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首页> 外文期刊>The FEBS journal >The subcellular organization of strictosidine biosynthesis in Catharanthus roseus epidermis highlights several trans-tonoplast translocations of intermediate metabolites
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The subcellular organization of strictosidine biosynthesis in Catharanthus roseus epidermis highlights several trans-tonoplast translocations of intermediate metabolites

机译：长春花表皮中严格糖苷生物合成的亚细胞组织突出了中间代谢物的几个反式-成膜细胞易位

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摘要

Catharanthus roseus synthesizes a wide range of valuable monoterpene indole alkaloids, some of which have recently been recognized as functioning in plant defence mechanisms. More specifically, in aerial organ epidermal cells, vacuole-accumulated strictosidine displays a dual fate, being either the precursor of all monoterpene indole alkaloids after export from the vacuole, or the substrate for a defence mechanism based on the massive protein cross-linking, which occurs subsequent to organelle membrane disruption during biotic attacks. Such a mechanism relies on a physical separation between the vacuolar strictosidine-synthesizing enzyme and the nucleus-targeted enzyme catalyzing its activation through deglucosylation. In the present study, we carried out the spatial characterization of this mechanism by a cellular and subcellular study of three enzymes catalyzing the synthesis of the two strictosidine precursors (i.e. tryptamine and secologanin). Using RNA in situ hybridization, we demonstrated that loganic acid O-methyltransferase transcript, catalysing the penultimate step of secologanin synthesis, is specifically localized in the epidermis. A combination of green fluorescent protein imaging, bimolecular fluorescence complementation assays and yeast two-hybrid analysis enabled us to establish that both loganic acid O-methyltransferase and the tryptamine-producing enzyme, tryptophan decarboxylase, form homodimers in the cytosol, thereby preventing their passive diffusion to the nucleus. We also showed that the cytochrome P450 secologanin synthase is anchored to the endoplasmic reticulum via a N-teminal helix, thus allowing the production of secologanin on the cytosolic side of the endoplasmic reticulum membrane. Consequently, secologanin and tryptamine must be transported to the vacuole to achieve strictosidine biosynthesis, demonstrating the importance of trans-tonoplast translocation events during these metabolic processes.
机译：长春花（Catharanthus roseus）合成了多种有价值的单萜吲哚生物碱，其中一些最近被认为在植物防御机制中起作用。更具体地讲，在气生器官表皮细胞中，液泡积累的丁二糖苷具有双重命运，既是从液泡中输出的所有单萜吲哚生物碱的前体，又是基于大量蛋白质交联的防御机制的底物，发生在生物攻击期间细胞器膜破裂之后。这样的机制依赖于液泡中的缩丁嘧啶合成酶和通过脱糖基化催化其活化的核靶向酶之间的物理分离。在本研究中，我们通过对三种酶的细胞和亚细胞研究进行了空间表征，该酶催化了两种严格的尿苷前体（即色胺和secologanin）的合成。使用RNA原位杂交，我们证明了对数烷酸O-甲基转移酶转录本，催化仲硫蛋白合成的倒数第二个步骤，专门位于表皮中。绿色荧光蛋白成像，双分子荧光互补测定法和酵母菌双杂交分析相结合，使我们得以确定，对数酸O-甲基转移酶和产生色胺的酶色氨酸脱羧酶均在胞质溶胶中形成同型二聚体，从而防止其被动扩散到细胞核。我们还表明，细胞色素P450 secologanin合酶通过N-末端螺旋锚定到内质网，从而允许在内质网膜的胞质侧生成secologanin。因此，secologanin和色胺必须转运到液泡中以实现严格的核苷生物合成，这证明了在这些代谢过程中反式质膜转运事件的重要性。

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