The subcellular organization of strictosidine biosynthesis in Catharanthus roseus epidermis highlights several trans-tonoplast translocations of intermediate metabolites
The subcellular organization of strictosidine biosynthesis in Catharanthus roseus epidermis highlights several trans-tonoplast translocations of intermediate metabolites
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DOI：
10.1111/j.1742-4658.2010.07994.x
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发表时间：
2011-03-01
期刊：
影响因子：
5.4
通讯作者：
Courdavault, Vincent
中科院分区：
文献类型：
--
作者：
Guirimand, Gregory;Guihur, Anthony;Courdavault, Vincent
长春花合成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发挥作用。更具体地说，在气生器官表皮细胞，空泡积累的strictosidine显示出双重命运，无论是出口后的所有单萜吲哚生物碱的前体从空泡，或基板的防御机制的基础上大量的蛋白质交联，发生在细胞器膜破坏生物攻击。这种机制依赖于液泡中的异胡萝卜苷合成酶和通过去葡萄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，我们进行了这一机制的空间表征的细胞和亚细胞研究的三种酶催化合成的两个strictosidine前体（即色胺和secologanin）。使用RNA原位杂交，我们证明了马钱酸O-甲基转移酶转录，催化secologanin合成的倒数第二步，是专门定位于表皮。结合绿色荧光蛋白成像，双分子荧光互补分析和酵母双杂交分析，使我们能够建立两个落木酸O-甲基转移酶和色胺产生酶，色氨酸脱羧酶，形成同源二聚体在胞质溶胶中，从而防止其被动扩散到细胞核。我们还表明，细胞色素P450裂叶甘露醇合酶是锚定的内质网通过一个N-tetrahydroxide螺旋，从而允许生产裂叶甘露醇的内质网膜的胞质侧。因此，secologanin和色胺必须被运送到液泡，以实现strictosidine的生物合成，证明在这些代谢过程中的trans-tonoplast易位事件的重要性。
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.