NAC61 regulates late and post-ripening associated processes in grape comprising a NAC60-NAC dependent regulatory network
NACs (NAM/ATAF/CUC) proteins are plant-specific transcription factors with key roles in plant development and stress responses. Here, Vitis vinifera NAC61 was selected for its functional characterization as one of the key candidates of the grape post-harvest withering process. VviNAC61 is particularly expressed at veraison and post-veraison berry stages and also shows a high expression in lignified tissues such as roots, tendrils, seeds and rachis. In leaves, VviNAC61 achieves its highest expression at senescence stage. In line with this observation, a NAC family multispecies phylogenetic tree revealed that VviNAC61 clusters with VviNAC33 and AtANAC046, both involved in regulating senescence processes. VviNAC61 was transiently overexpressed in N. benthamiana leaves, showing wilting/water-soaking symptoms correlated to low stomatal conductance, high leaf temperature, increased loss of the membrane integrity and accumulation of hydrogen peroxide and water. Transcriptomic analysis of cultivar 'Thompson Seedless' leaves transiently overexpressing VviNAC61 and the search of TF-bound genes by DAP-seq was allowed us to narrow down a list of VviNAC61-regulated genes. The 'secondary metabolism', 'transcription factor regulation' and 'oxidative stress' functional categories were found to be the most enriched among the candidate target genes. More in detail, several genes encoding for the phenylpropanoid pathway-related enzymes, several MYBs and NAC genes (including VviNAC61), were bound in their promoter region by this TF and were among the upregulated genes. Direct regulation of the stilbene synthase regulator VviMYB14, the abiotic stress-related gene DHN1b, the Botrytis cinerea susceptibility gene VviWRKY52 and the VviNAC61 self-regulation were validated through dual luciferase assays. Lastly, the upstream regulation mechanism of VviNAC61 was further investigated demonstrating that VviNAC61 is controlled by and interacts with VviNAC60, a proposed master regulator of grapevine organ maturation. Overall design: Identification of binding sites for NAC61 transcription factor in Vitis vinifera by direct sequencing of affinity purified genomic DNA fragments.