Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, “Nongda146” and “Jimai6358”, by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying.
Using a series of multiplexed experiments we studied the quantitative changes in protein abundance of three Australian bread wheat cultivars (Triticum aestivum L.) in response to a drought stress. Three cultivars differing in their ability to maintain grain yield during drought, Kukri (intolerant), Excalibur (tolerant), and RAC875 (tolerant), were grown in the glasshouse with cyclic drought treatment that mimicked conditions in the field. Proteins were isolated from leaves of mature plants and isobaric tags were used to follow changes in the relative protein abundance of 159 proteins. This is the first shotgun proteomics study in wheat, providing important insights into protein responses to drought as well as identifying the largest number of wheat proteins (1,299) in a single study. The changes in the three cultivars at the different time points reflected their differing physiological responses to drought, with the two drought tolerant varieties (Excalibur and RAC875) differing in their protein responses. Excalibur lacked significant changes in proteins during the initial onset of the water deficit in contrast to RAC875 that had a large number of significant changes. All three cultivars had changes consistent with an increase in oxidative stress metabolism and reactive O2 species (ROS) scavenging capacity seen through increases in superoxide dismutases and catalases as well as ROS avoidance through the decreases in proteins involved in photosynthesis and the Calvin cycle.
Triticum aestivum; bread wheat; drought; quantitative proteomics; iTRAQ
The classical Osborne wheat protein fractions (albumins, globulins, gliadins, and glutenins), as well as several proteins from each of the four subunits of gliadin using SDS-PAGE analyses, were determined in the grain of five bread (T. aestivum L.) and five durum wheat (T. durum Desf.) genotypes. In addition, content of tryptophan and wet gluten were analyzed. Gliadins and glutenins comprise from 58.17% to 65.27% and 56.25% to 64.48% of total proteins and as such account for both quantity and quality of the bread and durum wheat grain proteins, respectively. The ratio of gliadin/total glutenin varied from 0.49 to 1.01 and 0.57 to 1.06 among the bread and durum genotypes, respectively. According to SDS-PAGE analysis, bread wheat genotypes had a higher concentration of α + β + γ-subunits of gliadin (on average 61.54% of extractable proteins) than durum wheat (on average 55.32% of extractable proteins). However, low concentration of ω-subunit was found in both bread (0.50% to 2.53% of extractable proteins) and durum (3.65% to 6.99% of extractable proteins) wheat genotypes. On average, durum wheat contained significantly higher amounts of tryptophan and wet gluten (0.163% dry weight (d.w.) and 26.96% d.w., respectively) than bread wheat (0.147% d.w. and 24.18% d.w., respectively).
bread wheat; durum wheat; protein fractions and subunits; tryptophan; wet gluten
The effect of high temperatures (above 25°C) on starch concentration and the morphology of starch granules in the grains of wheat (Triticum aestivum L.) were studied. Wheat plants of cultivars Yangmai 9 (weak-gluten) and Yangmai 12 (medium-gluten) were treated with high temperatures for 3 days at different times after anthesis. The results showed that the starch concentration of grains given a heat-shock treatment above 30°C were lower than those developing at normal temperature in both cultivars. High temperature lowered starch concentration due to the decrease of amylopectin. Under the same temperature, the effect of heat shock from 6 to 8 days after anthesis (DAA) was the greatest, whereas from 36 to 38 DAA the effect was the least. The effects of high temperatures after anthesis on starch-pasting properties were similar to those on starch concentration, especially after 35–40°C treatments. The size, shape and structure of starch granules in wheat grains (determined by electron microscopy) after heat shock were visibly different from the control. When given heat shock during development, the starch granules in mature wheat grains were ellipsoid in shape and bound loosely with a protein sheath in Yangmai 9, while they were damaged and compressed with fissures in Yangmai 12, indicating the differences in resistance to high temperature between cultivars. Ratios of large (type-A) and small (type-B) starch granules significantly decreased under heat shock, which limited the potential sink size for dry matter deposition in the grain.
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.
Few genes are available to develop drought-tolerant bread wheat (Triticum aestivum L.) cultivars. One way to enhance bread wheat’s genetic diversity would be to take advantage of the diversity of wild species by creating synthetic hexaploid wheat (SW) with the genomic constitution of bread wheat. In this study, we compared the expression of traits encoded at different ploidy levels and evaluated the applicability of Aegilops tauschii drought-related traits using 33 Ae. tauschii accessions along with their corresponding SW lines under well-watered and drought conditions. We found wide variation in Ae. tauschii, and even wider variation in the SW lines. Some SW lines were more drought-tolerant than the standard cultivar Cham 6. Aegilops tauschii from some regions gave better performing SW lines. The traits of Ae. tauschii were not significantly correlated with their corresponding SW lines, indicating that the traits expressed in wild diploid relatives of wheat may not predict the traits that will be expressed in SW lines derived from them. We suggest that, regardless of the adaptability and performance of the Ae. tauschii under drought, production of SW could probably result in genotypes with enhanced trait expression due to gene interactions, and that the traits of the synthetic should be evaluated in hexaploid level.
wheat; Aegilops tauschii; synthetic hexaploid wheat; wild relatives of wheat
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
Among health-promoting phytochemicals in whole grains, phenolic compounds have gained attention as they have strong antioxidant properties and can protect against many degenerative diseases. Aim of this study was to profile grain phenolic extracts of one modern and five old common wheat (Triticum aestivum L.) varieties and to evaluate their potential antiproliferative or cytoprotective effect in different cell culture systems.
Wheat extracts were characterized in terms of antioxidant activity and phenolic composition (HPLC/ESI-TOF-MS profile, polyphenol and flavonoid contents). Results showed that antioxidant activity (FRAP and DPPH) is mostly influenced by flavonoid (both bound and free) content and by the ratio flavonoids/polyphenols. Using a leukemic cell line, HL60, and primary cultures of neonatal rat cardiomyocytes, the potential antiproliferative or cytoprotective effects of different wheat genotypes were evaluated in terms of intracellular reactive oxygen species levels and cell viability. All tested wheat phenolic extracts exerted dose-dependent cytoprotective and antiproliferative effects on cardiomyocytes and HL60 cells, respectively. Due to the peculiar phenolic pattern of each wheat variety, a significant genotype effect was highlighted. On the whole, the most relevant scavenging effect was found for the old variety Verna. No significant differences in terms of anti-proliferative activities among wheat genotypes was observed.
Results reported in this study evidenced a correspondence between the in vitro antioxidant activity and potential healthy properties of different extracts. This suggests that an increased intake of wheat grain derived products could represent an effective strategy to achieve both chemoprevention and protection against oxidative stress related diseases.
Water stress during grain filling has a marked effect on grain yield, leading to a reduced endosperm cell number and thus sink capacity to accumulate dry matter. The bread wheat cultivar Chinese Spring (CS), a Chinese Spring terminal deletion line (CS_5AL-10) and the durum wheat cultivar Creso were subjected to transcriptional profiling after exposure to mild and severe drought stress at the grain filling stage to find evidences of differential stress responses associated to different wheat genome regions.
The transcriptome analysis of Creso, CS and its deletion line revealed 8,552 non redundant probe sets with different expression levels, mainly due to the comparisons between the two species. The drought treatments modified the expression of 3,056 probe sets. Besides a set of genes showing a similar drought response in Creso and CS, cluster analysis revealed several drought response features that can be associated to the different genomic structure of Creso, CS and CS_5AL-10. Some drought-related genes were expressed at lower level (or not expressed) in Creso (which lacks the D genome) or in the CS_5AL-10 deletion line compared to CS. The chromosome location of a set of these genes was confirmed by PCR-based mapping on the D genome (or the 5AL-10 region). Many clusters were characterized by different level of expression in Creso, CS and CS_AL-10, suggesting that the different genome organization of the three genotypes may affect plant adaptation to stress. Clusters with similar expression trend were grouped and functional classified to mine the biological mean of their activation or repression. Genes involved in ABA, proline, glycine-betaine and sorbitol pathways were found up-regulated by drought stress. Furthermore, the enhanced expression of a set of transposons and retrotransposons was detected in CS_5AL-10.
Bread and durum wheat genotypes were characterized by a different physiological reaction to water stress and by a substantially different molecular response. The genome organization accounted for differences in the expression level of hundreds of genes located on the D genome or controlled by regulators located on the D genome. When a genomic stress (deletion of a chromosomal region) was combined with low water availability, a molecular response based on the activation of transposons and retrotransposons was observed.
In the South Australian wheat belt, cyclic drought is a frequent event represented by intermittent periods of rainfall which can occur around anthesis and post-anthesis in wheat. Three South Australian bread wheat (Triticum aestivum L.) cultivars, Excalibur, Kukri, and RAC875, were evaluated in one greenhouse and two growth-room experiments. In the first growth-room experiment, where plants were subjected to severe cyclic water-limiting conditions, RAC875 and Excalibur (drought-tolerant) showed significantly higher grain yield under cyclic water availability compared to Kukri (drought-susceptible), producing 44% and 18% more grain compared to Kukri, respectively. In the second growth-room experiment, where plants were subjected to a milder drought stress, the differences between cultivars were less pronounced, with only RAC875 showing significantly higher grain yield under the cyclic water treatment. Grain number per spike and the percentage of aborted tillers were the major components that affected yield under cyclic water stress. Excalibur and RAC875 adopted different morpho-physiological traits and mechanisms to reduce water stress. Excalibur was most responsive to cyclic water availability and showed the highest level of osmotic adjustment (OA), high stomatal conductance, lowest ABA content, and rapid recovery from stress under cyclic water stress. RAC875 was more conservative and restrained, with moderate OA, high leaf waxiness, high chlorophyll content, and slower recovery from stress. Within this germplasm, the capacity for osmotic adjustment was the main physiological attribute associated with tolerance under cyclic water stress which enabled plants to recover from water deficit.
Cyclic drought; osmotic adjustment; wheat
A comparative proteomic analysis of drought-responsive proteins during grain development of two wheat varieties Kauz (strong resistance to drought stress) and Janz (sensitive to drought stress) was performed by using linear and nonlinear 2-DE and MALDI-TOF mass spectrometry technologies. Results revealed that the nonlinear 2-DE had much higher resolution than the linear 2-DE. A total of 153 differentially expressed protein spots were detected by both 2-DE maps, of which 122 protein spots were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry. The identified differential proteins were mainly involved in carbohydrate metabolism (26%), detoxification and defense (23%), and storage proteins (17%). Some key proteins demonstrated significantly different expression patterns between the two varieties. In particular, catalase isozyme 1, WD40 repeat protein, LEA and alpha-amylase inhibitors displayed an upregulated expression pattern in Kauz, whereas they were downregulated or unchanged in Janz. Small and large subunit ADP glucose pyrophosphorylase, ascorbate peroxidase and G beta-like protein were all downregulated under drought stress in Janz, but had no expression changes in Kauz. Sucrose synthase and triticin precursor showed an upregulated expression pattern under water deficits in both varieties, but their upregulation levels were much higher in Kauz than in Janz. These differentially expressed proteins could be related to the biochemical pathways for stronger drought resistance of Kauz.
linear and nonlinear 2-DE; MALDI-TOF/TOF MS; comparative proteomics; drought stress; wheat grains
There is a general concern that changes in plant productivity and composition caused by increase in atmospheric CO2 concentration will alter the chemical composition of the grain. This review describes the impact of rising atmospheric CO2 on the grain characteristics in wheat, rice, brassica, mungbean and soybean, which are significantly responsive to the elevated CO2 for their growth, physiology and biochemical processes. The synthesis of the CO2 induced changes in the chemical composition and nutritional qualities of their grains has been discussed. It was demonstrated that the rise in atmospheric CO2 affects the nutritional and industrial application properties of the grains of crop plants. The grain proteins and other nutritionally important constituents significantly reduced, adversely affecting the nutritional and bread making quality in wheat. However, there are evidences suggesting the sustenance of the bread making properties by fertilizer application. Similarly, the CO2 induced changes in the composition of starch in rice grains, result into easy gelatinization and higher viscosity on cooking. These grains bring firmness due to increase in amylose content. Adequately larger size of grains was the outcome of the elevated CO2 effects, in Brassica species. It increased the oil content due to greater acetyl Co A enzyme activity and also help in regulating fatty acid biosynthesis. Some of the nutritionally undesirable fatty acids were significantly reduced in this process, making this oil less harmful for heart patients. The adequate use of fertilizer application and selection pressure of breeders may significantly contribute in developing cultivars, which will counter the adverse effect of rising atmospheric CO2 on grain quality.
Carbon dioxide; Grain quality; Fatty acids; Wheat; Rice; Brassica
The responses to low red light/far-red light (R/FR) ratios simulating dense stands were evaluated in wheat (Triticum aestivum L) cultivars released at different times in the 20th century and consequently resulting from an increasingly prolonged breeding and selection history. While tillering responses to the R/FR ratio were unaffected by the cultivars, low R/FR ratios reduced grain yield per plant (primarily grain number and secondarily grain weight per plant) particularly in modern cultivars. Low R/FR ratios delayed spike growth and development, reduced the expression of spike marker genes, accelerated the development of florets already initiated, and reduced the number of fertile florets at anthesis. It is noteworthy that low R/FR ratios did not promote stem or leaf sheath growth and therefore the observed reduction of yield cannot be accounted for as a consequence of divergence of resources towards increased plant stature. It is proposed that the regulation of yield components by the R/FR ratio could help plants to adjust to the limited availability of resources under crop conditions.
Grain yield; phytochrome; red/far-red ratio; shade avoidance; spike; tillering; wheat
Nitrogen uptake and the efficient absorption and metabolism of nitrogen are essential elements in attempts to breed improved cereal cultivars for grain or silage production. One of the enzymes related to nitrogen metabolism is glutamine-2-oxoglutarate amidotransferase (GOGAT). Together with glutamine synthetase (GS), GOGAT maintains the flow of nitrogen from NH4+ into glutamine and glutamate, which are then used for several aminotransferase reactions during amino acid synthesis.
The aim of the present work was to identify and analyse the structure of wheat NADH-GOGAT genomic sequences, and study the expression in two durum wheat cultivars characterized by low and high kernel protein content. The genomic sequences of the three homoeologous A, B and D NADH-GOGAT genes were obtained for hexaploid Triticum aestivum and the tetraploid A and B genes of Triticum turgidum ssp. durum. Analysis of the gene sequences indicates that all wheat NADH-GOGAT genes are composed of 22 exons and 21 introns. The three hexaploid wheat homoeologous genes have high conservation of sequence except intron 13 which shows differences in both length and sequence. A comparative analysis of sequences among di- and mono-cotyledonous plants shows both regions of high conservation and of divergence. qRT-PCR performed with the two durum wheat cvs Svevo and Ciccio (characterized by high and low protein content, respectively) indicates different expression levels of the two NADH-GOGAT-3A and NADH-GOGAT-3B genes.
The three hexaploid wheat homoeologous NADH-GOGAT gene sequences are highly conserved – consistent with the key metabolic role of this gene. However, the dicot and monocot amino acid sequences show distinctive patterns, particularly in the transit peptide, the exon 16–17 junction, and the C-terminus. The lack of conservation in the transit peptide may indicate subcellular differences between the two plant divisions - while the sequence conservation within enzyme functional domains remains high. Higher expression levels of NADH-GOGAT are associated with higher grain protein content in two durum wheats.
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.
Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4+ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD).
The effects of deleting individual gluten loci on both the level of T-cell stimulatory epitopes in the gluten proteome and the technological properties of the flour were analyzed using a set of deletion lines of Triticum aestivum cv. Chinese Spring. The reduction of T-cell stimulatory epitopes was analyzed using monoclonal antibodies that recognize T-cell epitopes present in gluten proteins. The deletion lines were technologically tested with respect to dough mixing properties and dough rheology. The results show that removing the α-gliadin locus from the short arm of chromosome 6 of the D-genome (6DS) resulted in a significant decrease in the presence of T-cell stimulatory epitopes but also in a significant loss of technological properties. However, removing the ω-gliadin, γ-gliadin, and LMW-GS loci from the short arm of chromosome 1 of the D-genome (1DS) removed T-cell stimulatory epitopes from the proteome while maintaining technological properties.
The consequences of these data are discussed with regard to reducing the load of T-cell stimulatory epitopes in wheat, and to contributing to the design of CD-safe wheat varieties.
Mineral nutrition during wheat grain development has large effects on wheat flour protein content and composition, which in turn affect flour quality and immunogenic potential for a commodity of great economic value. However, it has been difficult to define the precise effects of mineral nutrition on protein composition because of the complexity of the wheat flour proteome. Recent improvements in the identification of flour proteins by tandem mass spectrometry (MS/MS) and the availability of a comprehensive proteome map of flour from the US wheat Butte 86 now make it possible to document changes in the proportions of individual flour proteins that result from the application of mineral nutrition.
Plants of Triticum aestivum 'Butte 86' were grown with or without post-anthesis fertilization (PAF) and quantitative 2-dimensional gel electrophoresis (2-DE) was used to analyze protein composition of the resulting flour. Significant changes in the proportions of 54 unique proteins were observed as a result of the treatment. Most omega-gliadins, high molecular weight glutenin subunits (HMW-GS) and serpins as well as some alpha-gliadins increased in proportion with PAF. In contrast, alpha-amylase/protease inhibitors, farinins, purinins and puroindolines decreased in proportion. Decreases were also observed in several low molecular weight glutenin subunits (LMW-GS), globulins, defense proteins and enzymes. The ratio of HMW-GS to LMW-GS in the flour increased from 0.61 to 0.95 and the ratio of gliadins to glutenins increased from 1.02 to 1.30 with PAF. Because flour protein content doubled with PAF from 7 to 14%, most protein types actually increased in absolute amount (μg/mg flour protein). Data further suggest that flour proteins change with PAF according to their content of sulfur-containing amino acids Cys + Met.
A 2-DE approach revealed changes in the wheat flour proteome due to PAF that are important for flour quality and immunogenic potential. The work forms a baseline for further studies of the effects of environmental variables on flour protein composition and provides clues about the regulation of specific flour protein genes. The study also is important for identifying targets for breeding programs and biotechnology efforts aimed at improving flour quality.
gliadins; glutenins; gluten proteins; nitrogen; sulfur
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
Serial Analysis of Gene Expression (SAGE) is a powerful tool for genome-wide transcription studies. Unlike microarrays, it has the ability to detect novel forms of RNA such as alternatively spliced and antisense transcripts, without the need for prior knowledge of their existence. One limitation of using SAGE on an organism with a complex genome and lacking detailed sequence information, such as the hexaploid bread wheat Triticum aestivum, is accurate annotation of the tags generated. Without accurate annotation it is impossible to fully understand the dynamic processes involved in such complex polyploid organisms. Hence we have developed and utilised novel procedures to characterise, in detail, SAGE tags generated from the whole grain transcriptome of hexaploid wheat.
Examination of 71,930 Long SAGE tags generated from six libraries derived from two wheat genotypes grown under two different conditions suggested that SAGE is a reliable and reproducible technique for use in studying the hexaploid wheat transcriptome. However, our results also showed that in poorly annotated and/or poorly sequenced genomes, such as hexaploid wheat, considerably more information can be extracted from SAGE data by carrying out a systematic analysis of both perfect and "fuzzy" (partially matched) tags. This detailed analysis of the SAGE data shows first that while there is evidence of alternative polyadenylation this appears to occur exclusively within the 3' untranslated regions. Secondly, we found no strong evidence for widespread alternative splicing in the developing wheat grain transcriptome. However, analysis of our SAGE data shows that antisense transcripts are probably widespread within the transcriptome and appear to be derived from numerous locations within the genome. Examination of antisense transcripts showing sequence similarity to the Puroindoline a and Puroindoline b genes suggests that such antisense transcripts might have a role in the regulation of gene expression.
Our results indicate that the detailed analysis of transcriptome data, such as SAGE tags, is essential to understand fully the factors that regulate gene expression and that such analysis of the wheat grain transcriptome reveals that antisense transcripts maybe widespread and hence probably play a significant role in the regulation of gene expression during grain development.
Genetic differences among major types of wheat are well characterized; however, little is known about how these distinctions affect the small molecule profile of the wheat seed. Ethanol/water (65% v/v) extracts of seed from 45 wheat lines representing 3 genetically distinct classes, tetraploid durum (Triticum turgidum subspecies durum) (DW) and hexaploid hard and soft bread wheat (T. aestivum subspecies aestivum) (BW) were subjected to ultraperformance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-TOF-MS). Discriminant analyses distinguished DW from BW with 100% accuracy due to differences in expression of nonpolar and polar ions, with differences attributed to sterol lipids/fatty acids and phospholipids/glycerolipids, respectively. Hard versus soft BW was distinguished with 100% accuracy by polar ions, with differences attributed to heterocyclic amines and polyketides versus phospholipid ions, respectively. This work provides a foundation for identification of metabolite profiles associated with desirable agronomic and human health traits and for assessing how environmental factors impact these characteristics.
Study was done to compare the response of Triticum aestivum (hexaploid), Triticum durum (tetraploid) and Triticum monococcum (diploid) wheat species to the elevated CO2 using Free Air CO2 Enrichment (FACE) facility. It was demonstrated that the modern cultivar of wheat Triticum aestivum (hexaploid) was largely sink limited. It appeared to have less photosynthesis per unit leaf area than Triticum monococcum (diploid wheat). While leaf size, grain weight and amylase activity increased with the ploidy level from diploid to hexaploid wheat forms, the photosynthetic rate was reduced significantly. These wheat species responded differentially to the elevated CO2. The larger leaf area and greater seed weight and presence of 38 KDa protein band caused by elevated CO2 had additive effect in improving the productivity of hexaploid wheat by changing the source sink ratio. Whereas, such a source sink balance was not induced by elevated CO2 in diploid wheat. The increasing CO2 may present opportunities to breeders and possibly allow them to select for cultivars responsive to the elevated CO2 with better sink potential.
Elevated CO2; FACE technology; Photosynthesis; Seed weight; Source sink ratio; Triticum
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
A pot experiment was conducted to investigate the effect of the non-protein amino acid, β-aminobutyric acid (BABA), on the homeostasis between reactive oxygen species (ROS) and antioxidant defence during progressive soil drying, and its relationship with the accumulation of abscisic acid (ABA), water use, grain yield, and desiccation tolerance in two spring wheat (Triticum aestivum L.) cultivars released in different decades and with different yields under drought. Drenching the soil with 100 µM BABA increased drought-induced ABA production, leading to a decrease in the lethal leaf water potential (Ψ) used to measure desiccation tolerance, decreased water use, and increased water use efficiency for grain (WUEG) under moderate water stress. In addition, at severe water stress levels, drenching the soil with BABA reduced ROS production, increased antioxidant enzyme activity, and reduced the oxidative damage to lipid membranes. The data suggest that the addition of BABA triggers ABA accumulation that acts as a non-hydraulic root signal, thereby closing stomata, and reducing water use at moderate stress levels, and also reduces the production of ROS and increases the antioxidant defence enzymes at severe stress levels, thus increasing the desiccation tolerance. However, BABA treatment had no effect on grain yield of wheat when water availability was limited. The results suggest that there are ways of effectively priming the pre-existing defence pathways, in addition to genetic means, to improve the desiccation tolerance and WUEG of wheat.
Abscisic acid; β-aminobutyric acid; desiccation resistance; non-hydraulic signals; reactive oxygen species; transpiration efficiency for grain; Triticum aestivum L.; water use efficiency for grain
The relationship between chromosome deletion in wheat and protein expression were investigated using Chinese Spring and fine deletion line 3BS-8. Through 2-DE (2-D electrophoresis) analysis, no differentially expressed proteins (DEPs) were found in leaf samples; however, 47 DEPs showed at least two-fold abundance variation (p < 0.05) in matured wheat grains and 21 spots were identified by tandem MALDI-TOF/TOF-MS. Among the identified spots, four were cultivar-specific, including three (spots B15, B16, and B21) in Chinese Spring and one in 3BS-8 (spot B10). Among variety-different DEPs between Chinese Spring and 3BS-8, most spots showed a higher express profile in CS; only four spots showed up-regulated expression tendency in 3BS-8. An interesting observation was that more than half of the identified protein spots were involved in storage proteins, of which 11 spots were identified as globulins. According to these results, we can presume that the encoded genes of protein spots B15, B16, and B21 were located on the chromosome segment deleted in 3BS-8.
Chinese spring; 3BS-8; albumins; globulins; proteome
The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homoeologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species.
Eight new non-homoeologous wheat genes were cloned and characterized. The nine PDI and PDI-like sequences of wheat were located in chromosome regions syntenic to those in rice and assigned to eight plant phylogenetic groups. The nine wheat genes differed in their sequences, genomic organization as well as in the domain composition and architecture of their deduced proteins; conversely each of them showed high structural conservation with genes from other plant species in the same phylogenetic group. The extensive quantitative RT-PCR analysis of the nine genes in a set of 23 wheat samples, including tissues and developmental stages, showed their constitutive, even though highly variable expression.
The nine wheat genes showed high diversity, while the members of each phylogenetic group were highly conserved even between taxonomically distant plant species like the moss Physcomitrella patens. Although constitutively expressed the nine wheat genes were characterized by different expression profiles reflecting their different genomic organization, protein domain architecture and probably promoter sequences; the high conservation among species indicated the ancient origin and diversification of the still evolving gene family. The comprehensive structural and expression characterization of the complete set of PDI and PDI-like wheat genes represents a basis for the functional characterization of this gene family in the hexaploid context of bread wheat.