Misra Rajesh Chandra, Garg Anchal, Roy Sudeep, Chanotiya Chandan Singh, Vasudev Prema G, Ghosh Sumit
Biotechnology Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India.
Chemical Sciences Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India.
Plant Sci. 2015 Nov;240:50-64. doi: 10.1016/j.plantsci.2015.08.016. Epub 2015 Aug 28.
Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance.
对映-贝壳杉烷型二萜（ent-LRD）是药用植物穿心莲（Andrographis paniculata）的一类特殊（即次生）代谢产物，长期以来因其多种药理活性而为人所知。然而，我们对ent-LRD生物合成途径的了解在很大程度上仍不完整。由于ent-LRDs在叶片中积累，我们使用叶片和根组织进行了比较转录分析，鉴定出389个差异表达的转录本，其中包括223个在叶片组织中优先表达的转录本。对这些转录本的分析揭示了各种特殊代谢途径，包括ent-LRD生物合成途径的转录本。我们鉴定出了两个II类二萜合酶（ApCPS1和ApCPS2），以及一个（ApCPS1'）和两个（ApCPS2'和ApCPS2″）转录变体，它们分别是前体mRNA可变剪接和可变转录终止的结果。ApCPS1和ApCPS2分别编码832和817个氨基酸的蛋白质，在系统发育上与双子叶植物对映-贝壳杉烯二磷酸合酶（ent-CPSs）相关。ent-LRD代谢产物积累和基因表达的时空模式表明，ApCPS1可能在一般（即初级）代谢中发挥作用，可能是通过为植物激素赤霉素（GA）的生物合成提供前体。然而，ApCPS2可能参与ent-LRD特殊代谢产物的组织特异性积累。细菌表达的重组ApCPS2催化了二萜的通用前体（E,E,E）-香叶基香叶基二磷酸（GGPP）转化为ent-LRDs的前体对映-贝壳杉烯二磷酸（ent-CPP）。综上所述，这些结果推进了我们对具有药用重要性的特殊ent-LRDs组织特异性积累的理解。
