Heat stress severely restricts the growth and fruit development of apple (Malus domestica). Little is known about the involvement of WRKY proteins in the heat tolerance mechanism in apple. In this study, we found that the apple transcription factor MdWRKY75 responds to heat and positively regulates basal thermotolerance. Apple plants that overexpressed MdWRKY75 were more tolerant to heat stress, while silencing MdWRKY75 caused the opposite phenotype. RNA-seq and reverse transcription quantitative PCR showed that heat shock transcription factor genes (MdHsfs) could be the potential targets of MdWRKY75. Electrophoretic mobility shift, yeast one-hybrid, β-glucuronidase, and dual-luciferase assays showed that MdWRKY75 can bind to the promoters of MdHsf4, MdHsfB2a, and MdHsfA1d and activate their expression. Apple plants that overexpressed MdHsf4, MdHsfB2a, and MdHsfA1d exhibited heat tolerance and rescued the heat sensitive phenotype of MdWRKY75-Ri3. In addition, apple heat shock cognate 70 (MdHSC70) interacts with MdWRKY75, as shown by yeast two-hybrid, split luciferase, bimolecular fluorescence complementation, and pull-down assays. MdHSC70 acts as a negative regulator of the heat stress response. Apple plants that overexpressed MdHSC70 were sensitive to heat, while virus-induced gene silencing of MdHSC70 enhanced heat tolerance. Additional research showed that MdHSC70 exhibits heat sensitivity by interacting with MdWRKY75 and inhibiting MdHsfs expression. In summary, we proposed a mechanism for the response of apple to heat that is mediated by the ‘MdHSC70/MdWRKY75-MdHsfs’ molecular module, which enhances our understanding of apple thermotolerance regulated by WRKY transcription factors.

中文翻译：

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MdHSC70-MdWRKY75模块通过调节热休克因子基因的表达介导苹果基础耐热性


热胁迫严重限制了苹果(Malusdomestica)的生长和果实发育。关于 WRKY 蛋白在苹果耐热机制中的作用知之甚少。在这项研究中，我们发现苹果转录因子 MdWRKY75 对热做出反应并正向调节基础耐热性。过度表达 MdWRKY75 的苹果植株对热胁迫具有更强的耐受性，而沉默 MdWRKY75 则会导致相反的表型。 RNA-seq和逆转录定量PCR表明热休克转录因子基因（MdHsfs）可能是MdWRKY75的潜在靶标。电泳迁移率变动、酵母单杂交、β-葡萄糖醛酸酶和双荧光素酶测定表明，MdWRKY75 可以与 MdHsf4、MdHsfB2a 和 MdHsfA1d 的启动子结合并激活它们的表达。过表达 MdHsf4、MdHsfB2a 和 MdHsfA1d 的苹果植株表现出耐热性，并挽救了 MdWRKY75-Ri3 的热敏感表型。此外，酵母双杂交、裂解荧光素酶、双分子荧光互补和 Pull-down 测定表明，苹果热休克同源 70 (MdHSC70) 与 MdWRKY75 相互作用。 MdHSC70 充当热应激反应的负调节剂。过表达 MdHSC70 的苹果植株对热敏感，而病毒诱导的 MdHSC70 基因沉默增强了耐热性。其他研究表明，MdHSC70 通过与 MdWRKY75 相互作用并抑制 MdHsfs 表达而表现出热敏感性。总之，我们提出了一种由“MdHSC70/MdWRKY75-MdHsfs”分子模块介导的苹果对热的响应机制，这增强了我们对WRKY转录因子调控苹果耐热性的理解。