Kalmegh(穿心莲)时空产生医学上重要的与labdane相关的二萜(ent-LRD);穿心莲内酯(AD),14-脱氧-11,12-二脱氢穿心莲内酯(DDAD),新穿心莲内酯(NAD)。ApCPS1和ApCPS2,即产生n-copalyl焦磷酸(ent-CPP)的II类二萜合酶(diTPP),但是他们对ent-LRD生物合成的ent-CPP前体供应的贡献尚不清楚。这里,我们表征了ApCPS4,一种额外的ent-CPP形成diTPS。Further,我们通过整合转录-代谢物共谱阐明了产生ent-CPP的diTPP(ApCPS1,2,4)的植物功能,生化分析和基因功能表征。ApCPS1,2,4定位于质体,在植物中发生二萜生物合成的地方,但ApCPS1,2,4转录本表达模式和ent-LRD含量显示,ApCPS2表达与心电中的ent-LRD积累有很强的相关性。ApCPS1,2,4上游序列在拟南芥和瞬时转化的kalmegh中差异激活的β-葡糖醛酸糖苷酶(GUS)。类似于ApCPS1在kalmegh茎中的较高表达,ApCPS1上游序列激活拟南芥和kalmegh茎/下胚轴中的GUS。然而,ApCPS2,4上游序列弱激活拟南芥中的GUS表达,这与kalmegh组织中的ApCPS2,4转录本表达没有很好的相关性。然而,ApCPS2,4上游序列可以在kalmegh叶片和根/花萼中激活GUS表达,分别,提示可能参与kalmegh特异性二萜途径的ApCPS2,4转录调节因子的参与。有趣的是,ApCPS2沉默的kalmegh显示AD的急剧减少,DDAD和NAD含量以及对昆虫食草动物斜纹夜蛾的防御受损。然而,ApCPS1或ApCPS4沉默的植物中的ent-LRD含量和草食动物防御能力基本保持不变。总的来说,这些结果表明,ApCPS2在生产用于药用ent-LRD生物合成和防御昆虫草食动物的ent-CPP中具有重要作用。
Kalmegh (Andrographis paniculata) spatiotemporally produces medicinally-important ent-labdane-related diterpenoids (ent-LRDs); andrographolide (AD), 14-deoxy-11,12-didehydroandrographolide (DDAD), neoandrographolide (NAD). ApCPS1 and ApCPS2, the ent-copalyl pyrophosphate (ent-CPP)-producing class II diterpene synthases (diTPSs) were identified, but their contributions to ent-CPP precursor supply for ent-LRD biosynthesis were not well understood. Here, we characterized ApCPS4, an additional ent-CPP-forming diTPS. Further, we elucidated in planta function of the ent-CPP-producing diTPSs (ApCPS1,2,4) by integrating transcript-metabolite co-profiles, biochemical analysis and gene functional characterization. ApCPS1,2,4 localized to the plastids, where diterpenoid biosynthesis occurs in plants, but ApCPS1,2,4 transcript expression patterns and ent-LRD contents revealed a strong correlation of ApCPS2 expression and ent-LRD accumulation in kalmegh. ApCPS1,2,4 upstream sequences differentially activated β-glucuronidase (GUS) in Arabidopsis and transiently-transformed kalmegh. Similar to higher expression of ApCPS1 in kalmegh stem, ApCPS1 upstream sequence activated GUS in stem/hypocotyl of Arabidopsis and kalmegh. However, ApCPS2,4 upstream sequences weakly activated GUS expression in Arabidopsis, which was not well correlated with ApCPS2,4 transcript expression in kalmegh tissues. Whereas, ApCPS2,4 upstream sequences could activate GUS expression at a considerable level in kalmegh leaf and roots/calyx, respectively, suggesting the involvement of transcriptional regulator(s) of ApCPS2,4 that might participate in kalmegh-specific diterpenoid pathway. Interestingly, ApCPS2-silenced kalmegh showed a drastic reduction in AD, DDAD and NAD contents and compromised defense against insect herbivore Spodoptera litura. However, ent-LRD contents and herbivore defense in ApCPS1 or ApCPS4-silenced plants remained largely unaltered. Overall, these results suggested an important role of ApCPS2 in producing ent-CPP for medicinal ent-LRD biosynthesis and defense against insect herbivore.