The VviNAC33 acts a molecular switch by activating leaf degreening and repressing vegetative growth during the immature-to-mature phase transition in grapevine

In this study we demonstrated that the grapevine NAC transcription factor VviNAC33 is directly involved in leaf degreening and organ growth. Through the application of DAP-seq approach we identified several putative targets of VviNAC33, among which the SGR1 involved in the breakdown of chlorophyll. Stable VviNAC33 overexpressing transgenic plants displayed an obvious degreening effect on leaves and an inhibition of leaf growth. Consistently, transgenic plant expressing a chimeric repressor of the VviNAC33 showed the opposite phenotypic alterations. Our results evidenced that VviNAC33 is a direct player of leaf senescence program and propose a blueprint of the complex transcriptional regulatory network that govern organ phase transition in grapevine. For transient transformation of Vitis vinifera cv. Sultana, the 35S:VviNAC33 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. Eight in vitro six-week-old plants of grapevine cv. Sultana for overexpression and seven 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.

本研究证实，葡萄NAC转录因子（NAC transcription factor）VviNAC33直接参与叶片褪绿与器官生长过程。通过DNA亲和纯化测序（DAP-seq）技术，本研究鉴定出VviNAC33的多个潜在靶标基因，其中包括参与叶绿素降解的持绿蛋白1（SGR1）。稳定过表达VviNAC33的转基因植株叶片表现出显著褪绿现象，且叶片生长受到抑制。与之一致的是，表达VviNAC33嵌合抑制子的转基因植株则呈现出相反的表型变化。本研究结果证明VviNAC33是叶片衰老程序的直接调控因子，并提出了调控葡萄器官阶段转变的复杂转录调控网络蓝图。针对葡萄品种苏丹娜（Vitis vinifera cv. Sultana）的瞬时转化实验中，本研究通过电转化法将35S:VviNAC33重组载体导入根癌农杆菌（Agrobacterium tumefaciens）C58C1菌株中。作为对照，同时将携带非编码序列的空载体pK7WG2导入农杆菌。各取8株6周龄的离体苏丹娜葡萄幼苗用于过表达组、7株用于对照组，将其浸入菌悬液后进行真空渗透处理（90kPa下处理2次，每次2分钟）。农杆菌浸润处理完成后，将幼苗在离体培养条件下恢复培养7天，随后收集材料用于RNA提取与转录组分析。