Peptidomics analysis of in vitro digested wheat breads: Effect of genotype and environment on protein digestibility and release of celiac disease and wheat allergy related epitopes.

Wheat proteins can trigger immunogenic reactions due to their resistance to digestion and immunostimulatory epitopes. Here, we investigated the peptidomic map of partially digested bread samples and the fingerprint of epitope diversity from 16 wheat genotypes grown in two environmental conditions. Flour protein content and composition were characterized; gastric and jejunal peptides were quantified using LC-MS/MS, and genotypes were classified into high or low bread protein digestibility. Differences in flour protein content and peptide composition distinguish high from low digestibility genotypes in both growing environments. No common peptide signature was found between high- and low-digestible genotypes; however, the celiac or allergen epitopes were noted not to be higher in low-digestible genotypes. Overall, this study established a peptidomic and epitope diversity map of digested wheat bread and provided new insights and correlations between weather conditions, genotypes, digestibility and wheat sensitivities such as celiac disease and wheat allergy.

Different wheat loci are associated to heritable free asparagine content in grain grown under different water and nitrogen availability.

KEY MESSAGE: Different wheat QTLs were associated to the free asparagine content of grain grown in four different conditions. Environmental effects are a key factor when selecting for low acrylamide-forming potential. The amount of free asparagine in grain of a wheat genotype determines its potential to form harmful acrylamide in derivative food products. Here, we explored the variation in the free asparagine, aspartate, glutamine and glutamate contents of 485 accessions reflecting wheat worldwide diversity to define the genetic architecture governing the accumulation of these amino acids in grain. Accessions were grown under high and low nitrogen availability and in water-deficient and well-watered conditions, and plant and grain phenotypes were measured. Free amino acid contents of grain varied from 0.01 to 1.02 mg g-1 among genotypes in a highly heritable way that did not correlate strongly with grain yield, protein content, specific weight, thousand-kernel weight or heading date. Mean free asparagine content was 4% higher under high nitrogen and 3% higher in water-deficient conditions. After genotyping the accessions, single-locus and multi-locus genome-wide association study models were used to identify several QTLs for free asparagine content located on nine chromosomes. Each QTL was associated with a single amino acid and growing environment, and none of the QTLs colocalised with genes known to be involved in the corresponding amino acid metabolism. This suggests that free asparagine content is controlled by several loci with minor effects interacting with the environment. We conclude that breeding for reduced asparagine content is feasible, but should be firmly based on multi-environment field trials. KEY MESSAGE: Different wheat QTLs were associated to the free asparagine content of grain grown in four different conditions. Environmental effects are a key factor when selecting for low acrylamide-forming potential.

Storage protein activator controls grain protein accumulation in bread wheat in a nitrogen dependent manner.

The expression of cereal grain storage protein (GSP) genes is controlled by a complex network of transcription factors (TFs). Storage protein activator (SPA) is a major TF acting in this network but its specific function in wheat (Triticum aestivum L.) remains to be determined. Here we generated an RNAi line in which expression of the three SPA homoeologs was reduced. In this line and its null segregant we analyzed GSP accumulation and expression of GSP and regulatory TF genes under two regimes of nitrogen availability. We show that down regulation of SPA decreases grain protein concentration at maturity under low but not high nitrogen supply. Under low nitrogen supply, the decrease in SPA expression also caused a reduction in the total quantity of GSP per grain and in the ratio of GSP to albumin-globulins, without significantly affecting GSP composition. The slight reduction in GSP gene expression measured in the SPA RNAi line under low nitrogen supply did not entirely account for the more significant decrease in GSP accumulation, suggesting that SPA regulates additional levels of GSP synthesis. Our results demonstrate a clear role of SPA in the regulation of grain nitrogen metabolism when nitrogen is a limiting resource.

Wheat DOF transcription factors TaSAD and WPBF regulate glutenin gene expression in cooperation with SPA.

Grain storage proteins (GSPs) quantity and composition determine the end-use value of wheat flour. GSPs consists of low-molecular-weight glutenins (LMW-GS), high-molecular-weight glutenins (HMW-GS) and gliadins. GSP gene expression is controlled by a complex network of DNA-protein and protein-protein interactions, which coordinate the tissue-specific protein expression during grain development. The regulatory network has been most extensively studied in barley, particularly the two transcription factors (TFs) of the DNA binding with One Finger (DOF) family, barley Prolamin-box Binding Factor (BPBF) and Scutellum and Aleurone-expressed DOF (SAD). They activate hordein synthesis by binding to the Prolamin box, a motif in the hordein promoter. The BPBF ortholog previously identified in wheat, WPBF, has a transcriptional activity in expression of some GSP genes. Here, the wheat ortholog of SAD, named TaSAD, was identified. The binding of TaSAD to GSP gene promoter sequences in vitro and its transcriptional activity in vivo were investigated. In electrophoretic mobility shift assays, recombinant TaSAD and WPBF proteins bound to cis-motifs like those located on HMW-GS and LMW-GS gene promoters known to bind DOF TFs. We showed by transient expression assays in wheat endosperms that TaSAD and WPBF activate GSP gene expression. Moreover, co-bombardment of Storage Protein Activator (SPA) with WPBF or TaSAD had an additive effect on the expression of GSP genes, possibly through conserved cooperative protein-protein interactions.

Sulfur in determining seed protein composition: present understanding of its interaction with abiotic stresses and future directions.

Improving and stabilizing the quality of seed proteins are of growing interest in the current food and agroecological transitions. Sulfur is a key determinant of this quality since it is essential for the synthesis of sulfur-rich proteins in seeds. A lack of sulfur provokes drastic changes in seed protein composition, negatively impacting the nutritional and functional properties of proteins, and leading in some cases to diseases or health problems in humans. Sulfur also plays a crucial role in stress tolerance through the synthesis of antioxidant or protective molecules. In the context of climate change, questions arise regarding the trade-off between seed yield and seed quality with respect to sulfur availability and use by crops that represent important sources of proteins for human nutrition. Here, we review recent work obtained in legumes, cereals, as well as in Arabidopsis, that present major advances on: (i) the interaction between sulfur nutrition and environmental or nutritional stresses with regard to seed yield and protein composition; (ii) metabolic pathways that merit to be targeted to mitigate negative impacts of environmental stresses on seed protein quality; and (iii) the importance of sulfur homeostasis for the regulation of seed protein composition and its interplay with seed redox homeostasis.

Bread wheat (T. aestivum) variability: Phenotypic and genotypic data from 75 varieties.

Most bread wheat is consumed after processing, which mainly depends on the quantity and quality of protein in the grain. Storage protein content and composition particularly influence the end use quality of milled grain products. Storage proteins are components of the gluten network that confer dough viscoelasticity, an essential property for processing. To explore grain storage protein diversity, 75 bread wheat accessions were grown with two replicates each at two locations. Grains were harvested at maturity and samples were phenotyped for each site and each replicate plant. Grain hardness, thousand-kernel weight and grain nitrogen content were measured. The protein composition of flour from each replicate was characterised by reverse phase-high performance liquid chromatography (RP-HPLC). The molecular distribution of flour polymers was determined by asymmetric flow field-flow fractionation (AF4) and dough technological properties were assessed using a Glutomatic system and a Chopin alveograph. In addition, the 75 accessions were genotyped by the BreedWheat 35k genotyping array (Axiom TaBW35K) containing 34,746 single nucleotide polymorphism markers (SNPs). The dataset produced by this work includes six files with raw data, two files with protocols and figures. Data show the genotypic and phenotypic variabilities of the material used and can be used to explore genetic and environmental effects on traits involved in grain protein quality. This dataset is associated to the research article "Differences in bread protein digestibility traced to wheat cultivar traits" [1].

Breeding for Economically and Environmentally Sustainable Wheat Varieties: An Integrated Approach from Genomics to Selection.

There is currently a strong societal demand for sustainability, quality, and safety in bread wheat production. To address these challenges, new and innovative knowledge, resources, tools, and methods to facilitate breeding are needed. This starts with the development of high throughput genomic tools including single nucleotide polymorphism (SNP) arrays, high density molecular marker maps, and full genome sequences. Such powerful tools are essential to perform genome-wide association studies (GWAS), to implement genomic and phenomic selection, and to characterize the worldwide diversity. This is also useful to breeders to broaden the genetic basis of elite varieties through the introduction of novel sources of genetic diversity. Improvement in varieties particularly relies on the detection of genomic regions involved in agronomical traits including tolerance to biotic (diseases and pests) and abiotic (drought, nutrient deficiency, high temperature) stresses. When enough resolution is achieved, this can result in the identification of candidate genes that could further be characterized to identify relevant alleles. Breeding must also now be approached through in silico modeling to simulate plant development, investigate genotype × environment interactions, and introduce marker-trait linkage information in the models to better implement genomic selection. Breeders must be aware of new developments and the information must be made available to the world wheat community to develop new high-yielding varieties that can meet the challenge of higher wheat production in a sustainable and fluctuating agricultural context. In this review, we compiled all knowledge and tools produced during the BREEDWHEAT project to show how they may contribute to face this challenge in the coming years.

Deciphering carbohydrate metabolism during wheat grain development via integrated transcriptome and proteome dynamics.

Grain development of Triticum aestivum is being studied extensively using individual OMICS tools. However, integrated transcriptome and proteome studies are limited mainly due to complexity of genome. Current study focused to unravel the transcriptome-proteome coordination of key mechanisms underlying carbohydrate metabolism during whole wheat grain development. Wheat grains were manually dissected to obtain grain tissues for proteomics and transcriptomics analyses. Differentially expressed proteins and transcripts at the 11 stages of grain development were compared. Computational workflow for integration of two datasets related to carbohydrate metabolism was designed. For CM proteins, output peptide sequences of proteomic analyses (via LC-MS/MS) were used as source to search corresponding transcripts. The transcript that turned out with higher number of peptides was selected as bona fide ribonucleotide sequence for respective protein synthesis. More than 90% of hits resulted in successful identification of respective transcripts. Comparative analysis of protein and transcript expression profiles resulted in overall 32% concordance between these two series of data. However, during grain development correlation of two datasets gradually increased up to ~ tenfold from 152 to 655 °Cd and then dropped down. Proteins involved in carbohydrate metabolism were divided in five categories in accordance with their functions. Enzymes involved in starch and sucrose biosynthesis showed the highest correlations between proteome-transcriptome profiles. High percentage of identification and validation of protein-transcript hits highlighted the power of omics data integration approach over existing gene functional annotation tools. We found that correlation of two datasets is highly influenced by stage of grain development. Further, gene regulatory networks would be helpful in unraveling the mechanisms underlying the complex and significant traits such as grain weight and yield.

Omics Data Reveal Putative Regulators of Einkorn Grain Protein Composition under Sulfur Deficiency.

Understanding the molecular mechanisms controlling the accumulation of grain storage proteins in response to nitrogen (N) and sulfur (S) nutrition is essential to improve cereal grain nutritional and functional properties. Here, we studied the grain transcriptome and metabolome responses to postanthesis N and S supply for the diploid wheat einkorn (Triticum monococcum). During grain filling, 848 transcripts and 24 metabolites were differentially accumulated in response to N and S availability. The accumulation of total free amino acids per grain and the expression levels of 241 genes showed significant modifications during most of the grain filling period and were upregulated in response to S deficiency. Among them, 24 transcripts strongly responded to S deficiency and were identified in coexpression network analyses as potential coordinators of the grain response to N and S supply. Sulfate transporters and genes involved in sulfate and Met metabolism were upregulated, suggesting regulation of the pool of free amino acids and of the grain N-to-S ratio. Several genes highlighted in this study might limit the impact of S deficiency on the accumulation of grain storage proteins.

SNP markers for early identification of high molecular weight glutenin subunits (HMW-GSs) in bread wheat.

KEY MESSAGE: A set of eight SNP markers was developed to facilitate the early selection of HMW-GS alleles in breeding programmes. In bread wheat (Triticum aestivum), the high molecular weight glutenin subunits (HMW-GSs) are the most important determinants of technological quality. Known to be very diverse, HMW-GSs are encoded by the tightly linked genes Glu-1-1 and Glu-1-2. Alleles that improve the quality of dough have been identified. Up to now, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of grain proteins is the most widely used for their identification. To facilitate the early selection of HMW-GS alleles in breeding programmes, we developed DNA-based molecular markers. For each accession of a core collection (n = 364 lines) representative of worldwide bread wheat diversity, HMW-GSs were characterized by both genotyping and SDS-PAGE. Based on electrophoresis, we observed at least 8, 22 and 9 different alleles at the Glu-A1, Glu-B1 and Glu-D1 loci, respectively, including new variants. We designed a set of 17 single-nucleotide polymorphism (SNP) markers that were representative of the most frequent SDS-PAGE alleles at each locus. At Glu-A1 and Glu-D1, two and three marker-based haplotypes, respectively, captured the diversity of the SDS-PAGE alleles rather well. Discrepancies were found mainly for the Glu-B1 locus. However, statistical tests revealed that two markers at each Glu-B1 gene and their corresponding haplotypes were more significantly associated with the rheological properties of the dough than were the relevant SDS-PAGE alleles. To conclude, this study demonstrates that the SNP markers developed provide additional information on HMW-GS diversity. Two markers at Glu-A1, four at Glu-B1 and two at Glu-D1 constitute a useful toolbox for breeding wheat to improve end-use value.

Proteomic Data Integration Highlights Central Actors Involved in Einkorn (Triticum monococcum ssp. monococcum) Grain Filling in Relation to Grain Storage Protein Composition.

Albumins and globulins (AGs) of wheat endosperm represent about 20% of total grain proteins. Some of these physiologically active proteins can influence the synthesis of storage proteins (SPs) (gliadins and glutenins) and consequently, rheological properties of wheat flour and processing. To identify such AGs, data, (published by Bonnot et al., 2017) concerning abundance in 352 AGs and in the different seed SPs during grain filling and in response to different nitrogen (N) and sulfur (S) supply, were integrated with mixOmics R package. Relationships between AGs and SPs were first unraveled using the unsupervised method sparse Partial Least Square, also known as Projection to Latent Structure (sPLS). Then, data were integrated using a supervised approach taking into account the nutrition and the grain developmental stage. We used the block.splda procedure also referred to as DIABLO (Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies). These approaches led to the identification of discriminant and highly correlated features from the two datasets (AGs and SPs) which are not necessarily differentially expressed during seed development or in response to N or S supply. Eighteen AGs were correlated with the quantity of SPs per grain. A statistical validation of these proteins by genetic association analysis confirmed that 5 out of this AG set were robust candidate proteins able to modulate the seed SP synthesis. In conclusion, this latter result confirmed that the integrative strategy is an adequate way to reduce the number of potentially relevant AGs for further functional validation.

Gluten-free diet in French adults without coeliac disease: sociodemographic characteristics, motives and dietary profile.

The number of people avoiding gluten is growing in many Western countries. However, little information is available on their sociodemographic and dietary profiles. We aimed to describe sociodemographic, behavioural and dietary profiles of participants avoiding gluten in the NutriNet-Santé cohort. Participants of the NutriNet-Santé cohort - excluding coeliac patients - who completed a questionnaire about food exclusions, with complete data on sociodemographic characteristics and dietary intake were included (n 20 456). Food group consumptions and nutrient intakes according to self-reported avoidance of gluten were estimated using ANCOVA adjusted for age, sex and daily energy intake. Based on principal component analysis, three dietary patterns (DP) were identified. Association between DP and avoidance of gluten was investigated using multivariate logistic regression. All data were weighted on the French census. A total of 10·31 (95 % CI 9·90, 10·73) % of the participants declared avoiding gluten, of which 1·65 % totally. They were more likely to be women, older persons, non-smokers, to have a lower educational level and declared more food intolerances. They had higher consumption of fruit, vegetables and lower consumption of dairy products, salty/sweet and fatty foods and alcohol. After adjustments on confounders, a healthy dietary pattern was positively associated with total gluten avoidance (ORQuintile5vsQuintile1 = 14·44, 95 % CI 8·62, 24·19). Our study highlighted that, in this population, individuals who avoid gluten from their diet tend to have a diet more favourable to health. These results can serve as a basis for future studies investigating the potential consequences of a gluten-free diet in non-coeliac population.

Genome-wide analysis, expansion and expression of the NAC family under drought and heat stresses in bread wheat (T. aestivum L.).

The NAC family is one of the largest plant-specific transcription factor families, and some of its members are known to play major roles in plant development and response to biotic and abiotic stresses. Here, we inventoried 488 NAC members in bread wheat (Triticum aestivum). Using the recent release of the wheat genome (IWGS RefSeq v1.0), we studied duplication events focusing on genomic regions from 4B-4D-5A chromosomes as an example of the family expansion and neofunctionalization of TaNAC members. Differentially expressed TaNAC genes in organs and in response to abiotic stresses were identified using publicly available RNAseq data. Expression profiling of 23 selected candidate TaNAC genes was studied in leaf and grain from two bread wheat genotypes at two developmental stages in field drought conditions and revealed insights into their specific and/or overlapping expression patterns. This study showed that, of the 23 TaNAC genes, seven have a leaf-specific expression and five have a grain-specific expression. In addition, the grain-specific genes profiles in response to drought depend on the genotype. These genes may be considered as potential candidates for further functional validation and could present an interest for crop improvement programs in response to climate change. Globally, the present study provides new insights into evolution, divergence and functional analysis of NAC gene family in bread wheat.

The bZIP transcription factor SPA Heterodimerizing Protein represses glutenin synthesis in Triticum aestivum.

The quality of wheat grain is mainly determined by the quantity and composition of its grain storage proteins (GSPs). Grain storage proteins consist of low- and high-molecular-weight glutenins (LMW-GS and HMW-GS, respectively) and gliadins. The synthesis of these proteins is essentially regulated at the transcriptional level and by the availability of nitrogen and sulfur. The regulation network has been extensively studied in barley where BLZ1 and BLZ2, members of the basic leucine zipper (bZIP) family, activate the synthesis of hordeins. To date, in wheat, only the ortholog of BLZ2, Storage Protein Activator (SPA), has been identified as playing a major role in the regulation of GSP synthesis. Here, the ortholog of BLZ1, named SPA Heterodimerizing Protein (SHP), was identified and its involvement in the transcriptional regulation of the genes coding for GSPs was analyzed. In gel mobility shift assays, SHP binds cis-motifs known to bind to bZIP family transcription factors in HMW-GS and LMW-GS promoters. Moreover, we showed by transient expression assays in wheat endosperm that SHP acts as a repressor of the activity of these gene promoters. This result was confirmed in transgenic lines overexpressing SHP, which were grown with low and high nitrogen supply. The phenotype of SHP-overexpressing lines showed a lower quantity of both LMW-GS and HMW-GS, while the quantity of gliadin was unchanged, whatever the nitrogen availability. Thus, the gliadin/glutenin ratio was increased, which suggests that gliadin and glutenin genes may be differently regulated.

Grain subproteome responses to nitrogen and sulfur supply in diploid wheat Triticum monococcum ssp. monococcum.

Wheat grain storage proteins (GSPs) make up most of the protein content of grain and determine flour end-use value. The synthesis and accumulation of GSPs depend highly on nitrogen (N) and sulfur (S) availability and it is important to understand the underlying control mechanisms. Here we studied how the einkorn (Triticum monococcum ssp. monococcum) grain proteome responds to different amounts of N and S supply during grain development. GSP composition at grain maturity was clearly impacted by nutrition treatments, due to early changes in the rate of GSP accumulation during grain filling. Large-scale analysis of the nuclear and albumin-globulin subproteomes during this key developmental phase revealed that the abundance of 203 proteins was significantly modified by the nutrition treatments. Our results showed that the grain proteome was highly affected by perturbation in the N:S balance. S supply strongly increased the rate of accumulation of S-rich α/ß-gliadin and γ-gliadin, and the abundance of several other proteins involved in glutathione metabolism. Post-anthesis N supply resulted in the activation of amino acid metabolism at the expense of carbohydrate metabolism and the activation of transport processes including nucleocytoplasmic transit. Protein accumulation networks were analyzed. Several central actors in the response were identified whose variation in abundance was related to variation in the amounts of many other proteins and are thus potentially important for GSP accumulation. This detailed analysis of grain subproteomes provides information on how wheat GSP composition can possibly be controlled in low-level fertilization condition.

Wheat glutenin: the "tail" of the 1By protein subunits.

Gluten-forming storage proteins play a major role in the viscoelastic properties of wheat dough through the formation of a continuous proteinaceous network. The high-molecular-weight glutenin subunits represent a functionally important subgroup of gluten proteins by promoting the formation of large glutenin polymers through interchain disulphide bonds between glutenin subunits. Here, we present evidences that y-type glutenin subunits encoded at the Glu-B1 locus are prone to proteolytic processing at the C-terminus tail, leading to the loss of the unique cysteine residue present at the C-terminal domain. Results obtained by intact mass measurement and immunochemistry for each proteoform indicate that the proteolytic cleavage appears to occur at the carboxyl-side of two conserved asparagine residues at the C-terminal domain start. Hence, we hypothesize that the responsible enzymes are a class of cysteine endopeptidases - asparaginyl endopeptidases - described in post-translational processing of other storage proteins in wheat. Biological significance The reported study provides new insights into wheat storage protein maturation. In view of the importance of gluten proteins on dough viscoelastic properties and end-product quality, the reported C-terminal domain cleavage of high-molecular-weight glutenin subunits is of particular interest, since this domain possesses a unique conserved cysteine residue which is assumed to participate in gluten polymerization.

(1)H-NMR screening for the high-throughput determination of genotype and environmental effects on the content of asparagine in wheat grain.

Free asparagine in cereals is known to be the precursor of acrylamide, a neurotoxic and carcinogenic product formed during cooking processes. Thus, the development of crops with lower asparagine is of considerable interest to growers and the food industry. In this study, we describe the development and application of a rapid (1)H-NMR-based analysis of cereal flour, that is, suitable for quantifying asparagine levels, and hence acrylamide-forming potential, across large numbers of samples. The screen was applied to flour samples from 150 bread wheats grown at a single site in 2005, providing the largest sample set to date. Additionally, screening of 26 selected cultivars grown for two further years in the same location and in three additional European locations in the third year (2007) provided six widely different environments to allow estimation of the environmental (E) and G x E effects on asparagine levels. Asparagine concentrations in the 150 genotypes ranged from 0.32 to 1.56 mg/g dry matter in wholemeal wheat flours. Asparagine levels were correlated with plant height and therefore, due to recent breeding activities to produce semi-dwarf varieties, a negative relationship with the year of registration of the cultivar was also observed. The multisite study indicated that only 13% of the observed variation in asparagine levels was heritable, whilst the environmental contribution was 36% and the GxE component was 43%. Thus, compared to some other phenotypic traits, breeding for low asparagine wheats presents a difficult challenge.

Transcriptional and metabolic alternations rebalance wheat grain storage protein accumulation under variable nitrogen and sulfur supply.

Wheat (Triticum aestivum L.) grain storage proteins (GSPs) are major determinants of flour end-use value. Biological and molecular mechanisms underlying the developmental and nutritional determination of GSP accumulation in cereals are as yet poorly understood. Here we timed the accumulation of GSPs during wheat grain maturation relative to changes in metabolite and transcript pools in different conditions of nitrogen (N) and sulfur (S) availability. We found that the N/S supply ratio modulated the duration of accumulation of S-rich GSPs and the rate of accumulation of S-poor GSPs. These changes are likely to be the result of distinct relationships between N and S allocation, depending on the S content of the GSP. Most developmental and nutritional modifications in GSP synthesis correlated with the abundance of structural gene transcripts. Changes in the expression of transport and metabolism genes altered the concentrations of several free amino acids under variable conditions of N and S supply, and these amino acids seem to be essential in determining GSP expression. The comprehensive data set generated and analyzed here provides insights that will be useful in adapting fertilizer use to variable N and S supply, or for breeding new cultivars with balanced and robust GSP composition.

RulNet: A Web-Oriented Platform for Regulatory Network Inference, Application to Wheat -Omics Data.

With the increasing amount of -omics data available, a particular effort has to be made to provide suitable analysis tools. A major challenge is that of unraveling the molecular regulatory networks from massive and heterogeneous datasets. Here we describe RulNet, a web-oriented platform dedicated to the inference and analysis of regulatory networks from qualitative and quantitative -omics data by means of rule discovery. Queries for rule discovery can be written in an extended form of the RQL query language, which has a syntax similar to SQL. RulNet also offers users interactive features that progressively adjust and refine the inferred networks. In this paper, we present a functional characterization of RulNet and compare inferred networks with correlation-based approaches. The performance of RulNet has been evaluated using the three benchmark datasets used for the transcriptional network inference challenge DREAM5. Overall, RulNet performed as well as the best methods that participated in this challenge and it was shown to behave more consistently when compared across the three datasets. Finally, we assessed the suitability of RulNet to analyze experimental -omics data and to infer regulatory networks involved in the response to nitrogen and sulfur supply in wheat (Triticum aestivum L.) grains. The results highlight putative actors governing the response to nitrogen and sulfur supply in wheat grains. We evaluate the main characteristics and features of RulNet as an all-in-one solution for RN inference, visualization and editing. Using simple yet powerful RulNet queries allowed RNs involved in the adaptation of wheat grain to N and S supply to be discovered. We demonstrate the effectiveness and suitability of RulNet as a platform for the analysis of RNs involving different types of -omics data. The results are promising since they are consistent with what was previously established by the scientific community.

Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits.

The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression.