NAC61 regulates late-and post-ripening associated processes in grapes involving a NAC60-dependent regulatory network

During late-and post-ripening stages, grape berry undergoes profound biochemical and physiological changes whose molecular control is poorly understood. Here, we report the role of NAC61, a grapevine NAC transcription factor, in regulating different processes featuring the berry ripening progression. NAC61 is highly expressed during post-harvest berry dehydration and its expression pattern is closely related to sugar concentration. The ectopic expression of NAC61 in Nicotiana benthamiana leaves determines low stomatal conductance, high leaf temperature, tissue collapse and a higher relative water content. Transcriptome analysis of grapevine leaves transiently overexpressing NAC61, and DNA affinity purification and sequencing analyses allowed us to narrow down a list of NAC61-regulated genes. Direct regulation of the stilbene synthase regulator MYB14 , the osmotic stress-related gene DHN1b , the Botrytis cinerea susceptibility gene WRKY52 and the NAC61 itself, is validated. We also demonstrate that NAC61 interacts with NAC60, a proposed master regulator of grapevine organ maturation, in the activation of MYB14 and NAC61 expression. Overall, our findings establish NAC61 as a key player in a regulative network that governs stilbenoid metabolism and osmotic, oxidative and biotic stress responses in grape berry during late-and post-ripening. Highlights NAC61 regulates stilbene biosynthesis and abiotic/biotic stress responses that hallmark late-and post-ripening developmental stages in grapevine berry. NAC61 participates in a NAC60-dependent regulatory network, also triggering its self-activation. ### Competing Interest Statement The authors have declared no competing interest. * ### Abbreviations (DAB) : 3,3’-Diaminobenzidine (ABA) : Abscisic Acid (BiFC) : Bimolecular Fluorescence Complementation Assay (CDS) : Coding Sequence (DEGs) : Control (CTRL), Differentially Expressed Genes (DAP-seq) : DNA Affinity Purification and sequencing (DLRA) : Dual Luciferase Reporter Assay (LUC) : Firefly Luciferase (FC) : Fold Change (GCNs) : Gene Co-expression Networks (GRN) : Gene Regulatory Network (HCTs) : High Confident Targets (PHW) : Post Harvest Withering (ROS) : Reactive Oxygen species (RT-qPCR) : Real Time quantitative Polymerase Chain Reaction (RWC) : Relative Water Content (REN) : Renilla reniformis Luciferase (TF) : Tissue-independent (TI), Transcription Factor (TSS) : Transcription Start Site (YFP) : Yellow Fluorescent Protein.