A novel approach to identify genes that determine grain protein deviation in cereals
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ABSTRACT: Grain yield and protein content were determined for six wheat cultivars grown over three years at multiple sites and at multiple N-fertilizer inputs. Although grain protein was negatively correlated with yield, some grain samples had higher protein contents than expected based on their yields, a trait referred to as grain protein deviation (GPD). We used novel statistical approaches to calculate GPD across environment and to correlate gene expression in the developing caryopsis with this trait. The yield and protein content were initially adjusted for nitrogen fertilizer inputs, and then adjusted for yield (to remove the negative correlation) resulting in environmental corrected GPD. The transcriptome data for all samples were subjected to Principal Component Analysis (PCA) and ANOVA to identify individual Principal Components (PCs) correlating with GPD alone. Scores of the selected PCs significantly related to cultivar differences and GPD but not to the yield or protein content were identified as reflecting a multivariate pattern of gene expression related to genetic variation in GPD. Sets of genes significant for these PCs and hence GPD were identified as candidate genes determining cultivar differences in GPD. Microarray profiling has been used to identify the links between gene expression and grain protein content in 6 different varietes of wheat grown at 2 different sites, 3 N levels and during 3 growth seasons.
Project description:Grain yield and protein content were determined for six wheat cultivars grown over three years at multiple sites and at multiple N-fertilizer inputs. Although grain protein was negatively correlated with yield, some grain samples had higher protein contents than expected based on their yields, a trait referred to as grain protein deviation (GPD). We used novel statistical approaches to calculate GPD across environment and to correlate gene expression in the developing caryopsis with this trait. The yield and protein content were initially adjusted for nitrogen fertilizer inputs, and then adjusted for yield (to remove the negative correlation) resulting in environmental corrected GPD. The transcriptome data for all samples were subjected to Principal Component Analysis (PCA) and ANOVA to identify individual Principal Components (PCs) correlating with GPD alone. Scores of the selected PCs significantly related to cultivar differences and GPD but not to the yield or protein content were identified as reflecting a multivariate pattern of gene expression related to genetic variation in GPD. Sets of genes significant for these PCs and hence GPD were identified as candidate genes determining cultivar differences in GPD. Microarray profiling has been used to identify the links between gene expression and grain protein content in 6 different varietes of wheat grown at 2 different sites, 3 N levels and during 3 growth seasons. Six UK cultivars (Istabraq , Hereward, Marksman, Cordiale, Malacca and Xi 19) were grown over three seasons (2008-9, 2009-10 and 2010-11) at Rothamsted . In the present publication, data from two sites are included: Rothamsted and RAGTResearch (Harpenden, UK) and at four other sites in the south-east of the UK (RAGT, Ickleton, Cambridge; Limagrain, Woolpit, Suffolk; Syngenta, Whittlesford, Cambridge; KWS-UK, Thriplow, Hertfordshire) in 2009-10 and 2010-11 only. Three replicate plots were grown at three N levels: 100kg/ha, (N100), 200kg/ha (N200) and 350 kg/ha (N350) (see Barraclough et al., 2010, Chope at al., 2014). Developing heads (10 per plot) were tagged and caryopses were harvested from the Rothamsted (2009, 2010 and 2011) and RAGT (2010 and 2011) sites at 21 days after anthesis (mid-grain filling) to measure gene expression using Affymetrix wheat microarrays giving a total of 161 samples. Six UK cultivars (Istabraq , Hereward, Marksman, Cordiale, Malacca and Xi 19) were grown over three seasons (2008-9, 2009-10 and 2010-11) at Rothamsted Research (Harpenden, UK) and at four other sites in the south-east of the UK (RAGT, Ickleton, Cambridge; Limagrain, Woolpit, Suffolk; Syngenta, Whittlesford, Cambridge; KWS-UK, Thriplow, Hertfordshire) in 2009-10 and 2010-11 only. Three replicate plots were grown at three N levels: 100kg/ha, (N100), 200kg/ha (N200) and 350 kg/ha (N350) (see Barraclough et al., 2010, Chope at al., 2014). Developing heads (10 per plot) were tagged and caryopses were harvested from the Rothamsted (2009, 2010 and 2011) and RAGT (2010 and 2011) sites at 21 days after anthesis (mid-grain filling) to measure gene expression using Affymetrix wheat microarrays giving a total of 161 samples.
Project description:It is well documented that biostimulants could play an important role in agriculture. Additionally, increased fertilizer use efficiency is essential for maintaining both yield and grain quality, especially for bread wheat, which is a major global crop. In the present study, we explored the effects of mixing urea-ammonium-nitrate fertilizer with Glutacetine® on the physiological responses, agronomic traits and grain quality of winter wheat. Grain proteome analysis revealed that Glutacetine strongly reduced 11 proteins including storage proteins. Indeed, 2 alpha-gliadins and 2 avenin-like proteins decreased after Glutacetine application, which were good for celiac disease patients. Moreover, 2 glutenin HMW subunit were reduced, changing the gliadin/glutenin ratio and the HMW/LMW ratio, thus modifying the wheat flour dough quality. Our investigation reveals the important role of these formulations in achieving significant increases in seed yield and grain quality.
Project description:Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.
Project description:Waxy starch has an important influence on bread dough and the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. The waxy and non-waxy wheats had similar starch granule morphological features and developmental patterns, and similar amylopectin quality in the grain. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Dynamic transcriptional expression profiling of starch biosynthesis-related genes indicated similar transcriptional expression profiles in both cultivars. Our results revealed that phosphorylation modifications played critical roles in amylose and amylopectin biosynthesis, but the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.
Project description:Drought is among the most limiting factors for sustainable agricultural production. Water shortage at the onset of flowering severely affects the quality and quantity of grain yield of bread wheat (Triticum aestivum). Herein, we measured oxidative stress and photosynthesis-related parameters upon applying transient drought on contrasting wheat cultivars at the flowering initiation stage of ontogenesis. The sensitive cultivar showed ineffective water management and a more severe decline of photosynthesis. Apparently, the tolerant genotype used photorespiration to dissipate excessive light energy. The tolerant cultivar sooner induced superoxide dismutase and showed less inhibited photosynthesis. Such protective effect resulted in less affected yield and spectrum of seed proteome. The tolerant cultivar had a more stable gluten profile, which defines bread-making quality, upon drought. Drought caused the accumulation of medically relevant proteins: (i) components of gluten in the sensitive cultivar and (ii) metabolic proteins in the tolerant cultivar. We propose specific proteins as markers of drought tolerance for guiding efficient breeding: thaumatin-like protein, 14-3-3 protein, peroxiredoxins, peroxidase, FBD domain protein, and Ap2/ERF plus B3 domain protein.
Project description:Nitrogen (N) fertilization is essential in order to insure wheat bread yield and quality. Improving nitrogen use efficiency is therefore crucial for wheat grain protein quality. In the present work, we analysed the effects of new biostimulants containing Glutacetine® or derivate formulations that have been mixed with urea-ammonium-nitrate fertilizer (UAN) on winter wheat grain proteome. A largescale quantitative proteomics analysis of 2 wheat flour fractions led to a dataset of 4369 identified proteins. Quantitative analysis revealed 9, 39 and 96 proteins with a significantly varying abundance after Glutacetine®, VNT1 and VNT4 treatment, respectively, with 11 proteins (or homologue) which were affected by 2 different biostimulants. Major effects affected proteins involved in regulation processes with transcription regulator proteins, in stress responses with biotic and abiotic stress defence proteins, in flowering efficiency with proteins involved in pollen development, in storage functions with the gluten protein alpha-gliadins and starch synthase and in seed development with proteins implied in transport, proteases activity, energy machinery and N pathway. Altogether, our controlled conditions study showed that Glutacetine®, VNT1 and VNT4 biostimulants positively affected protein composition for grain quality.
Project description:Regulation of grain size is a crucial strategy for improving crop yield and is also a fundamental aspect of developmental biology. However, the underlying molecular mechanisms governing grain development in wheat remain largely unknown. In this study, we identified a wheat atypical basic helix-loop-helix (bHLH) transcription factor, TabHLH489, which is tightly associated with grain length through genome-wide association study and map-based cloning. Knockout of TabHLH489 and its homologous genes resulted in increased grain length and weight, whereas overexpression led to decreased grain length and weight. TaSnRK1α1, the α-catalytic subunit of plant energy sensor SnRK1, interacted with and phosphorylated TabHLH489 to induce its degradation, thereby promoting wheat grain development. Sugar treatment induced TaSnRK1α1 protein accumulation while reducing TabHLH489 protein levels. Moreover, brassinosteroid (BR) promotes grain development by decreasing TabHLH489 expression through the transcription factor BRASSINAZOLE RESISTANT1 (BZR1). Importantly, natural variations in the promoter region of TabHLH489 affect the TaBZR1 binding ability, thereby influencing TabHLH489 expression. Taken together, our findings reveal that the TaSnRK1α1-TabHLH489 regulatory module integrates BR and sugar signaling to regulate grain length, presenting potential targets for enhancing grain size in wheat.
Project description:Heat stress is a major limiting factor for grain yield and grain quality in wheat production. In crops, abiotic stresses have transgenerational effects and the mechanistic basis of stress memory is associated with epigenetic regulation. The current study presents the first systematic analysis of the transgenerational effects of post-anthesis heat stress in tetraploid wheat. Genotype-dependent response patterns to parental and progeny heat stress were found for the leaf physiological traits, harvest components, and grain quality traits measured. Parental heat stress had positive influence on the offspring under re-occurring stress for traits like chlorophyll content, grain weight, grain number and grain total starch content. Integrated sequencing analysis of the small RNAome, mRNA transcriptome, and mRNA degradome provided the first description of the molecular networks mediating heat stress adaption under transgenerational influence. The expression profile of 1771 microRNAs (733 being novel) and 66,559 genes was provided, with differentially expressed microRNAs and genes identified subject to the progeny treatment, parental treatment and tissue type factors. Gene Ontology and KEGG pathway annotation of stress responsive microRNAs-mRNA modules provided further information on their functional roles in biological processes like hormone homeostasis, signal transduction, and protein stabilization. Our results provide new sights on the molecular basis of transgenerational heat stress adaptation, which can be used for improving thermos-tolerance in breeding.
Project description:Wheat seed development is a very important stage in the cereal crops seed life cycle. The accumulation reserves of wheat mature seeds provide not only the food for human and livestock feed, but also the energy for the seed germination.However, due to the large genome size, many studies related to wheat seed are very complex and uncompleted. Transcriptome analysis of elite Chinses bread wheat cultivar Jimai 20 may provides a comprehensive understanding of wheat seed development. Seed development involves in the regulation of large number of genes, whether these genes are normal activated or not is very important to seed development. We performed microarray analysis using the Affymetrix Gene Chip to reveal the gene expression profiles in the phases of wheat cultivar Jimai 20 grain filling. Our results provide a new insights into the thoroughly metabolic changes of seed development as well as the key differentially expressed genes involved in wheat grain development.