As major contributors to flavor in many fruit species, volatile esters are useful for investigating the regulation of the biosynthesis pathways of volatile aroma compounds in plants. Ethylene promotes the biosynthesis of volatile esters during fruit storage while accelerating fruit ripening; thus, the ethylene perception inhibitor 1-Methylcyclopropene (1-MCP) is employed to prolong the shelf life of fruits. However, the mechanisms by which 1-MCP regulates volatiles synthesis remain unclear. In this study, we analyzed the pathway of 1-MCP-mediated volatile ester synthesis in ‘Nanguo’ pear (Pyrus ussuriensis). 1-MCP significantly decreased volatile ester synthesis during storage. Comparative transcriptome analysis showed that the genes encoding two transcription factors (PuNAC37, PuWRKY74) and a RING-type E3 ubiquitin ligase which have a domain of unknown function (PuRDUF2) were expressed at high levels, whereas ALCOHOL ACYLTRANSFERASE 1 (PuAAT1), encoding an enzyme responsible for volatile ester synthesis, was expressed at low levels in 1-MCP-treated fruit. Moreover, PuNAC37 inhibited the expression of PuWRKY74 via transcriptional regulation, whereas PuNAC37 and PuWRKY74, after directly binding to the promoter of PuAAT1, synergistically inhibited its expression in 1-MCP-treated fruit. In addition, in vitro and in vivo ubiquitination experiments revealed that PuRDUF2 functions as an E3 ubiquitin ligase that ubiquitinates PuAAT1, causing its degradation via the 26S proteasome pathway following 1-MCP treatment. Subsequent PuAAT1 degradation resulted in a reduction of volatile esters during fruit storage. Our findings provide insights into the complex transcriptional regulation of volatile ester formation in ‘Nanguo’ pears and contribute to future research on AAT protein ubiquitination in other species.

中文翻译：

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转录因子 PuNAC37/PuWRKY74 和 E3 泛素连接酶 PuRDUF2 抑制'南果'梨挥发性酯合成


作为许多水果物种风味的主要贡献者，挥发性酯可用于研究植物中挥发性香气化合物的生物合成途径的调节。乙烯在促进水果贮藏过程中挥发性酯的生物合成，同时加速水果成熟;因此，乙烯感知抑制剂 1-甲基环丙烯 （1-MCP） 被用来延长水果的保质期。然而，1-MCP 调节挥发物合成的机制仍不清楚。在本研究中，我们分析了 '南果' 梨 （Pyrus ussuriensis） 中 1-MCP 介导的挥发性酯合成途径。1-MCP 显著降低了贮藏过程中挥发性酯的合成。比较转录组分析显示，编码两种转录因子 （PuNAC37、PuWRKY74） 和一种功能域未知的 RING 型 E3 泛素连接酶 （PuRDUF2） 的基因表达水平较高，而编码负责挥发性酯合成的酶的醇酰基转移酶 1 （PuAAT1） 在 1-MCP 处理的果实中表达水平较低。此外，PuNAC37 通过转录调控抑制 PuWRKY74 的表达，而 PuNAC37 和 PuWRKY74 在直接与 PuAAT1 的启动子结合后，协同抑制其在 1-MCP 处理的果实中的表达。此外，体外和体内泛素化实验表明，PuRDUF2 作为泛素化 PuAAT1 的 E3 泛素连接酶发挥作用，导致其在 1-MCP 处理后通过 26S 蛋白酶体途径降解。随后的 PuAAT1 降解导致水果储存过程中挥发性酯的减少。 我们的研究结果为'南果'梨挥发性酯形成的复杂转录调控提供了见解，并为其他物种中 AAT 蛋白泛素化的未来研究做出了贡献。