Rho of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpenoid indole alkaloids (MIA) in Madagascar periwinkle (Catharanthus roseus), indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular, and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C-terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, their transcripts showed higher expression in most parts than other CrROPs. Protein-protein interaction studies revealed that CrROP3 and CrROP5, but not ΔCrROP3, ΔCrROP5, and CrROP2 lacking the CSIL motif, interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to the plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of ΔCrROP3 and ΔCrROP5 mutants devoid of sequence coding for the CSIL motif failed to enhance MIA biosynthesis. These results implicate that CrROP3 and CrROP5 have a positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus.

中文翻译：

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具有香叶基香叶基化基序的 ROP GTP 酶调节长春花中的生物碱生物合成

植物 Rho (ROP) GTP 酶作为分子开关，控制生长、发育和防御所必需的信号传导过程。然而，人们对它们在专门代谢中的作用知之甚少。此前，我们证明抑制蛋白质香叶基香叶基转移酶（PGGT-I）会对马达加斯加长春花（长春花）中单萜吲哚生物碱（MIA）的生物合成产生负面影响，表明异戊二烯化蛋白参与信号传导。在这里，我们通过生物化学、分子和植物方法证明，特定的香叶基香叶基化 ROPs 调节玫瑰花 MIA 生物合成。在六种玫瑰花 ROP GTP 酶 (CrROP) 中，只有具有 C 末端 CSIL 基序的 CrROP3 和 CrROP5 被 PGGT-I 特异性异戊二烯化。此外，它们的转录本在大部分部位表现出比其他 CrROP 更高的表达。蛋白质-蛋白质相互作用研究表明，CrROP3 和 CrROP5，但缺乏 CSIL 基序的 ΔCrROP3、ΔCrROP5 和 CrROP2 不会与 CrPGGT-I 相互作用。此外，CrROP3和CrROP5表现出核定位，而CrROP2定位于质膜。在植物中的功能研究表明，CrROP3 和 CrROP5 的沉默会对 MIA 生物合成产生负面影响，而它们的过度表达则会上调 MIA 的形成。相反，CrROP2 的沉默和过度表达对 MIA 生物合成没有影响。此外，缺乏CSIL基序编码序列的ΔCrROP3和ΔCrROP5突变体的过度表达未能增强MIA生物合成。这些结果表明 CrROP3 和 CrROP5 对 MIA 生物合成具有积极的调节作用，从而揭示了香叶基香叶基化的 ROP GTPases 如何介导玫瑰花中专门代谢的调节。