MAIN CONCLUSION Exogenous SA treatment at appropriate concentrations promotes adventitious root formation in cucumber hypocotyls, via competitive inhibiting the IAA-Asp synthetase activity of CsGH3.5, and increasing the local free IAA level. Adventitious root formation is critical for the cutting propagation of horticultural plants. Indole-3-acetic acid (IAA) has been shown to play a central role in regulating this process, while for salicylic acid (SA), its exact effects and regulatory mechanism have not been elucidated. In this study, we showed that exogenous SA treatment at the concentrations of both 50 and 100 µM promoted adventitious root formation at the base of the hypocotyl of cucumber seedlings. At these concentrations, SA could induce the expression of CYCLIN and Cyclin-dependent Kinase (CDK) genes during adventitious rooting. IAA was shown to be involved in SA-induced adventitious root formation in cucumber hypocotyls. Exposure to exogenous SA led to a slight increase in the free IAA content, and pre-treatment with the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) almost completely abolished the inducible effects of SA on adventitious root number. SA-induced IAA accumulation was also associated with the enhanced expression of Gretchen Hagen3.5 (CsGH3.5). The in vitro enzymatic assay indicated that CsGH3.5 has both IAA- and SA-amido synthetase activity and prefers aspartate (Asp) as the amino acid conjugate. The Asp concentration dictated the functional activity of CsGH3.5 on IAA. Both affinity and catalytic efficiency (Kcat/Km) increased when the Asp concentration increased from 0.3 to 1 mM. In contrast, CsGH3.5 showed equal catalytic efficiency for SA at low and high Asp concentrations. Furthermore, SA functioned as a competitive inhibitor of the IAA-Asp synthetase activity of CsGH3.5. During adventitious formation, SA application indeed repressed the IAA-Asp levels in the rooting zone. These data show that SA plays an inducible role in adventitious root formation in cucumber through competitive inhibition of the auxin conjugation enzyme CsGH3.5. SA reduces the IAA conjugate levels, thereby increasing the local free IAA level and ultimately enhancing adventitious root formation.

中文翻译：

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水杨酸通过竞争性抑制黄瓜下胚轴中的生长素结合酶 CsGH3.5 调节不定根的形成

主要结论 适当浓度的外源SA处理通过竞争性抑制CsGH3.5的IAA-Asp合成酶活性和增加局部游离IAA水平促进黄瓜下胚轴中不定根的形成。不定根的形成对于园艺植物的扦插繁殖至关重要。吲哚-3-乙酸（IAA）已被证明在调节这一过程中发挥核心作用，而对于水杨酸（SA），其确切作用和调节机制尚未阐明。在这项研究中，我们表明，50 和 100 µM 浓度的外源 SA 处理促进了黄瓜幼苗下胚轴基部不定根的形成。在这些浓度下，SA 可以在不定根期间诱导 CYCLIN 和细胞周期蛋白依赖性激酶 (CDK) 基因的表达。IAA 被证明参与 SA 诱导的黄瓜下胚轴中不定根的形成。暴露于外源 SA 导致游离 IAA 含量略有增加，并且用生长素转运抑制剂 1-萘基邻苯二甲酸 (NPA) 预处理几乎完全消除了 SA 对不定根数的诱导作用。SA 诱导的 IAA 积累也与 Gretchen Hagen3.5 (CsGH3.5) 的表达增强有关。体外酶促测定表明，CsGH3.5 具有 IAA-和 SA-酰胺合成酶活性，并且首选天冬氨酸 (Asp) 作为氨基酸缀合物。Asp 浓度决定了 CsGH3.5 对 IAA 的功能活性。当 Asp 浓度从 0.3 增加到 1 mM 时，亲和力和催化效率 (Kcat/Km) 都会增加。相比之下，CsGH3。图 5 显示了在低和高 Asp 浓度下对 SA 的相同催化效率。此外，SA 作为 CsGH3.5 的 IAA-Asp 合成酶活性的竞争性抑制剂起作用。在不定形成过程中，SA 应用确实抑制了生根区的 IAA-Asp 水平。这些数据表明，SA 通过竞争性抑制生长素结合酶 CsGH3.5 在黄瓜的不定根形成中发挥诱导作用。SA 降低了 IAA 结合物水平，从而增加了局部游离 IAA 水平并最终增强了不定根的形成。这些数据表明，SA 通过竞争性抑制生长素结合酶 CsGH3.5 在黄瓜的不定根形成中发挥诱导作用。SA 降低了 IAA 结合物水平，从而增加了局部游离 IAA 水平并最终增强了不定根的形成。这些数据表明，SA 通过竞争性抑制生长素结合酶 CsGH3.5 在黄瓜的不定根形成中发挥诱导作用。SA 降低了 IAA 结合物水平，从而增加了局部游离 IAA 水平并最终增强了不定根的形成。