperformance Liquid Chromatography (UPLC)

Low molecular weight glutenin subunits (LMW-GS), as important seed storage proteins, together with HMW-GS significantly define unique dough viscoelastic properties. In this study, a rapid ultraperformance liquid chromatography (UPLC) method for the separation and characterization of LMW-GS in wheat was optimized and established. The fast, reproducible, and high-resolution UPLC separation of LMW-GS could be obtained by gradually increasing eluting gradient from 21 to 47% in 30 min at flow rate of 0.55 mL/min and 60 °C for separation temperature. By this method, analysis of one sample could be completed in <20 min, significantly less time than the traditional reversed-phase high-performance liquid chromatography (RP-HPLC) method. Under the optimized conditions, the genetic features of LMW-GS and genotype × environmental interaction were successfully analyzed, leading to a fast identification of 17 main LMW-GS alleles that were related to different quality properties in wheat. The results demonstrated that UPLC could be a powerful and alternative tool for genetic and proteomic studies of wheat grain proteins and fast identification or screening of desirable LMW-GS alleles in wheat quality improvement.

Molecular characterization of LMW-GS genes in Brachypodium distachyon L. reveals highly conserved Glu-3 loci in Triticum and related species

BackgroundBrachypodium distachyon L. is a newly emerging model plant system for temperate cereal crop species. However, its grain protein compositions are still not clear. In the current study, we carried out a detailed proteomics and molecular genetics study on grain glutenin proteins in B. distachyon.ResultsSDS-PAGE and RP-HPLC analysis of grain proteins showed that Brachypodium has few gliadins and high molecular weight glutenin subunits. In contrast the electrophoretic patterns for the albumin, globulin and low molecular weight glutenin subunit (LMW-GS) fractions of the grain protein were similar to those in wheat. In particular, the LMW-C type subunits in Brachypodium were more abundant than the equivalent proteins in common wheat. Southern blotting analysis confirmed that Brachypodium has 4–5 copies of LMW-GS genes. A total of 18 LMW-GS genes were cloned from Brachypodium by allele specific PCR. LMW-GS and 4 deduced amino acid sequences were further confirmed by using Western-blotting and MALDI-TOF-MS. Phylogenetic analysis indicated that Brachypodium was closer to Ae. markgrafii and Ae. umbellulata than to T. aestivum.ConclusionsBrachypodium possessed a highly conserved Glu-3 locus that is closely related to Triticum and related species. The presence of LMW-GS in B. distachyon grains indicates that B. distachyon may be used as a model system for studying wheat quality attributes.

Fast identification of wheat 1BL.1RS translocation by reversed-phase ultra-performance liquid chromatography (RP-UPLC)

Abstract. The 1BL.1RS chromosomal translocation in wheat is the result of replacement of the short arm of chromosome 1B of wheat by the short arm of chromosome 1R of rye, which had been widely used as a parental line in worldwide wheat breeding, resulting in a high percentage of wheat cultivars containing this translocation. A fast and reliable approach to identify this translocation is highly desirable in modern wheat breeding. This study compared reversed-phase ultra-performance liquid chromatography (RP-UPLC), acidic polyacrylamide gel electrophoresis (A-PAGE), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), allelic-specific PCR, and reversed-phase high-performance liquid chromatography (RP-HPLC) approaches to identify the 1BL.1RS translocation in 76 bread wheat cultivars. Two gliadin bands in the Gli-B1 region of A-PAGE separation were confirmed by LC-MS/MS to be omega secalins from the 1BL.1RS translocation, and they can be used as reliable protein markers for identifying the translocation. A few specific minor peaks eluted at 12–13 min on the RP-UPLC patterns can readily differentiate the 1BL.1RS translocation. Of the 76 wheat cultivars tested, 40 were identified as carrying the 1BL.1RS translocation by RP-UPLC, which was consistent with the results of A-PAGE, HPLC, and PCR. Compared with other established methods, RP-UPLC showed a clear advantage in fast identification of the 1BL.1RS translocation with higher reliability and lower costs, and it is therefore ideal for large-scale screening of the 1BL.1RS translocation in wheat breeding.

Impact of mid-season sulphur deficiency on wheat nitrogen metabolism and biosynthesis of grain protein

Wheat (Triticum aestivum) quality is mainly determined by grain storage protein compositions. Sulphur availability is essential for the biosynthesis of the main wheat storage proteins. In this study, the impact of different sulphur fertilizer regimes on a range of agronomically important traits and associated gene networks was studied. High-performance liquid chromatography was used to analyse the protein compositions of grains grown under four different sulphur treatments. Results revealed that sulphur supplementation had a significant effect on grain yield, harvest index, and storage protein compositions. Consequently, two comparative sulphur fertilizer treatments (0 and 30 kg ha−1 sulphur, with 50 kg ha−1 nitrogen) at seven days post-anthesis were selected for a transcriptomics analysis to screen for differentially expressed genes (DEGs) involved in the regulation of sulphur metabolic pathways. The International Wheat Genome Sequencing Consortium chromosome survey sequence was used as reference. Higher sulphur supply led to one up-regulated DEG and sixty-three down-regulated DEGs. Gene ontology enrichment showed that four down-regulated DEGs were significantly enriched in nitrogen metabolic pathway related annotation, three of which were annotated as glutamine synthetase. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment identified three significantly enriched pathways involved in nitrogen and amino acid metabolism.

The α-gliadin genes from Brachypodium distachyon L. provide evidence for a significant gap in the current genome assembly

Brachypodium distachyon, is a new model plant for most cereal crops while gliadin is a class of wheat storage proteins related with wheat quality attributes. In the published B. distachyon genome sequence databases, no gliadin gene is found. In the current study, a number of gliadin genes in B. distachyon were isolated, which is contradictory to the results of genome sequencing projects. In our study, the B. distachyon seeds were found to have no gliadin protein expression by gel electrophoresis, reversed-phase high-performance liquid chromatography and Western blotting analysis. However, Southern blotting revealed a presence of more than ten copies of α-gliadin coding genes in B. distachyon. By means of AS-PCR amplification, four novel full-ORF α-gliadin genes, and 26 pseudogenes with at least one stop codon as well as their promoter regions were cloned and sequenced from different Brachypodium accessions. Sequence analysis revealed a few of single-nucleotide polymorphisms among these genes. Most pseudogenes were resulted from a C to T change, leading to the generation of TAG or TAA in-frame stop codon. To compare both the full-ORFs and the pseudogenes among Triticum and Triticum-related species, their structural characteristics were analyzed. Based on the four T cell stimulatory toxic epitopes and two ployglutamine domains, Aegilops, Triticum, and Brachypodium species were found to be more closely related. The phylogenetic analysis further revealed that B. distachyon was more closely related to Aegilops tauschii, Aegilops umbellulata, and the A or D genome of Triticum aestivum. The α-gliadin genes were able to express successfully in E. coli using the functional T7 promoter. The relative and absolute quantification of the transcripts of α-gliadin genes in wheat was much higher than that in B. distachyon. The abundant pseudogenes may affect the transcriptional and/or posttranscriptional level of the α-gliadin in B. distachyon.

Rapid separation of seed gliadins by reversed-phase ultra performance liquid chromatography (RP-UPLC) and its application in wheat cultivar and germplasm identification

To separate gliadin from wheat flour, a novel and stability-indicating reversed-phase ultra performance liquid chromatography (RP-UPLC) method is established and optimized. A comparative analysis of routine capillary electrophoresis (CE), reversed-phase high-performance liquid chromatography (RP-HPLC), and RP-UPLC was performed and the results showed that the resolution and efficiency of RP-UPLC were significantly higher than those of CE and RP-HPLC. Characteristic RP-UPLC patterns of different bread wheat variety and related species were readily identified. These results demonstrated that our RP-UPLC procedure resulted in significant improvements in sensitivity, speed, and resolution, and thus is highly useful in wheat cultivar and germplasm identification. The novel UP-UPLC method showing high resolution and efficiency in the separation of seed gliadins might be applied to wheat variety and germplasm identification.

High-Throughput Sequencing Reveals H2O2 Stress-Associated MicroRNAs and a Potential Regulatory Network in Brachypodium distachyon Seedlings

Oxidative stress in plants can be triggered by many environmental stress factors, such as drought and salinity. Brachypodium distachyon is a model organism for the study of biofuel plants and crops, such as wheat. Although recent studies have found many oxidative stress response-related proteins, the mechanism of microRNA (miRNA)-mediated oxidative stress response is still unclear. Using next generation high-throughput sequencing technology, the small RNAs were sequenced from the model plant B. distachyon 21 (Bd21) under H2O2 stress and normal growth conditions. In total, 144 known B. distachyon miRNAs and 221 potential new miRNAs were identified. Further analysis of potential new miRNAs suggested that 36 could be clustered into known miRNA families, while the remaining 185 were identified as B. distachyon-specific new miRNAs. Differential analysis of miRNAs from the normal and H2O2 stress libraries identified 31 known and 30 new H2O2 stress responsive miRNAs. The expression patterns of seven representative miRNAs were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, which produced results consistent with those of the deep sequencing method. Moreover, we also performed RT-qPCR analysis to verify the expression levels of 13 target genes and the cleavage site of 5 target genes by known or novel miRNAs were validated experimentally by 5′ RACE. Additionally, a miRNA-mediated gene regulatory network for H2O2 stress response was constructed. Our study identifies a set of H2O2-responsive miRNAs and their target genes and reveals the mechanism of oxidative stress response and defense at the post-transcriptional regulatory level.

Allelic variation of LMW-GS composition in Chinese wheat landraces of the Yangtze-River region detected by MALDI-TOF-MS

Low molecular weight glutenin subunits are important components of wheat storage proteins, which play an important role in determining end-use quality of common wheat. A newly established matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) procedure was used to analyze 478 landraces of bread wheat collected from the Yangtze-River region in China. Results indicated that 17 alleles at three loci: Glu-A3, Glu-B3 and Glu-D3 were identified, resulting in 87 different allele combinations. Of the 17 alleles detected at all the Glu-3 loci, five belonged to Glu-A3, seven to Glu-B3 and five to Glu-D3 locus. MALDI-TOF-MS indicated Glu-A3a/c was present in 72.8%, Glu-A3b in 8.4%, Glu-A3d in 8.4%, Glu-A3f in 5.2% and Glu-A3e in 3.6% lines. Seven types of alleles were identified at the Glu-B3 locus: Glu-B3d/i (25.5%), Glu-B3b (21.3%), Glu-B3c (16.9%), Glu-B3h (13.8%), Glu-B3f (8.4%), Glu-B3a (8.2%), and Glu-B3g (5.2%). Five types of Glu-D3 alleles were detected: Glu-D3a (58.4%), Glu-D3c (22.6%), Glu-D3d (15.5%), Glu-D3b (3.3%) and Glu-D3f (0.2%). Four new alleles that showed abnormal MALDI-TOF spectrum patterns were identified at the Glu-A3 and Glu-B3 loci. A detailed study is needed to further characterize these alleles and their potential usage for wheat improvement.

Applications of capillary electrophoresis for rapidly separating and characterizing water-soluble proteins of wheat grains

Separation and analysis of water-soluble proteins (WSP) are important in understanding wheat grain proteome fundamentals. However, due to their high degree of heterogeneity and complexity in the compositions, separating WSP is generally difficult and relevant methodologies are not efficiently developed yet. Capillary electrophoresis (CE) is one of the analytical methods currently used for protein separation and characterization. In the present study, a CE method is established for rapidly separating and characterizing WSP of wheat grains. The established method was tested in various applications including wheat variety and germplasm identification as well as protein synthesis and accumulation studies during different grain development stages subject to genotypic and environmental variations. As results, the characteristic CE patterns of a range of bread wheat cultivars and related species were readily identified. The synthesis and accumulation patterns of wheat WSP during developing grains as well as their stabilities in different environments were also investigated. The technical advancements present in this article appear to be useful for wheat cultivar and germplasm identification as well as genetics and breeding research.