Tracing the biosynthetic origin of limonoids and their functional groups through stable isotope labeling and inhibition in neem tree (Azadirachta indica) cell suspension.
Tracing the biosynthetic origin of limonoids and their functional groups through stable isotope labeling and inhibition in neem tree (Azadirachta indica) cell suspension.
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DOI：
10.1186/s12870-018-1447-6
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发表时间：
2018-10-11
影响因子：
5.3
通讯作者：
Thulasiram HV
中科院分区：
文献类型：
--
作者：
Aarthy T;Mulani FA;Pandreka A;Kumar A;Nandikol SS;Haldar S;Thulasiram HV
印楝树是被称为柠檬苦素的三萜类化合物的聚宝盆，由于它们具有不同的生物活性，人类对它们有着深刻的兴趣。然而，对于这棵神奇的树来说，植物用于生产柠檬苦素的生物合成途径仍然没有被探索。在这篇文章中，我们报道了利用印木细胞悬浮液中葡萄糖的13C同位素来示踪柠檬苦素的生物合成途径。建立了印尼树幼苗生长发育特异的柠檬苦素谱带和细胞系随时间变化的柠檬苦素生物合成特性。为了进一步了解甲丙酮酸(MVA)和甲基赤藓糖醇磷酸(MEP)途径在柠檬苦素类化合物生物合成中的作用，我们利用超高效液相色谱(UPLC)-串联质谱仪建立了柠檬苦素类化合物及其同位素同系物的结构-片段关系。对标记的柠檬苦素萃取物的分析导致鉴定出与印楝素和其他柠檬苦素生物合成有关的特征异戊二烯单位，这些单位被发现是通过甲氧戊酸途径形成的。MVA途径的特异性抑制剂梅维诺林处理细胞悬液后，柠檬苦素水平显著降低，而胞浆中4-磷酸甲基赤藓糖醇(MEP)途径的抑制不影响柠檬苦素的生物合成。MVA途径限速酶3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)在不同组织和体外培养细胞中的表达水平均高于MEP途径的脱氧果糖-磷酸合成酶(DXS)。因此，本研究将对柠檬苦素的生物合成途径有一个全面的了解，其中MVA途径和MEP途径具有不同的贡献。通过柠檬酸类化合物的定量测定和~(13)C柠檬苦素类化合物的同位素分析，揭示了印楝树和细胞系柠檬苦素的生物合成。印迹悬浮液的未分化细胞系产生一种类似于亲本组织、核的C-Seco柠檬苦素。印楝素是一种C-Seco柠檬苦素类化合物，在植物幼嫩的叶片中产生，而在树木的硬成熟叶片中，环状完整的柠檬苦素尼莫西诺含量较高。此外，通过稳定的同位素标记证明，甲氧戊酸途径只贡献柠檬苦素类化合物中的异戊二烯单位，并且在甲氧戊酸途径功能障碍的情况下，没有观察到MEP途径的互补。本文的在线版本(10.1186/s12870-0181447-6)包含补充材料，可供授权用户使用。
Neem tree serves as a cornucopia for triterpenoids called limonoids that are of profound interest to humans due to their diverse biological activities. However, the biosynthetic pathway that plant employs for the production of limonoids remains unexplored for this wonder tree. Herein, we report the tracing of limonoid biosynthetic pathway through feeding experiments using 13C isotopologues of glucose in neem cell suspension. Growth and development specific limonoid spectrum of neem seedling and time dependent limonoid biosynthetic characteristics of cell lines were established. Further to understand the role of mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways in limonoid biosynthesis, Ultra Performance Liquid Chromatography (UPLC)- tandem mass spectrometry based structure-fragment relationship developed for limonoids and their isotopologues have been utilized. Analyses of labeled limonoid extract lead to the identification of signature isoprenoid units involved in azadirachtin and other limonoid biosynthesis, which are found to be formed through mevalonate pathway. This was further confirmed by treatment of cell suspension with mevinolin, a specific inhibitor for MVA pathway, which resulted in drastic decrease in limonoid levels whereas their biosynthesis was unaffected with fosmidomycin mediated plastidial methylerythritol 4-phosphate (MEP) pathway inhibition. This was also conspicuous, as the expression level of genes encoding for the rate-limiting enzyme of MVA pathway, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) was comparatively higher to that of deoxyxylulose-phosphate synthase (DXS) of MEP pathway in different tissues and also in the in vitro grown cells. Thus, this study will give a comprehensive understanding of limonoid biosynthetic pathway with differential contribution of MVA and MEP pathways. Limonoid biosynthesis of neem tree and cell lines have been unraveled through comparative quantification of limonoids with that of neem tree and through 13C limonoid isotopologues analysis. The undifferentiated cell lines of neem suspension produced a spectrum of C-seco limonoids, similar to parental tissue, kernel. Azadirachtin, a C-seco limonoid is produced in young tender leaves of plant whereas in the hard mature leaves of tree, ring intact limonoid nimocinol accumulates in high level. Furthermore, mevalonate pathway exclusively contributes for isoprene units of limonoids as evidenced through stable isotope labeling and no complementation of MEP pathway was observed with mevalonate pathway dysfunction, using chemical inhibitors. The online version of this article (10.1186/s12870-018-1447-6) contains supplementary material, which is available to authorized users.
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