Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus
Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus
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DOI：
10.1111/j.1365-313x.2005.02557.x
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发表时间：
2005-11-01
期刊：
影响因子：
7.2
通讯作者：
De Luca, V
中科院分区：
文献类型：
--
作者：
Murata, J;De Luca, V
马达加斯加长春花（Catharanthus roseus）产生众所周知的和非常复杂的抗癌二聚生物碱长春碱和长春新碱，它们是由文多灵和长春质碱单体偶联得到的。最近的原位RNA杂交和免疫定位的数据表明，在叶片内的组合细胞工厂参与文多灵的生物合成。在这项研究中，负责vindoline生物合成的细胞类型进行了鉴定，通过激光捕获显微切割/RNA分离/RT-PCR显示，香叶醇羟化酶，裂叶甘露醇合酶，色氨酸脱羧酶，strictosidine合酶，strictosidine β-葡萄糖苷酶和tabersonine 16-羟化酶可以检测到优先在表皮细胞。一个新的和互补的应用carborescent abrasion（CA）技术的开发，以获得表皮丰富的叶提取物，可用于测量生物碱代谢物水平，酶活性和基因表达。CA技术表明，他勃松宁和16-甲氧基他勃松宁，以及16-羟基他勃松宁-16-O-甲基转移酶，主要存在于长春花叶表皮，与文多灵，长春质碱和文多灵生物合成中的后期酶促步骤相反。结果表明，叶表皮细胞具有生物合成能力，能够产生色胺和裂叶甘露醇前体，这些前体通过许多酶促转化而转化为16-甲氧基他勃松宁。这种生物碱或其2，3二氢衍生物然后被运输到长春花叶内的细胞（叶肉/异胚细胞/乳管）以完成最后三或四种酶促转化以制造文多灵。
The Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex anticancer dimeric alkaloids vinblastine and vincristine, which are derived by the coupling of vindoline and catharanthine monomers. Recent data from in situ RNA hybridization and immunolocalization suggest that combinatorial cell factories within the leaf are involved in vindoline biosynthesis. In this study, the cell types responsible for vindoline biosynthesis were identified by laser-capture microdissection/RNA isolation/RT-PCR to show that geraniol hydroxylase, secologanin synthase, tryptophan decarboxylase, strictosidine synthase, strictosidine ss-glucosidase and tabersonine 16-hydroxylase can be detected preferentially in epidermal cells. A new and complementary application of the carborundum abrasion (CA) technique was developed to obtain epidermis-enriched leaf extracts that can be used to measure alkaloid metabolite levels, enzyme activities and gene expression. The CA technique showed that tabersonine and 16-methoxytabersonine, together with 16-hydroxytabersonine-16-O-methyltransferase, are found predominantly in Catharanthus leaf epidermis, in contrast to vindoline, catharanthine and later enzymatic steps in vindoline biosynthesis. The results show that leaf epidermal cells are biosynthetically competent to produce tryptamine and secologanin precursors that are converted via many enzymatic transformations to make 16-methoxytabersonine. This alkaloid or its 2,3 dihydro-derivative is then transported to cells (mesophyll/idioblast/laticifer) within Catharanthus leaves to complete the last three or four enzymatic transformations to make vindoline.