National Research Center for Apple Engineering and Technology, Shandong Collaborative Innovation Center of Fruit & Vegetable Quality and Efficient Production, College of Horticultural Science and Engineering, Shandong Agricultural University, Tai'an, Shandong, China.
College of Horticulture, Agricultural University of Hebei, Baoding, Hebei, China.
Plant Biotechnol J. 2024 Dec;22(12):3244-3261. doi: 10.1111/pbi.14445. Epub 2024 Aug 23.
Softening in fruit adversely impacts their edible quality and commercial value, leading to substantial economic losses during fruit ripening, long-term storage, long-distance transportation, and marketing. As the apple fruit demonstrates climacteric respiration, its firmness decreases with increasing ethylene release rate during fruit ripening and postharvest storage. However, the molecular mechanisms underlying ethylene-mediated regulation of fruit softening in apple remain poorly understood. In this study, we identified a WRKY transcription factor (TF) MdWRKY31, which is repressed by ethylene treatment. Using transgenic approaches, we found that overexpression of MdWRKY31 delays softening by negatively regulating xyloglucan endotransglucosylase/hydrolases 2 (MdXTH2) expression. Yeast one-hybrid (Y1H), electrophoretic mobility shift (EMSA), and dual-luciferase assays further suggested that MdWRKY31 directly binds to the MdXTH2 promoter via a W-box element and represses its transcription. Transient overexpression of ethylene-induced MdNAC7, a NAC TF, in apple fruit promoted softening by decreasing cellulose content and increasing water-soluble pectin content in fruit. MdNAC7 interacted with MdWRKY31 to form a protein complex, and their interaction decreased the transcriptional repression of MdWRKY31 on MdXTH2. Furthermore, MdNAC7 does not directly regulate MdXTH2 expression, but the protein complex formed with MdWRKY31 hinders MdWRKY31 from binding to the promoter of MdXTH2. Our findings underscore the significance of the regulatory complex NAC7-WRKY31 in ethylene-responsive signalling, connecting the ethylene signal to XTH2 expression to promote fruit softening. This sheds light on the intricate mechanisms governing apple fruit firmness and opens avenues for enhancing fruit quality and reducing economic losses associated with softening.
果实软化会降低其食用品质和商业价值，导致果实成熟、长期贮藏、长途运输和销售过程中的重大经济损失。由于苹果果实表现出呼吸跃变，其硬度随着果实成熟和采后贮藏过程中乙烯释放率的增加而降低。然而，乙烯调控苹果果实软化的分子机制尚不清楚。本研究鉴定了一个受乙烯处理抑制的 WRKY 转录因子（TF）MdWRKY31。通过转基因方法，我们发现过表达 MdWRKY31 通过负调控木葡聚糖内转糖苷酶/水解酶 2（MdXTH2）的表达来延迟软化。酵母单杂交（Y1H）、电泳迁移率变动分析（EMSA）和双荧光素酶报告基因检测进一步表明，MdWRKY31 通过 W-box 元件直接结合到 MdXTH2 启动子上，并抑制其转录。瞬时过表达乙烯诱导的苹果果实 MdNAC7（一种 NAC TF）通过降低纤维素含量和增加果实中水溶性果胶含量来促进软化。MdNAC7 与 MdWRKY31 相互作用形成蛋白复合物，其相互作用降低了 MdWRKY31 对 MdXTH2 的转录抑制。此外，MdNAC7 不直接调控 MdXTH2 的表达，而是与 MdWRKY31 形成的蛋白复合物阻碍了 MdWRKY31 与 MdXTH2 启动子的结合。我们的研究结果强调了 NAC7-WRKY31 调控复合物在乙烯响应信号中的重要性，将乙烯信号与 XTH2 表达联系起来，促进果实软化。这揭示了调控苹果果实硬度的复杂机制，并为提高果实品质和减少软化相关经济损失提供了新的途径。
