NAC61 regulates late- and post-ripening osmotic, oxidative, and biotic stress responses in grapevine

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 that hallmark late- and post-ripening grape stages. For transient transformation of Vitis vinifera cv. Thompson Seedless, the 35S:NAC61 construct was transferred to Agrobacterium tumefaciens strain C58C1 by electroporation. As control, Agrobacterium was also transformed with an empty pK7WG2 containing a non-coding sequence. Seven in vitro six-week-old plants of grapevine cv. Thompson Seedless for overexpression and five for the control were immersed in each bacterial suspension and vacuum infiltrated (2 X 2 min at 90kPa). After agroinfiltration, plantlets were allowed to recover in vitro for seven days before collecting material for RNA extraction and transcriptomic analysis.