ABSTRACT

Autophagy, an evolutionally conserved cellular degradation process, plays critical roles in plant development and stress response. Despite the wealth of information on the vital role of autophagy in responses to environmental stresses, little is known about the regulation of autophagy. In this study, we demonstrated that spermidine (Spd), a kind of polyamine, was involved in the regulation of salt tolerance through activating the expression of ATG (autophagy-related) genes and the formation of autophagosomes in cucumber under salt stress. Furthermore, NADPH oxidase-derived apoplastic H₂O₂-mediated Spd-induced salt tolerance and autophagy. Exogenous Spd significantly increased the tolerance to salt stress and inhibited the accumulation and ubiquitination of insoluble proteins. Foliar application of Spd promoted the transcript levels of ATG genes and autophagosomes formation. Besides, Spd induced the expression of RBOH (respiratory burst oxidase homolog), and the accumulation of H₂O₂ both in leaves and roots. However, either pretreatment with dimethylthiourea (DMTU, an H₂O₂ scavenger) or diphenyleneiodonium chloride (DPI, an inhibitor of NADPH oxidase) reduced Spd-induced accumulation of apoplastic H₂O₂. Importantly, Spd-induced salt tolerance and autophagy were compromised when plants were pretreated with DMTU or DPI. Furthermore, the silencing of ATG4 and ATG7 reduced Spd-induced salt tolerance and autophagosomes formation. Taken together, these results revealed that RBOH-dependent H₂O₂ mediated the Spd-induced autophagy and salt tolerance in cucumber.

Abbreviations: Asat: light-saturated rate of CO₂ assimilation; ATG: autophagy-related; DCF-DA: 2, 7-dichlorofluorescein diacetate; DMTU: dimethylthiourea; DPI: diphenyleneiodonium chloride; DW: dry weight; EL: electrolyte leakage; FW: fresh weight; Fv/Fm: the maximum quantum yield of photosystem II; GFP: green fluorescent protein; MDC: monodansylcadaverine; PDS: phytoene desaturase; PE: phosphatidylethanolamine; PLD: phospholipase D; RBOH: respiratory burst oxidase homolog; ROS: reactive oxygen species; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIN1: salt induced NAC1; Spd: spermidine; TOR: target of rapamycin; VIGS: virus-induced gene silencing.

摘要

自噬是一种进化上保守的细胞降解过程，在植物发育和应激反应中起关键作用。尽管有大量关于自噬在应对环境压力中的重要作用的信息，但对自噬的调节知之甚少。在这项研究中，我们证明了亚精胺（Spd）是一种多胺，在盐胁迫下通过激活ATG（自噬相关）基因的表达和黄瓜自噬体的形成参与了耐盐性的调节。此外，NADPH 氧化酶衍生的质外体 H ₂ O ₂-介导的 Spd 诱导的耐盐性和自噬。外源Spd显着提高了对盐胁迫的耐受性，并抑制了不溶性蛋白质的积累和泛素化。叶面喷施 Spd 促进了ATG基因的转录水平和自噬体的形成。此外，Spd诱导RBOH（呼吸爆发氧化酶同源物）的表达，以及H ₂ O ₂在叶和根中的积累。然而，无论是用二甲基硫脲（DMTU，一种 H ₂ O ₂清除剂）还是二亚苯基碘鎓氯化物（DPI，一种 NADPH 氧化酶抑制剂）预处理，都能减少 Spd 诱导的质外体 H ₂ O _2积累. 重要的是，当植物用 DMTU 或 DPI 预处理时，Spd 诱导的耐盐性和自噬受到损害。此外，ATG4和ATG7的沉默降低了 Spd 诱导的耐盐性和自噬体的形成。总之，这些结果表明RBOH依赖性H ₂ O ₂介导了Spd诱导的黄瓜自噬和耐盐性。

缩写： Asat：CO ₂同化的光饱和速率；ATG：自噬相关；DCF-DA：2, 7-二氯荧光素二乙酸酯；DMTU：二甲基硫脲；DPI：二苯碘鎓氯化物；DW：干重；EL：电解液泄漏；FW：鲜重；Fv/Fm：光系统 II 的最大量子产率；GFP：绿色荧光蛋白；MDC：单丹磺酰尸胺；PDS：八氢番茄红素去饱和酶；PE：磷脂酰乙醇胺；PLD：磷脂酶D；RBOH：呼吸爆发氧化酶同系物；ROS：活性氧；SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；SIN1：盐诱导的 NAC1；Spd：亚精胺；TOR：雷帕霉素靶点；VIGS：病毒诱导的基因沉默。