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Proteomic analysis of middle and late stages of bread wheat (Triticum aestivum L.) grain development.

ABSTRACT: Proteomic approaches were applied in four grain developmental stages of the Chinese bread wheat Yunong 201 and its ethyl methanesulfonate (EMS) mutant line Yunong 3114. 2-DE and tandem MALDI-TOF/TOF-MS analyzed proteome characteristics during middle and late grain development of the Chinese bread wheat Yunong 201 and its EMS mutant line Yunong 3114 with larger grain sizes. We identified 130 differentially accumulated protein spots representing 88 unique proteins, and four main expression patterns displayed a dynamic description of middle and late grain formation. Those identified protein species participated in eight biochemical processes: stress/defense, carbohydrate metabolism, protein synthesis/assembly/degradation, storage proteins, energy production and transportation, photosynthesis, transcription/translation, signal transduction. Comparative proteomic characterization demonstrated 12 protein spots that co-accumulated in the two wheat cultivars with different expression patterns, and six cultivar-specific protein spots including serpin, small heat shock protein, ?-amylase, ?-amylase inhibitor, dimeric ?-amylase inhibitor precursor, and cold regulated protein. These cultivar-specific protein spots possibly resulted in differential yield-related traits of the two wheat cultivars. Our results provide valuable information for dissection of molecular and genetics basis of yield-related traits in bread wheat and the proteomic characterization in this study could also provide insights in the biology of middle and late grain development.

SUBMITTER: Zhang N

PROVIDER: S-EPMC4569854 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Proteomic analysis of middle and late stages of bread wheat (Triticum aestivum L.) grain development.

Zhang Ning N Chen Feng F Huo Wang W Cui Dangqun D

Frontiers in plant science 20150915

Proteomic approaches were applied in four grain developmental stages of the Chinese bread wheat Yunong 201 and its ethyl methanesulfonate (EMS) mutant line Yunong 3114. 2-DE and tandem MALDI-TOF/TOF-MS analyzed proteome characteristics during middle and late grain development of the Chinese bread wheat Yunong 201 and its EMS mutant line Yunong 3114 with larger grain sizes. We identified 130 differentially accumulated protein spots representing 88 unique proteins, and four main expression pattern ...[more]

PMID: 26442048

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Project description:Background and aimsGluten proteins are the major storage protein fraction in the mature wheat grain. They are restricted to the starchy endosperm, which forms white flour on milling, and interact during grain development to form large polymers which form a continuous proteinaceous network when flour is mixed with water to give dough. This network confers viscosity and elasticity to the dough, enabling the production of leavened products. The starchy endosperm is not a homogeneous tissue and quantitative and qualitative gradients exist for the major components: protein, starch and cell wall polysaccharides. Gradients in protein content and composition are the most evident and are of particular interest because of the major role played by the gluten proteins in determining grain processing quality.MethodsProtein gradients in the starchy endosperm were investigated using antibodies for specific gluten protein types for immunolocalization in developing grains and for western blot analysis of protein extracts from flour fractions obtained by sequential abrasion (pearling) to prepare tissue layers.Key resultsDifferential patterns of distribution were found for the high-molecular-weight subunits of glutenin (HMW-GS) and γ-gliadins when compared with the low-molecular-weight subunits of glutenin (LMW-GS), ω- and α-gliadins. The first two types of gluten protein are more abundant in the inner endosperm layers and the latter more abundant in the subaleurone. Immunolocalization also showed that segregation of gluten proteins occurs both between and within protein bodies during protein deposition and may still be retained in the mature grain.ConclusionsQuantitative and qualitative gradients in gluten protein composition are established during grain development. These gradients may be due to the origin of subaleurone cells, which unlike other starchy endosperm cells derive from the re-differentiation of aleurone cells, but could also result from the action of specific regulatory signals produced by the maternal tissue on specific domains of the gluten protein gene promoters.

Project description:BACKGROUND:High post-anthesis (p.a) temperatures reduce mature grain weights in wheat and other cereals. However, the causes of this reduction are not entirely known. Control of grain expansion by the maternally derived pericarp of the grain has previously been suggested, although this interaction has not been investigated under high p.a. temperatures. Down-regulation of pericarp localised genes that regulate cell wall expansion under high p.a. temperatures may limit expansion of the encapsulated endosperm due to a loss of plasticity in the pericarp, reducing mature grain weight. Here the effect of high p.a. temperatures on the transcriptome of the pericarp and endosperm of the wheat grain during early grain-filling was investigated via RNA-Seq and is discussed alongside grain moisture dynamics during early grain development and mature grain weight. RESULTS:High p.a. temperatures applied from 6-days after anthesis (daa) and until 18daa reduced the grain's ability to accumulate water, with total grain moisture and percentage grain moisture content being significantly reduced from 14daa onwards. Mature grain weight was also significantly reduced by the same high p.a. temperatures applied from 6daa for 4-days or more, in a separate experiment. Comparison of our RNA-Seq data from whole grains, with existing data sets from isolated pericarp and endosperm tissues enabled the identification of subsets of genes whose expression was significantly affected by high p.a. temperature and predominantly expressed in either tissue. Hierarchical clustering and gene ontology analysis resulted in the identification of a number of genes implicated in the regulation of cell wall expansion, predominantly expressed in the pericarp and significantly down-regulated under high p.a. temperatures, including endoglucanase, xyloglucan endotransglycosylases and a ?-expansin. An over-representation of genes involved in the 'cuticle development' functional pathway that were expressed in the pericarp and affected by high p.a. temperatures was also observed. CONCLUSIONS:High p.a. temperature induced down-regulation of genes involved in regulating pericarp cell wall expansion. This concomitant down-regulation with a reduction in total grain moisture content and grain weight following the same treatment period, adds support to the theory that high p.a. temperatures may cause a reduction in mature grain weight as result of decreased pericarp cell wall expansion.

Project description:Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M(2) mutants in a common wheat cultivar 'Jinmai 47'. Numerous phenotypes with altered morphological and agronomic traits were observed from the M(2) and M(3) lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.

Project description:BackgroundThe analyses of protein synthesis, accumulation and regulation during grain development in wheat are more complex because of its larger genome size compared to model plants such as Arabidopsis and rice. In this study, grains from two wheat cultivars Jimai 20 and Zhoumai 16 with different gluten quality properties were harvested at five development stages, and were used to displayed variable expression patterns of grain proteins.ResultsProteome characterization during grain development in Chinese bread wheat cultivars Jimai 20 and Zhoumai 16 with different quality properties was investigated by 2-DE and tandem MALDI-TOF/TOF-MS. Identification of 117 differentially accumulated protein spots representing 82 unique proteins and five main expression patterns enabled a chronological description of wheat grain formation. Significant proteome expression differences between the two cultivars were found; these included 14 protein spots that accumulated in both cultivars but with different patterns and 27 cultivar-different spots. Among the cultivar-different protein spots, 14 accumulated in higher abundance in Jimai 20 than in Zhoumai 16, and included NAD-dependent isocitrate dehydrogenase, triticin precursor, LMW-s glutenin subunit and replication factor C-like protein. These proteins are likely to be associated with superior gluten quality. In addition, some proteins such as class II chitinase and peroxidase 1 with isoforms in developing grains were shown to be phosphorylated by Pro-Q Diamond staining and phosphorprotein site prediction. Phosphorylation could have important roles in wheat grain development. qRT-PCR analysis demonstrated that transcriptional and translational expression patterns of many genes were significantly different.ConclusionsWheat grain proteins displayed variable expression patterns at different developmental stages and a considerable number of protein spots showed differential accumulation between two cultivars. Differences in seed storage proteins were considered to be related to different quality performance of the flour from these wheat cultivars. Some proteins with isoforms were phosphorylated, and this may reflect their importance in grain development. Our results provide new insights into proteome characterization during grain development in different wheat genotypes.

Project description:BACKGROUND:Celiac disease (CD) is caused by specific sequences of gluten proteins found in cereals such as bread wheat (Triticum aestivum ssp. aestivum) and spelt (T. aestivum ssp. spelta). Among them, the ?-gliadins display the highest immunogenicity, with four T-cell stimulatory epitopes. The toxicity of each epitope sequence can be reduced or even suppressed according to the allelic form of each sequence. One way to address the CD problem would be to make use of this allelic variability in breeding programs to develop safe varieties, but tools to track the presence of toxic epitopes are required. The objective of this study was to develop a tool to accurately detect and quantify the immunogenic content of expressed ?-gliadins of spelt and bread wheat. RESULTS:Four TaqMan probes that only hybridize to the canonical-i.e. toxic-form of each of the four epitopes were developed and their specificity was demonstrated. Six TaqMan probes targeting stable reference genes were also developed and constitute a tool to normalize qPCR data. The probes were used to measure the epitope expression levels of 11 contrasted spelt accessions and three ancestral diploid accessions of bread wheat and spelt. A high expression variability was highlighted among epitopes and among accessions, especially in Asian spelts, which showed lower epitope expression levels than the other spelts. Some discrepancies were identified between the canonical epitope expression level and the global amount of expressed ?-gliadins, which makes the designed TaqMan probes a useful tool to quantify the immunogenic potential independently of the global amount of expressed ?-gliadins. CONCLUSIONS:The results obtained in this study provide useful tools to study the immunogenic potential of expressed ?-gliadin sequences from Triticeae accessions such as spelt and bread wheat. The application of the designed probes to contrasted spelt accessions revealed a high variability and interesting low canonical epitope expression levels in the Asian spelt accessions studied.

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Proteomic analysis of middle and late stages of bread wheat (Triticum aestivum L.) grain development.

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Proteomic analysis of middle and late stages of bread wheat (Triticum aestivum L.) grain development.

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