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Storage proteins (SP) are the product of genes whose expression is regulated at the transcriptional level by the availability of nitrogen and sulphur assimilates. Transcriptional regulation involves several transcription factors (TFs) that bind at the level of cis-motifs located in the promoter regions of SP genes. Studies carried out in barley have shown the involvement of 5 cis-motifs and 8 TFs in the activation of hordein synthesis, including MCB1. This regulatory network is conserved between species, not only between monocotyledons but also between dicotyledons. In bread wheat, the cis-motifs identified in barley are present on the promoters of the genes coding for SP. Orthologous TFs in barley have also been found in bread wheat and Triticum monococcum (einkorn). Furthermore, in order to study the impact of nutrition on the response of T. monococcum grain and the accumulation of storage proteins, an integrative analysis of omics data (transcriptomics, proteomics, metabolomics) was carried out within the team. It enabled the identification of metabolic pathways potentially involved in the synthesis of SP and key players, including a TF from the NAC family (NAC22). Finally, an association genetics analysis revealed that TaMCB1 and NAC22 are both associated with glutenin composition, with NAC22 also being associated with parameters of nitrogen allocation to certain protein fractions.

In wheat, the roles in transcriptional regulation of SP synthesis of these TFs has not yet been studied. The thesis therefore proposes to elucidate the role of these two original candidates in the synthesis of SP in wheat to enrich their regulation scheme. To answer this research question, the involvement of TaMCB1 and NAC22 in the regulation of SP synthesis will be functionally characterized and validated. For this, the student will be in charge of:

1/ to study in vitro by gel mobility shift assays (EMSA) the interactions between candidate TFs and the cis-motifs of promoters of SP genes.

2/ to evaluate the regulatory activity of TFs in vivo by transient expression on immature wheat endosperms. The impact of nitrogen and sulphur nutrition on regulatory activity will also be studied after endosperm transformation by modulating amino acid supply for 24 hours.

3/ to validate in planta the role of these TFs by studying transgenic plants over- and under-expressing them in response to nitrogen and sulphur nutrition. The SP content and composition of the grains at maturity and RNAseq analyses will be carried out. Grains of T. monococcum T1 RNAi and OE from several transformation events are already available. T. monococcum is a good study model due to its diploid genome which is very close to subgenome A of bread wheat (hexaploid species) and that it possesses all the SP of bread wheat. Moreover, its growth cycle is faster than that of hexaploid wheat.