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, β-amylase, α-amylase inhibitor, dimeric α-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.
Triticum aestivum L.; EMS; grain development; grain size; proteome
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.
Wheat; Grain proteome; iTRAQ; Phosphoproteins; qRT-PCR
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.
Wheat; Glu-A3a; Molecular cloning; Dough strength; Breadmaking quality
Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences.
Grain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alpha-gliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%.
This is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar-specific contigs helped to overcome the difficulties of matching peptides to gene sequences for members of highly similar, rapidly evolving storage protein families. Prospects for simplifying this process for routine analyses are discussed. The ability to measure expression levels for individual flour protein genes complements information gained from efforts to sequence the wheat genome and is essential for studies of effects of environment on gene expression.
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.
Bread wheat; Seed germination; Transcriptome; qRT-PCR
Wheat seeds provide a staple food and an important protein source for the world’s population. Seed germination is vital to wheat growth and development and directly affects grain yield and quality. In this study, we performed the first comparative proteomic analysis of wheat embryo and endosperm during seed germination.
The proteomic changes in embryo and endosperm during the four different seed germination stages of elite Chinese bread wheat cultivar Zhengmai 9023 were first investigated. In total, 74 and 34 differentially expressed protein (DEP) spots representing 63 and 26 unique proteins were identified in embryo and endosperm, respectively. Eight common DEP were present in both tissues, and 55 and 18 DEP were specific to embryo and endosperm, respectively. These identified DEP spots could be sorted into 13 functional groups, in which the main group was involved in different metabolism pathways, particularly in the reserves necessary for mobilization in preparation for seed germination. The DEPs from the embryo were mainly related to carbohydrate metabolism, proteometabolism, amino acid metabolism, nucleic acid metabolism, and stress-related proteins, whereas those from the endosperm were mainly involved in protein storage, carbohydrate metabolism, inhibitors, stress response, and protein synthesis. During seed germination, both embryo and endosperm had a basic pattern of oxygen consumption, so the proteins related to respiration and energy metabolism were up-regulated or down-regulated along with respiration of wheat seeds. When germination was complete, most storage proteins from the endosperm began to be mobilized, but only a small amount was degraded during germination. Transcription expression of six representative DEP genes at the mRNA level was consistent with their protein expression changes.
Wheat seed germination is a complex process with imbibition, stirring, and germination stages, which involve a series of physiological, morphological, and proteomic changes. The first process is a rapid water uptake, in which the seed coat becomes softer and the physical state of storage materials change gradually. Then the germinated seed enters the second process (a plateau phase) and the third process (the embryonic axes elongation). Seed embryo and endosperm display distinct differentially expressed proteins, and their synergistic expression mechanisms provide a basis for the normal germination of wheat seeds.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0471-z) contains supplementary material, which is available to authorized users.
Wheat seeds; Germination; Embryo; Endosperm; Proteome; qRT-PCR
Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD.
A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 ‘unique deduced protein fragments’ of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes.
The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains.
Wheat grains accumulate a variety of low molecular weight proteins that are inhibitors of alpha-amylases and proteases and play an important protective role in the grain. These proteins have more balanced amino acid compositions than the major wheat gluten proteins and contribute important reserves for both seedling growth and human nutrition. The alpha-amylase/protease inhibitors also are of interest because they cause IgE-mediated occupational and food allergies and thereby impact human health.
The complement of genes encoding alpha-amylase/protease inhibitors expressed in the US bread wheat Butte 86 was characterized by analysis of expressed sequence tags (ESTs). Coding sequences for 19 distinct proteins were identified. These included two monomeric (WMAI), four dimeric (WDAI), and six tetrameric (WTAI) inhibitors of exogenous alpha-amylases, two inhibitors of endogenous alpha-amylases (WASI), four putative trypsin inhibitors (CMx and WTI), and one putative chymotrypsin inhibitor (WCI). A number of the encoded proteins were identical or very similar to proteins in the NCBI database. Sequences not reported previously included variants of WTAI-CM3, three CMx inhibitors and WTI. Within the WDAI group, two different genes encoded the same mature protein. Based on numbers of ESTs, transcripts for WTAI-CM3 Bu-1, WMAI Bu-1 and WTAI-CM16 Bu-1 were most abundant in Butte 86 developing grain. Coding sequences for 16 of the inhibitors were unequivocally associated with specific proteins identified by tandem mass spectrometry (MS/MS) in a previous proteomic analysis of milled white flour from Butte 86. Proteins corresponding to WDAI Bu-1/Bu-2, WMAI Bu-1 and the WTAI subunits CM2 Bu-1, CM3 Bu-1 and CM16 Bu-1 were accumulated to the highest levels in flour.
Information on the spectrum of alpha-amylase/protease inhibitor genes and proteins expressed in a single wheat cultivar is central to understanding the importance of these proteins in both plant defense mechanisms and human allergies and facilitates both breeding and biotechnology approaches for manipulating the composition of these proteins in plants.
allergens; expressed sequence tags; plant defense proteins; tandem mass spectrometry
Grain texture is one of the most important characteristics in bread wheat (Triticum aestivum L.). Puroindoline-D1 genes play the main role in controlling grain texture and are intimately associated with the milling and processing qualities in bread wheat.
A series of diagnostic molecular markers and dCAPS markers were used to characterize Pina-D1 and Pinb-D1 in 493 wheat cultivars from diverse geographic locations. A primer walking strategy was used to characterize PINA-null alleles at the DNA level. Results indicated that Chinese landraces encompassing 12 different Puroindoline-D1 allelic combinations showed the highest diversity, while CIMMYT wheat cultivars containing 3 different Puroindoline-D1 allelic combinations showed the lowest diversity amongst wheat cultivars from the five countries surveyed. Two novel Pina-D1 alleles, designated Pina-D1s with a 4,422-bp deletion and Pina-D1u with a 6,460-bp deletion in the Ha (Hardness) locus, were characterized at the DNA level by a primer walking strategy, and corresponding molecular markers Pina-N3 and Pina-N4 were developed for straightforward identification of the Pina-D1s and Pina-D1u alleles. Analysis of the association of Puroindoline-D1 alleles with grain texture indicated that wheat cultivars with Pina-null/Pinb-null allele, possessing an approximate 33-kb deletion in the Ha locus, have the highest SKCS hardness index amongst the different genotypes used in this study. Moreover, wheat cultivars with the PINA-null allele have significantly higher SKCS hardness index than those of Pinb-D1b and Pinb-D1p alleles.
Molecular characterization of the Puroindoline-D1 allele was investigated in bread wheat cultivars from five geographic regions, resulting in the discovery of two new alleles - Pina-D1s and Pina-D1u. Molecular markers were developed for both alleles. Analysis of the association of the Puroindoline-D1 alleles with grain texture showed that cultivars with PINA-null allele possessed relatively high SKCS hardness index. This study can provide useful information for the improvement of wheat quality, as well as give a deeper understanding of the molecular and genetic processes controlling grain texture in bread wheat.
Bread wheat (Triticum aestivum L.); Puroindoline-D1 genes; Grain texture; Primer walking; Functional marker
In northern China, large-spike wheat (Triticum aestivum L) is considered to have significant potential for increasing yields due to its greater single-plant productivity despite its lower percentage of effective tillers, and increasing the plant density is an effective means of achieving a higher grain yield. However, with increases in plant density, the amount of solar radiation intercepted by lower strata leaves is decreased and the rate of leaf senescence is accelerated. Row spacing can be manipulated to optimize the plant spatial distribution under high plant density, therefore improving light conditions within the canopy. Consequently, field experiments were conducted from 2010 to 2012 to investigate whether changes in row spacing under high plant density led to differences in canopy apparent photosynthesis (CAP), individual leaf photosynthesis and grain yield. Two different spike-type winter wheat cultivars, Jimai22 (a small-spike cultivar as a control cultivar) and Wennong6 (a large-spike cultivar), were grown at a constant plant density of 3,600,000 plants ha–1 (a relatively higher plant density) over a wide range of row spacing as follows: 5-cm row spacing (R0), 15-cm row spacing (R1), 25-cm conventional row spacing (R2), and 35-cm row spacing (R3). The two-year investigations revealed that increased row spacing exhibited a significantly higher light transmission ratio (LT), which improved light conditions within the canopy; however, excessive light leakage losses in R2 and R3 treatments were not favorable to improved irradiation energy utilization efficiency. Aboveground biomass accumulation was influenced by row spacing. Two spike-type wheat accumulated greater biomass under 15-cm row spacing compared to other row spacing treatments, although a markedly improved photosynthetic rate (PN), effective quantum yield of photosystem II (ΦPSII) and maximal efficiency of photosystem II photochemistry (Fv/Fm) in the penultimate and third leaves were observed in R2 and R3 treatments. At the same time, a longer duration of CAP and green leaf area was maintained in R1 during grain filling. Compared with conventional row spacing, Wennong6 in R1 treatment obtained 21.0% and 19.1% higher grain yield in 2011 and 2012, respectively, while for Jimai22 it increased by 11.3% and 11.4%, respectively. A close association of yield with CAP and LAI at mid-grain filling was observed. In conclusion, for the tested growing conditions, decreasing the row spacing to an optimal distance (15 cm) maintained a longer duration of LAI and CAP during grain filling, made a better coordination of group and individual leaf photosynthesis, and accumulated higher aboveground biomass, leading to a greater grain yield. In addition, Wennong6 had a more rational canopy architecture than Jimai22 (improved LT and higher LAI) and CAP under 15-cm row spacing, leading to a higher grain yield, which indicated that the large-spike type cultivar has the potential to obtain higher yields by increasing plant density through optimum row spacing allocation (15 cm).
Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated.
Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction.
This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the analysis revealed additional layers of complexity to the wheat flour proteome that should be considered when evaluating quantitative 2-DE data.
Chain-terminating gliadins; Gluten polymer; Size-exclusion chromatography; Wheat flour quality
A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences.
Flour quality is largely determined by the gluten proteins, a complex mixture of proteins consisting of high molecular weight-glutenin subunits (HMW-GS), low molecular weight-glutenin subunits (LMW-GS), and α-, γ-, and ω-gliadins. Detailed proteomic analyses of the effects of fertilizer and high temperature on individual gliadin and glutenin protein levels are needed to determine how these environmental factors influence flour quality.
Wheat plants (Triticum aestivum L. cv. Butte 86) were grown in greenhouses under moderate and high temperature regimens with and without post-anthesis fertilizer. Quantitative two-dimensional gel electrophoresis was used to construct accumulation profiles in developing endosperm for the entire complement of gluten proteins identified previously by tandem mass spectrometry. Amounts of individual gliadins and glutenins were also determined in flour produced under each of the regimens. Under all environmental regimens, most HMW-GS, LMW-GS, γ- and ω-gliadins accumulated rapidly during early stages of grain development and leveled off during middle stages of development. A subset of LMW-GS showed a second distinct profile, accumulating throughout development, while α-gliadins showed a variety of accumulation profiles. In flour, fourteen distinct gluten proteins responded similarly to fertilizer, high temperature, and high temperature plus fertilizer. The majority of HMW-GS and ω-gliadins and some α-gliadins increased while two LMW-GS and a minor γ-gliadin decreased. Fertilizer did not influence gluten protein accumulation under high temperature conditions. Additionally, the effects of fertilizer and high temperature were not additive; very few changes were observed when plants that received fertilizer were subjected to high temperature.
Although post-anthesis temperature and fertilizer have very different effects on grain development and yield, the two treatments elicit surprisingly similar effects on the accumulation of gluten proteins. The similarity of the responses to the different treatments is likely due to source-sink activities of nitrogen reserves in the wheat plant. Because each protein that showed a response in this study is linked to a gene sequence, the work sets the stage for transgenic studies that will better elucidate the roles of specific proteins in flour quality and in the response to the environment.
Endosperm; Fertilizer; Flour; Gliadins; Glutenins; Proteome; Temperature; Wheat
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.
Background and Aims
The ω-gliadin storage proteins of wheat are of interest in relation to their impact on grain processing properties and their role in food allergy, particularly the ω-5 sub-group and wheat-dependent exercise-induced anaphylaxis. The ω-gliadins are also known to be responsive to nitrogen application. This study therefore compares the effects of cultivar and nitrogen availability on the synthesis and deposition of ω-gliadins in wheat grown under field conditions in the UK, including temporal and spatial analyses at the protein and transcript levels.
SDS–PAGE, western blotting and N-terminal amino acid sequencing were used to compare the patterns of ω-gliadin components in mature grain of six British wheat (Triticum aestivum) cultivars and their accumulation during the development of grain grown in field plots with varying nitrogen supply. Changes in gene expression during development were determined using real-time reverse transcription–PCR (RT–PCR). Spatial patterns of gene expression and protein accumulation were determined by in situ hybridization and immunofluorescence microscopy, respectively.
Two patterns of ω-gliadins were identified in the six cultivars, including both monomeric ‘gliadin’ proteins and subunits present in polymeric ‘glutenin’ fractions. Increasing the level of nitrogen fertilizer in field plots resulted in increased expression of ω-gliadin transcripts and increased proportions of ω-5 gliadins. Nitrogen supply also affected the spatial patterns of ω-gliadin synthesis and deposition, which were differentially increased in the outer layers of the starchy endosperm with high levels of nitrogen.
Wheat ω-gliadins vary in amount and composition between cultivars, and in their response to nitrogen supply. Their spatial distribution is also affected by nitrogen supply, being most highly concentrated in the sub-aleurone cells of the starchy endosperm under higher nitrogen availability.
Wheat; Triticum aestivum; storage protein; nitrogen; ω-gliadin; RNA in situ hybridization; immunolocalization; protein bodies; wheat allergy
TaGW2-6A, cloned in earlier research, strongly influences wheat grain width and TKW. Here, we mainly analyzed haplotypes of TaGW2-6B and their effects on TKW and interaction with haplotypes at TaGW2-6A.
About 2.9 kb of the promoter sequences of TaGW2-6B and TaGW2-6D were cloned in 34 bread wheat cultivars. Eleven SNPs were detected in the promoter region of TaGW2-6B, forming 4 haplotypes, but no divergence was detected in the TaGW2-6D promoter or coding region. Three molecular markers including CAPS, dCAPS and ACAS, were developed to distinguish the TaGW2-6B haplotypes. Haplotype association analysis indicated that TaGW2-6B has a stronger influence than TaGW2-6A on TKW, and Hap-6B-1 was a favored haplotype increasing grain width and weight that had undergone strong positive selection in global wheat breeding. However, clear geographic distribution differences for TaGW2-6A haplotypes were found; Hap-6A-A was favored in Chinese, Australian and Russian cultivars, whereas Hap-6A-G was preferred in European, American and CIMMYT cultivars. This difference might be caused by a flowering and maturity time difference between the two haplotypes. Hap-6A-A is the earlier type. Haplotype interaction analysis between TaGW2-6A and TaGW2-6B showed additive effects between the favored haplotypes. Hap-6A-A/Hap-6B-1 was the best combination to increase TKW. Relative expression analysis of the three TaGW2 homoeologous genes in 22 cultivars revealed that TaGW2-6A underwent the highest expression. TaGW2-6D was the least expressed during grain development and TaGW2-6B was intermediate. Diversity of the three genes was negatively correlated with their effect on TKW.
Genetic effects, expression patterns and historic changes of haplotypes at three homoeologous genes of TaGW2 influencing yield were dissected in wheat cultivars. Strong and constant selection to favored haplotypes has been found in global wheat breeding during the past century. This research also provides a valuable case for understanding interaction of genes that control complex traits in polyploid species.
Triticum aestivum; TaGW2; Grain weight; Gene expression; Haplotype interaction
The processing properties of the wheat flour are largely determined by the structures and interactions of the grain storage proteins (also called gluten proteins) which form a continuous visco-elastic network in dough. Wheat gluten proteins are classically divided into two groups, the monomeric gliadins and the polymeric glutenins, with the latter being further classified into low molecular weight (LMW) and high molecular weight (HMW) subunits. The synthesis, folding and deposition of the gluten proteins take place within the endomembrane system of the plant cell. However, determination of the precise routes of trafficking and deposition of individual gluten proteins in developing wheat grain has been limited in the past by the difficulty of developing monospecific antibodies. To overcome this limitation, a single gluten protein (a LMW subunit) was expressed in transgenic wheat with a C-terminal epitope tag, allowing the protein to be located in the cells of the developing grain using highly specific antibodies. This approach was also combined with the use of wider specificity antibodies to compare the trafficking and deposition of different gluten protein groups within the same endosperm cells. These studies are in agreement with previous suggestions that two trafficking pathways occur in wheat, with the proteins either being transported via the Golgi apparatus into the vacuole or accumulating directly within the lumen of the ER. They also suggest that the same individual protein could be trafficked by either pathway, possibly depending on the stage of development, and that segregation of gluten proteins both between and within protein bodies may occur.
Epitope tagging; gluten; immunolocalization; protein bodies; protein trafficking; wheat grain
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.
Seed storage proteins in wheat endosperm, particularly high-molecular-weight glutenin subunits (HMW-GS), are primary determinants of dough properties, and affect both end-use quality and grain utilization of wheat (Triticum aestivum L). In order to investigate the interactive effects between the transgenically overexpressed 1Ax1 subunit with different HMW-GS on dough quality traits, we developed a set of 8 introgression lines (ILs) overexpressing the transgenic HMW-glutenin subunit 1Ax1 by introgression of this transgene from transgenic line B102-1-2/1 into an elite Chinese wheat variety Chuanmai107 (C107), using conventional crossing and backcrossing breeding technique. The donor C107 strain lacks 1Ax1 but contains the HMW-GS pairs 1Dx2+1Dy12 and 1Bx7+1By9. The resultant ILs showed robust and stable expression of 1Ax1 even after five generations of self-pollination, and crossing/backcrossing three times. In addition, overexpression of 1Ax1 was compensated by the endogenous gluten proteins. All ILs exhibited superior agronomic performance when compared to the transgenic parent line, B102-1-2/1. Mixograph results demonstrated that overexpressed 1Ax1 significantly improved dough strength, resistance to extension and over-mixing tolerance, in the targeted wheat cultivar C107. Further, comparisons among the ILs showed the interactive effects of endogenous subunits on dough properties when 1Ax1 was overexpressed: subunit pair 17+18 contributed to increased over-mixing tolerance of the dough; expression of the Glu-D1 allele maintained an appropriate balance between x-type and y-type subunits and thereby improved dough quality. It is consistent with ILs C4 (HMW-GS are 1, 17+18, 2+12) had the highest gluten index and Zeleny sedimentation value. This study demonstrates that wheat quality could be improved by using transgenic wheat overexpressing HMW-GS and the feasibility of using such transgenic lines in wheat quality breeding programs.
Lycopene β-cyclase (LCYB) is a key enzyme catalyzing the biosynthesis of β-carotene, the main source of provitamin A. However, there is no documented research about this key cyclase gene’s function and relationship with β-carotene content in wheat. Therefore, the objectives of this study were to clone TaLCYB and characterize its function and relationship with β-carotene biosynthesis in wheat grains. We also aimed to obtain more information about the endogenous carotenoid biosynthetic pathway and thus provide experimental support for carotenoid metabolic engineering in wheat.
In the present study, a lycopene β-cyclase gene, designated TaLCYB, was cloned from the hexaploid wheat cultivar Chinese Spring. The cyclization activity of the encoded protein was demonstrated by heterologous complementation analysis. The TaLCYB gene was expressed differentially in different tissues of wheat. Although TaLCYB had a higher expression level in the later stages of grain development, the β-carotene content still showed a decreasing tendency. The expression of TaLCYB in leaves was dramatically induced by strong light and the β-carotene content variation corresponded with changes of TaLCYB expression. A post-transcriptional gene silencing strategy was used to down-regulate the expression of TaLCYB in transgenic wheat, resulting in a decrease in the content of β-carotene and lutein, accompanied by the accumulation of lycopene to partly compensate for the total carotenoid content. In addition, changes in TaLCYB expression also affected the expression of several endogenous carotenogenic genes to varying degrees.
Our results suggest that TaLCYB is a genuine lycopene cyclase gene and plays a crucial role in β-carotene biosynthesis in wheat. Our attempt to silence it not only contributes to elucidating the mechanism of carotenoid accumulation in wheat but may also help in breeding wheat varieties with high provitamin A content through RNA interference (RNAi) to block specific carotenogenic genes in the wheat endosperm.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0514-5) contains supplementary material, which is available to authorized users.
Lycopene; Lycopene β-cyclase; β-carotene; Provitamin A; RNA interference; Wheat
Sodium dodecyl sulfate (SDS) sedimentation volume has long been used to characterize wheat flours and meals with the aim of predicting processing and end-product qualities. In order to survey the influence of low-molecular-weight glutenin subunits (LMW-GSs) at Glu-B3 locus on wheat SDS sedimentation volume, a total of 283 wheat (Triticum aestivum L.) varieties including landraces and improved and introduced cultivars were analyzed using 10 allele-specific PCR markers at the Glu-B3 locus. The highest allele frequency observed in the tested varieties was Glu-B3i with 21.9% in all varieties, 21.1% in landraces, 25.5% in improved cultivars, and 12% in introduced cultivars. Glu-B3 locus represented 8.6% of the variance in wheat SDS sedimentation volume, and Glu-B3b, Glu-B3g, and Glu-B3h significantly heightened the SDS sedimentation volume, but Glu-B3a, Glu-B3c, and Glu-B3j significantly lowered the SDS sedimentation volume. For the bread-making quality, the most desirable alleles Glu-B3b and Glu-B3g become more and more popular and the least desirable alleles Glu-B3a and Glu-B3c got less and less in modern improved cultivars, suggesting that wheat grain quality in China has been significantly improved through breeding effort.
Background and Aims
Gluten 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.
Protein 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.
Differential 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.
Quantitative 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.
Triticum aestivum; wheat grain; gluten proteins; bread wheat; immunolocalization; protein bodies; pearling
Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the α-gliadin fraction of gluten, γ-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic γ-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in γ-gliadin transcripts of developing bread wheat grains.
A detailed analysis of the genetic variation present in γ-gliadin transcripts of bread wheat (T. aestivum, allo-hexaploid, carrying the A, B and D genome), together with genomic γ-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic γ-gliadin loci, Gli-A1, Gli-B1 or Gli-D1. Almost half of the γ-gliadin transcripts of bread wheat (49%) was assigned to locus Gli-D1. Transcripts from each locus differed in CD epitope content and composition. The Gli-D1 transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the Gli-A1 transcripts (8.6) and the Gli-B1 transcripts (5.4). The natural variants of the major CD epitope from γ-gliadins, DQ2-γ-I, showed variation in their capacity to induce in vitro proliferation of a DQ2-γ-I specific and HLA-DQ2 restricted T-cell clone.
Evaluating the CD epitopes derived from γ-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat cultivars directly from their transcripts (cDNAs).
Wheat; Gluten; γ-gliadins; Celiac disease; T-cell epitopes
Fusarium head blight (FHB) caused by Fusarium species like F. graminearum is a devastating disease of wheat (Triticum aestivum) worldwide. Mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. Resistant varieties are the only effective way to control this disease, but the molecular events leading to FHB resistance are still poorly understood. Transcriptional profiling was conducted for the winter wheat cultivars Dream (moderately resistant) and Lynx (susceptible). The gene expressions at 32 and 72 h after inoculation with Fusarium were used to trace possible defence mechanisms and associated genes. A comparative qPCR was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars Dream and Sumai 3 (Chinese spring wheat).
Among 2,169 differentially expressed genes, two putative main defence mechanisms were found in the FHB-resistant Dream cultivar. Both are defined base on their specific mode of resistance. A non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and GDSL-like lipase genes. Additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to FHB. Another mechanism based on the targeted suppression of essential Fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. Moreover, similar inductions upon fungal infection were frequently observed among FHB-responsive genes of both mechanisms in the cultivars Dream and Sumai 3.
Especially ABC transporter, UDP-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. This was further supported in our qPCR experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars Dream and Sumai 3. Finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars. The respective mechanisms are attractive for advanced studies aiming at new resistance and toxin management strategies.
Wheat grain storage protein (GSP) content and composition are the main determinants of the end-use value of bread wheat (Triticum aestivum L.) grain. The accumulation of glutenins and gliadins, the two main classes of GSP in wheat, is believed to be mainly controlled at the transcriptional level through a network of transcription factors. This regulation network could lead to stable cross-environment allometric scaling relationships between the quantity of GSP classes/subunits and the total quantity of nitrogen per grain. This work conducted a genetic mapping study of GSP content and composition and allometric scaling parameters of grain N allocation using a bread wheat worldwide core collection grown in three environments. The core collection was genotyped with 873 markers for genome-wide association and 167 single nucleotide polymorphism markers in 51 candidate genes for candidate association. The candidate genes included 35 transcription factors (TFs) expressed in grain. This work identified 74 loci associated with 38 variables, of which 19 were candidate genes or were tightly linked with candidate genes. Besides structural GSP genes, several loci putatively trans-regulating GSP accumulation were identified. Seven candidate TFs, including four wheat orthologues of barley TFs that control hordein gene expression, were associated or in strong linkage disequilibrium with markers associated with the composition or quantity of glutenin or gliadin, or allometric grain N allocation parameters, confirming the importance of the transcriptional control of GSP accumulation. Genome-wide association results suggest that the genes regulating glutenin and gliadin compositions are mostly distinct from each other and operate differently.
Association study; bread wheat; ecophysiological model; grain composition; storage proteins; transcription factors; Triticum aestivum.