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author:("Yan, kunming")
1.  Deletion of the low-molecular-weight glutenin subunit allele Glu-A3a of wheat (Triticum aestivum L.) significantly reduces dough strength and breadmaking quality 
BMC Plant Biology  2014;14(1):367.
Low-molecular-weight glutenin subunits (LMW-GS), encoded by Glu-3 complex loci in hexaploid wheat, play important roles in the processing quality of wheat flour. To date, the molecular characteristics and effects on dough quality of individual Glu-3 alleles and their encoding proteins have been poorly studied. We used a Glu-A3 deletion line of the Chinese Spring (CS-n) wheat variety to conduct the first comprehensive study on the molecular characteristics and functional properties of the LMW-GS allele Glu-A3a.
The Glu-A3a allele at the Glu-A3 locus in CS and its deletion in CS-n were identified and characterized by proteome and molecular marker methods. The deletion of Glu-A3a had no significant influence on plant morphological and yield traits, but significantly reduced the dough strength and breadmaking quality compared to CS. The complete sequence of the Glu-A3a allele was cloned and characterized, which was found to encode a B-subunit with longer repetitive domains and an increased number of α-helices. The Glu-A3a-encoded B-subunit showed a higher expression level and accumulation rate during grain development. These characteristics of the Glu-A3a allele could contribute to achieving superior gluten quality and demonstrate its potential application to wheat quality improvement. Furthermore, an allele-specific polymerase chain reaction (AS-PCR) marker for the Glu-A3a allele was developed and validated using different bread wheat cultivars, including near-isogenic lines (NILs) and recombinant inbred lines (RILs), which could be used as an effective molecular marker for gluten quality improvement through marker-assisted selection.
This work demonstrated that the LMW-GS allele Glu-A3a encodes a specific LMW-i type B-subunit that significantly affects wheat dough strength and breadmaking quality. The Glu-A3a-encoded B-subunit has a long repetitive domain and more α-helix structures as well as a higher expression level and accumulation rate during grain development, which could facilitate the formation of wheat with a stronger dough structure and superior breadmaking quality.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0367-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4275963  PMID: 25524150
Wheat; Glu-A3a; Molecular cloning; Dough strength; Breadmaking quality
2.  Unusual tandem expansion and positive selection in subgroups of the plant GRAS transcription factor superfamily 
BMC Plant Biology  2014;14(1):373.
GRAS proteins belong to a plant transcription factor family that is involved with multifarious roles in plants. Although previous studies of this protein family have been reported for Arabidopsis, rice, Chinese cabbage and other species, investigation of expansion patterns and evolutionary rate on the basis of comparative genomics in different species remains inadequate.
A total of 289 GRAS genes were identified in Arabidopsis, B. distachyon, rice, soybean, S. moellendorffii, and P. patens and were grouped into seven subfamilies, supported by the similarity of their exon–intron patterns and structural motifs. All of tandem duplicated genes were found in group II except one cluster of rice, indicating that tandem duplication greatly promoted the expansion of group II. Furthermore, segment duplications were mainly found in the soybean genome, whereas no single expansion pattern dominated in other plant species indicating that GRAS genes from these five species might be subject to a more complex evolutionary mechanism. Interestingly, branch-site model analyses of positive selection showed that a number of sites were positively selected under foreground branches I and V. These results strongly indicated that these groups were experiencing higher positive selection pressure. Meanwhile, the site-specific model revealed that the GRAS genes were under strong positive selection in P. patens. DIVERGE v2.0 was used to detect critical amino acid sites, and the results showed that the shifted evolutionary rate was mainly attributed to the functional divergence between the GRAS genes in the two groups. In addition, the results also demonstrated the expression divergence of the GRAS duplicated genes in the evolution. In short, the results above provide a solid foundation for further functional dissection of the GRAS gene superfamily.
In this work, differential expression, evolutionary rate, and expansion patterns of the GRAS gene family in the six species were predicted. Especially, tandem duplication events played an important role in expansion of group II. Together, these results contribute to further functional analysis and the molecular evolution of the GRAS gene superfamily.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0373-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4279901  PMID: 25524588
3.  iTRAQ-based quantitative proteome and phosphoprotein characterization reveals the central metabolism changes involved in wheat grain development 
BMC Genomics  2014;15(1):1029.
Wheat (Triticum aestivum L.) is an economically important grain crop. Two-dimensional gel-based approaches are limited by the low identification rate of proteins and lack of accurate protein quantitation. The recently developed isobaric tag for relative and absolute quantitation (iTRAQ) method allows sensitive and accurate protein quantification. Here, we performed the first iTRAQ-based quantitative proteome and phosphorylated proteins analyses during wheat grain development.
The proteome profiles and phosphoprotein characterization of the metabolic proteins during grain development of the elite Chinese bread wheat cultivar Yanyou 361 were studied using the iTRAQ-based quantitative proteome approach, TiO2 microcolumns, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among 1,146 non-redundant proteins identified, 421 showed at least 2-fold differences in abundance, and they were identified as differentially expressed proteins (DEPs), including 256 upregulated and 165 downregulated proteins. Of the 421 DEPs, six protein expression patterns were identified, most of which were up, down, and up-down expression patterns. The 421 DEPs were classified into nine functional categories mainly involved in different metabolic processes and located in the membrane and cytoplasm. Hierarchical clustering analysis indicated that the DEPs involved in starch biosynthesis, storage proteins, and defense/stress-related proteins significantly accumulated at the late grain development stages, while those related to protein synthesis/assembly/degradation and photosynthesis showed an opposite expression model during grain development. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 12 representative genes encoding different metabolic proteins showed certain transcriptional and translational expression differences during grain development. Phosphorylated proteins analyses demonstrated that 23 DEPs such as AGPase, sucrose synthase, Hsp90, and serpins were phosphorylated in the developing grains and were mainly involved in starch biosynthesis and stress/defense.
Our results revealed a complex quantitative proteome and phosphorylation profile during wheat grain development. Numerous DEPs are involved in grain starch and protein syntheses as well as adverse defense, which set an important basis for wheat yield and quality. Particularly, some key DEPs involved in starch biosynthesis and stress/defense were phosphorylated, suggesting their roles in wheat grain development.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1029) contains supplementary material, which is available to authorized users.
PMCID: PMC4301063  PMID: 25427527
Wheat; Grain proteome; iTRAQ; Phosphoproteins; qRT-PCR
4.  Molecular cloning, phylogenetic analysis, and expression profiling of endoplasmic reticulum molecular chaperone BiP genes from bread wheat (Triticum aestivum L.) 
BMC Plant Biology  2014;14(1):260.
The 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.
Sequence 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.
The 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 material
The online version of this article (doi:10.1186/s12870-014-0260-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4189733  PMID: 25273817
Wheat; BiP; Cloning; Expression; HMW-GS silencing; Drought stress
5.  Dynamic development of starch granules and the regulation of starch biosynthesis in Brachypodium distachyon: comparison with common wheat and Aegilops peregrina 
BMC Plant Biology  2014;14(1):198.
Thorough understanding of seed starch biosynthesis and accumulation mechanisms is of great importance for agriculture and crop improvement strategies. We conducted the first comprehensive study of the dynamic development of starch granules and the regulation of starch biosynthesis in Brachypodium distachyon and compared the findings with those reported for common wheat (Chinese Spring, CS) and Aegilops peregrina.
Only B-granules were identified in Brachypodium Bd21, and the shape variation and development of starch granules were similar in the B-granules of CS and Bd21. Phylogenetic analysis showed that most of the Bd21 starch synthesis-related genes were more similar to those in wheat than in rice. Early expression of key genes in Bd21 starch biosynthesis mediate starch synthesis in the pericarp; intermediate-stage expression increases the number and size of starch granules. In contrast, these enzymes in CS and Ae. peregrina were mostly expressed at intermediate stages, driving production of new B-granules and increasing the granule size, respectively. Immunogold labeling showed that granule-bound starch synthase (GBSSI; related to amylose synthesis) was mainly present in starch granules: at lower levels in the B-granules of Bd21 than in CS. Furthermore, GBSSI was phosphorylated at threonine 183 and tyrosine 185 in the starch synthase catalytic domain in CS and Ae. peregrina, but neither site was phosphorylated in Bd21, suggesting GBSSI phosphorylation could improve amylose biosynthesis.
Bd21 contains only B-granules, and the expression of key genes in the three studied genera is consistent with the dynamic development of starch granules. GBSSI is present in greater amounts in the B-granules of CS than in Bd21; two phosphorylation sites (Thr183 and Tyr185) were found in Triticum and Aegilops; these sites were not phosphorylated in Bd21. GBSSI phosphorylation may reflect its importance in amylose synthesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0198-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4256708  PMID: 25095703
Brachypodium Bd21; B-granules; Starch biosynthesis; Expression profiling; GBSSI; Phosphorylation
6.  Molecular Characterization and Expression Profiling of the Protein Disulfide Isomerase Gene Family in Brachypodium distachyon L 
PLoS ONE  2014;9(4):e94704.
Protein disulfide isomerases (PDI) are involved in catalyzing protein disulfide bonding and isomerization in the endoplasmic reticulum and functions as a chaperone to inhibit the aggregation of misfolded proteins. Brachypodium distachyon is a widely used model plant for temperate grass species such as wheat and barley. In this work, we report the first molecular characterization, phylogenies, and expression profiles of PDI and PDI-like (PDIL) genes in B. distachyon in different tissues under various abiotic stresses. Eleven PDI and PDIL genes in the B. distachyon genome by in silico identification were evenly distributed across all five chromosomes. The plant PDI family has three conserved motifs that are involved in catalyzing protein disulfide bonding and isomerization, but a different exon/intron structural organization showed a high degree of structural differentiation. Two pairs of genes (BdPDIL4-1 and BdPDIL4-2; BdPDIL7-1 and BdPDIL7-2) contained segmental duplications, indicating each pair originated from one progenitor. Promoter analysis showed that Brachypodium PDI family members contained important cis-acting regulatory elements involved in seed storage protein synthesis and diverse stress response. All Brachypodium PDI genes investigated were ubiquitously expressed in different organs, but differentiation in expression levels among different genes and organs was clear. BdPDIL1-1 and BdPDIL5-1 were expressed abundantly in developing grains, suggesting that they have important roles in synthesis and accumulation of seed storage proteins. Diverse treatments (drought, salt, ABA, and H2O2) induced up- and down-regulated expression of Brachypodium PDI genes in seedling leaves. Interestingly, BdPDIL1-1 displayed significantly up-regulated expression following all abiotic stress treatments, indicating that it could be involved in multiple stress responses. Our results provide new insights into the structural and functional characteristics of the plant PDI gene family.
PMCID: PMC3991636  PMID: 24747843
7.  Soybean (Glycine max) expansin gene superfamily origins: segmental and tandem duplication events followed by divergent selection among subfamilies 
BMC Plant Biology  2014;14:93.
Expansins are plant cell wall loosening proteins that are involved in cell enlargement and a variety of other developmental processes. The expansin superfamily contains four subfamilies; namely, α-expansin (EXPA), β-expansin (EXPB), expansin-like A (EXLA), and expansin-like B (EXLB). Although the genome sequencing of soybeans is complete, our knowledge about the pattern of expansion and evolutionary history of soybean expansin genes remains limited.
A total of 75 expansin genes were identified in the soybean genome, and grouped into four subfamilies based on their phylogenetic relationships. Structural analysis revealed that the expansin genes are conserved in each subfamily, but are divergent among subfamilies. Furthermore, in soybean and Arabidopsis, the expansin gene family has been mainly expanded through tandem and segmental duplications; however, in rice, segmental duplication appears to be the dominant process that generates this superfamily. The transcriptome atlas revealed notable differential expression in either transcript abundance or expression patterns under normal growth conditions. This finding was consistent with the differential distribution of the cis-elements in the promoter region, and indicated wide functional divergence in this superfamily. Moreover, some critical amino acids that contribute to functional divergence and positive selection were detected. Finally, site model and branch-site model analysis of positive selection indicated that the soybean expansin gene superfamily is under strong positive selection, and that divergent selection constraints might have influenced the evolution of the four subfamilies.
This study demonstrated that the soybean expansin gene superfamily has expanded through tandem and segmental duplication. Differential expression indicated wide functional divergence in this superfamily. Furthermore, positive selection analysis revealed that divergent selection constraints might have influenced the evolution of the four subfamilies. In conclusion, the results of this study contribute novel detailed information about the molecular evolution of the expansin gene superfamily in soybean.
PMCID: PMC4021193  PMID: 24720629
8.  Transcriptome analysis during seed germination of elite Chinese bread wheat cultivar Jimai 20 
BMC Plant Biology  2014;14:20.
Wheat seed germination directly affects wheat yield and quality. Although transcriptome and proteome analyses during seed germination have been reported in some crop plant species, dynamic transcriptome characterization during wheat seed germination has not been conducted. We performed the first comprehensive dynamic transcriptome analysis during different seed germination stages of elite Chinese bread wheat cultivar Jimai 20 using the Affymetrix Wheat Genome Array.
A total of 61,703 probe sets representing 51,411 transcripts were identified during the five seed germination stages of Jimai 20, of which 2,825 differential expression probe sets corresponding to 2,646 transcripts with different functions were declared by ANOVA and a randomized variance model. The seed germination process included a rapid initial uptake phase (0–12 hours after imbibition [HAI]), a plateau phase (12–24 HAI), and a further water uptake phase (24–48 HAI), corresponding to switches from the degradation of small-molecule sucrose to the metabolism of three major nutrients and to photosynthesis. Hierarchical cluster and MapMan analyses revealed changes in several significant metabolism pathways during seed germination as well as related functional groups. The signal pathway networks constructed with KEGG showed three important genes encoding the phosphofructokinase family protein, with fructose-1, 6-bisphosphatase, and UTP-glucose-1-phosphate uridylyltransferase located at the center, indicating their pivotal roles in the glycolytic pathway, gluconeogenesis, and glycogenesis, respectively. Several significant pathways were selected to establish a metabolic pathway network according to their degree value, which allowed us to find the pathways vital to seed germination. Furthermore, 51 genes involved in transport, signaling pathway, development, lipid metabolism, defense response, nitrogen metabolism, and transcription regulation were analyzed by gene co-expression network with a k-core algorithm to determine which play pivotal roles in germination. Twenty-three meaningful genes were found, and quantitative RT-PCR analysis validated the expression patterns of 12 significant genes.
Wheat seed germination comprises three distinct phases and includes complicated regulation networks involving a large number of genes. These genes belong to many functional groups, and their co-regulations guarantee regular germination. Our results provide new insight into metabolic changes during seed germination and interactions between some significant genes.
PMCID: PMC3923396  PMID: 24410729
Bread wheat; Seed germination; Transcriptome; qRT-PCR
9.  Global Analysis of Differentially Expressed Genes and Proteins in the Wheat Callus Infected by Agrobacterium tumefaciens 
PLoS ONE  2013;8(11):e79390.
Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops.
PMCID: PMC3835833  PMID: 24278131
10.  The large soybean (Glycine max) WRKY TF family expanded by segmental duplication events and subsequent divergent selection among subgroups 
BMC Plant Biology  2013;13:148.
WRKY genes encode one of the most abundant groups of transcription factors in higher plants, and its members regulate important biological process such as growth, development, and responses to biotic and abiotic stresses. Although the soybean genome sequence has been published, functional studies on soybean genes still lag behind those of other species.
We identified a total of 133 WRKY members in the soybean genome. According to structural features of their encoded proteins and to the phylogenetic tree, the soybean WRKY family could be classified into three groups (groups I, II, and III). A majority of WRKY genes (76.7%; 102 of 133) were segmentally duplicated and 13.5% (18 of 133) of the genes were tandemly duplicated. This pattern was not apparent in Arabidopsis or rice. The transcriptome atlas revealed notable differential expression in either transcript abundance or in expression patterns under normal growth conditions, which indicated wide functional divergence in this family. Furthermore, some critical amino acids were detected using DIVERGE v2.0 in specific comparisons, suggesting that these sites have contributed to functional divergence among groups or subgroups. In addition, site model and branch-site model analyses of positive Darwinian selection (PDS) showed that different selection regimes could have affected the evolution of these groups. Sites with high probabilities of having been under PDS were found in groups I, II c, II e, and III. Together, these results contribute to a detailed understanding of the molecular evolution of the WRKY gene family in soybean.
In this work, all the WRKY genes, which were generated mainly through segmental duplication, were identified in the soybean genome. Moreover, differential expression and functional divergence of the duplicated WRKY genes were two major features of this family throughout their evolutionary history. Positive selection analysis revealed that the different groups have different evolutionary rates. Together, these results contribute to a detailed understanding of the molecular evolution of the WRKY gene family in soybean.
PMCID: PMC3850935  PMID: 24088323
11.  Correction: Molecular Mechanisms of HMW Glutenin Subunits from 1Sl Genome of Aegilops longissima Positively Affecting Wheat Breadmaking Quality 
PLoS ONE  2013;8(6):10.1371/annotation/d86a2c61-0953-4555-95bc-1ae9b8e3452c.
PMCID: PMC3734319
12.  Molecular Mechanisms of HMW Glutenin Subunits from 1Sl Genome of Aegilops longissima Positively Affecting Wheat Breadmaking Quality 
PLoS ONE  2013;8(4):e58947.
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.
PMCID: PMC3617193  PMID: 23593125
13.  Molecular characterization of LMW-GS genes in Brachypodium distachyon L. reveals highly conserved Glu-3 loci in Triticum and related species 
BMC Plant Biology  2012;12:221.
Brachypodium 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.
SDS-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.
Brachypodium 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.
PMCID: PMC3547698  PMID: 23171363
14.  Proteome characterization of developing grains in bread wheat cultivars (Triticum aestivum L.) 
BMC Plant Biology  2012;12:147.
The 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.
Proteome 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.
Wheat 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.
PMCID: PMC3480910  PMID: 22900893
Wheat; Grain proteome; Phosphorproteins; 2-DE; Tandem MS; qRT-PCR
15.  Screening and identification of seed-specific genes using digital differential display tools combined with microarray data from common wheat 
BMC Genomics  2011;12:513.
Wheat is one of the most important cereal crops for human beings, with seeds being the tissue of highly economic value. Various morphogenetic and metabolic processes are exclusively associated with seed maturation. The goal of this study was to screen and identify genes specifically expressed in the developing seed of wheat with an integrative utilization of digital differential display (DDD) and available online microarray databases.
A total of 201 unigenes were identified as the results of DDD screening and microarray database searching. The expressions of 6 of these were shown to be seed-specific by qRT-PCR analysis. Further GO enrichment analysis indicated that seed-specific genes were mainly associated with defense response, response to stress, multi-organism process, pathogenesis, extracellular region, nutrient reservoir activity, enzyme inhibitor activity, antioxidant activity and oxidoreductase activity. A comparison of this set of genes with the rice (Oryza sativa) genome was also performed and approximately three-fifths of them have rice counterparts. Between the counterparts, around 63% showed similar expression patterns according to the microarray data.
In conclusion, the DDD screening combined with microarray data analysis is an effective strategy for the identification of seed-specific expressed genes in wheat. These seed-specific genes screened during this study will provide valuable information for further studies about the functions of these genes in wheat.
PMCID: PMC3206523  PMID: 22003838
16.  Genome-scale identification of Soybean BURP domain-containing genes and their expression under stress treatments 
BMC Plant Biology  2010;10:197.
Multiple proteins containing BURP domain have been identified in many different plant species, but not in any other organisms. To date, the molecular function of the BURP domain is still unknown, and no systematic analysis and expression profiling of the gene family in soybean (Glycine max) has been reported.
In this study, multiple bioinformatics approaches were employed to identify all the members of BURP family genes in soybean. A total of 23 BURP gene types were identified. These genes had diverse structures and were distributed on chromosome 1, 2, 4, 6, 7, 8, 11, 12, 13, 14, and 18. Phylogenetic analysis suggested that these BURP family genes could be classified into 5 subfamilies, and one of which defines a new subfamily, BURPV. Quantitative real-time PCR (qRT-PCR) analysis of transcript levels showed that 15 of the 23 genes had no expression specificity; 7 of them were specifically expressed in some of the tissues; and one of them was not expressed in any of the tissues or organs studied. The results of stress treatments showed that 17 of the 23 identified BURP family genes responded to at least one of the three stress treatments; 6 of them were not influenced by stress treatments even though a stress related cis-element was identified in the promoter region. No stress related cis-elements were found in promoter region of any BURPV member. However, qRT-PCR results indicated that all members from BURPV responded to at least one of the three stress treatments. More significantly, the members from the RD22-like subfamily showed no tissue-specific expression and they all responded to each of the three stress treatments.
We have identified and classified all the BURP domain-containing genes in soybean. Their expression patterns in different tissues and under different stress treatments were detected using qRT-PCR. 15 out of 23 BURP genes in soybean had no tissue-specific expression, while 17 out of them were stress-responsive. The data provided an insight into the evolution of the gene family and suggested that many BURP family genes may be important for plants responding to stress conditions.
PMCID: PMC2956546  PMID: 20836857
17.  Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat 
BMC Plant Biology  2010;10:124.
Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in determining end-use quality of common wheat by influencing the viscoelastic properties of dough. Four different methods - sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE, IEF × SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR), were used to characterize the LMW-GS composition in 103 cultivars from 12 countries.
At the Glu-A3 locus, all seven alleles could be reliably identified by 2-DE and PCR. However, the alleles Glu-A3e and Glu-A3d could not be routinely distinguished from Glu-A3f and Glu-A3g, respectively, based on SDS-PAGE, and the allele Glu-A3a could not be differentiated from Glu-A3c by MALDI-TOF-MS. At the Glu-B3 locus, alleles Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3g, Glu-B3h and Glu-B3j could be clearly identified by all four methods, whereas Glu-B3ab, Glu-B3ac, Glu-B3ad could only be identified by the 2-DE method. At the Glu-D3 locus, allelic identification was problematic for the electrophoresis based methods and PCR. MALDI-TOF-MS has the potential to reliably identify the Glu-D3 alleles.
PCR is the simplest, most accurate, lowest cost, and therefore recommended method for identification of Glu-A3 and Glu-B3 alleles in breeding programs. A combination of methods was required to identify certain alleles, and would be especially useful when characterizing new alleles. A standard set of 30 cultivars for use in future studies was chosen to represent all LMW-GS allelic variants in the collection. Among them, Chinese Spring, Opata 85, Seri 82 and Pavon 76 were recommended as a core set for use in SDS-PAGE gels. Glu-D3c and Glu-D3e are the same allele. Two new alleles, namely, Glu-D3m in cultivar Darius, and Glu-D3n in Fengmai 27, were identified by 2-DE. Utilization of the suggested standard cultivar set, seed of which is available from the CIMMYT and INRA Clermont-Ferrand germplasm collections, should also promote information sharing in the identification of individual LMW-GS and thus provide useful information for quality improvement in common wheat.
PMCID: PMC3017774  PMID: 20573275

Results 1-17 (17)