wDBTF: an integrated database resource for studying wheat transcription factor families

<p>Abstract</p> <p>Background</p> <p>Transcription factors (TFs) regulate gene expression by interacting with promoters of their target genes and are classified into families based on their DNA-binding domains. Genes coding for TFs have been identified in the sequences of model plant genomes. The rice (<it>Oryza sativa </it>spp. <it>japonica</it>) genome contains 2,384 TF gene models, which represent the mRNA transcript of a locus, classed into 63 families.</p> <p>Results</p> <p>We have created an extensive list of wheat (<it>Triticum aestivum </it>L) TF sequences based on sequence homology with rice TFs identified and classified in the Database of Rice Transcription Factors (DRTF). We have identified 7,112 wheat sequences (contigs and singletons) from a dataset of 1,033,960 expressed sequence tag and mRNA (ET) sequences available. This number is about three times the number of TFs in rice so proportionally is very similar if allowance is made for the hexaploidy of wheat. Of these sequences 3,820 encode gene products with a DNA-binding domain and thus were confirmed as potential regulators. These 3,820 sequences were classified into 40 families and 84 subfamilies and some members defined orphan families. The results were compiled in the Database of Wheat Transcription Factor (wDBTF), an inventory available on the web <url>http://wwwappli.nantes.inra.fr:8180/wDBFT/</url>. For each accession, a link to its library source and its Affymetrix identification number is provided. The positions of Pfam (protein family database) motifs were given when known.</p> <p>Conclusions</p> <p>wDBTF collates 3,820 wheat TF sequences validated by the presence of a DNA-binding domain out of 7,112 potential TF sequences identified from publicly available gene expression data. We also incorporated <it>in silico </it>expression data on these TFs into the database. Thus this database provides a major resource for systematic studies of TF families and their expression in wheat as illustrated here in a study of DOF family members expressed during seed development.</p

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

International audienceGrain 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

Nucleotide Polymorphism in the Wheat Transcriptional Activator Spa Influences Its Pattern of Expression and Has Pleiotropic Effects on Grain Protein Composition, Dough Viscoelasticity, and Grain Hardness[W][OA]

Storage protein activator (SPA) is a key regulator of the transcription of wheat (Triticum aestivum) grain storage protein genes and belongs to the Opaque2 transcription factor subfamily. We analyzed the sequence polymorphism of the three homoeologous Spa genes in hexaploid wheat. The level of polymorphism in these genes was high particularly in the promoter. The deduced protein sequences of each homoeolog and haplotype show greater than 93% identity. Two major haplotypes were studied for each Spa gene. The three Spa homoeologs have similar patterns of expression during grain development, with a peak in expression around 300 degree days after anthesis. On average, Spa-B is 10 and seven times more strongly expressed than Spa-A and Spa-D, respectively. The haplotypes are associated with significant quantitative differences in Spa expression, especially for Spa-A and Spa-D. Significant differences were found in the quantity of total grain nitrogen allocated to the gliadin protein fractions for the Spa-A haplotypes, whereas the synthesis of glutenins is not modified. Genetic association analysis between Spa and dough viscoelasticity revealed that Spa polymorphisms are associated with dough tenacity, extensibility, and strength. Except for Spa-A, these associations can be explained by differences in grain hardness. No association was found between Spa markers and the average single grain dry mass or grain protein concentration. These results demonstrate that in planta Spa is involved in the regulation of grain storage protein synthesis. The associations between Spa and dough viscoelasticity and grain hardness strongly suggest that Spa has complex pleiotropic functions during grain development

Genetics of dietary fibre in bread wheat

Arabinoxylans (AX) are major components of cell walls in wheat endosperm. The water-extractable part, WEAX, is considered as dietary fibres with health promoting effects. AX exhibit large natural variations in their amount but few studies have been carried out on the genetics of WEAX content and structure in bread wheat. We first carried out a "forward'' quantitative genetic approach, using two recombinant populations derived from crosses between WEAX-high and WEAX-low parents and the viscosity of flour extract (WEAX-viscosity) as a predictor of WEAX content. This allowed us to identify two QTL regions, one of them, found on chromosome 6B in both populations, having a major effect with as much as 59% of the phenotypic variation explained by a single QTL. Then, we focused on key enzymes involved in the biosynthetic pathway of arabinoxylans. Out of 34 homoeologous candidate genes studied by sequencing a set of 46 lines, representing a worldwide diversity 179 show no polymorphi! sm. In the 16 polymorphic candidate genes, 80 SNP (in 15 genes) and 16 indels (in 4 genes) were detected. Then 27 SNP (in 13 genes) were genotyped in a larger collection of 156 lines (Healthgrain diversity screen). Eight associations were significant at an indicative 5% threshold, but only one, between COMT (Caffeic acid O methyltransferase) on chromosome 7A and A/X in WEAX was significant at a 1% level. The usefulness of these approaches and preliminary results for breeding is discussed

The anti-tumor efficacy of 3C23K, a glyco-engineered humanized anti-MISRII antibody, in an ovarian cancer model is mainly mediated by engagement of immune effector cells

International audienceOvarian cancer is the leading cause of death in women with gynecological cancers and despite recent advances, new and more efficient therapies are crucially needed. Müllerian Inhibiting Substance type II Receptor (MISRII, also named AMHRII) is expressed in most ovarian cancer subtypes and is a novel potential target for ovarian cancer immunotherapy. We previously developed and tested 12G4, the first murine monoclonal antibody (MAb) against human MISRII. Here, we report the humanization, affinity maturation and glyco-engineering steps of 12G4 to generate the Fc-optimized 3C23K MAb, and the evaluation of its in vivo anti-tumor activity. The epitopes of 3C23K and 12G4 were strictly identical and 3C23K affinity for MISRII was enhanced by a factor of about 14 (KD = 5.5 × 10-11 M vs 7.9 × 10-10 M), while the use of the EMABling® platform allowed the production of a low-fucosylated 3C23K antibody with a 30-fold KD improvement of its affinity to FcγRIIIa. In COV434-MISRII tumor-bearing mice, 3C23K reduced tumor growth more efficiently than 12G4 and its combination with carboplatin was more efficient than each monotherapy with a mean tumor size of 500, 1100 and 100 mm3 at the end of treatment with 3C23K (10 mg/kg, Q3-4D12), carboplatin (60 mg/kg, Q7D4) and 3C23K+carboplatin, respectively. Conversely, 3C23K-FcKO, a 3C23K form without affinity for the FcγRIIIa receptor, did not display any anti-tumor effect in vivo. These results strongly suggested that 3C23K mechanisms of action are mainly Fc-related. In vitro, antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) were induced by 3C23K, as demonstrated with human effector cells. Using human NK cells, 50% of the maximal lysis was obtained with a 46-fold lower concentration of low-fucosylated 3C23K (2.9 ng/ml) than of 3C23K expressed in CHO cells (133.35 ng/ml). As 3C23K induced strong ADCC with human PBMC but almost none with murine PBMC, antibody-dependent cell phagocytosis (ADCP) was then investigated. 3C23K-dependent (100 ng/ml) ADCP was more active with murine than human macrophages (only 10% of living target cells vs. about 25%). These in vitro results suggest that the reduced ADCC with murine effectors could be partially balanced by ADCP activity in in vivo experiments. Taken together, these preclinical data indicate that 3C23K is a new promising therapeutic candidate for ovarian cancer immunotherapy and justify its recent introduction in a phase I clinical trial