Data_Sheet_2_Terpene Synthase-b and Terpene Synthase-e/f Genes Produce Monoterpenes for Phalaenopsis bellina Floral Scent.pdf

Orchids are the most species-rich plants and highly interactive with pollinators via visual or olfactory cues. Biosynthesis and emission of volatile organic compounds (VOCs) to the atmosphere facilitate the olfactory cues and ensure successful pollination. Phalaenopsis bellina is a scented orchid with monoterpenes as major VOCs, comprising linalool, geraniol, and their derivatives. Comparative transcriptomics analysis identified four terpene synthase-b (TPS-b) genes and two TPS-e/f genes with differential gene expression between scented and scentless Phalaenopsis species. Here, we confirmed their differential expression between scented and scentless Phalaenopsis orchids and excluded one TPS-b candidate. We analyzed the temporal and spatial expression and functionally characterized these TPSs. Both TPS-b and TPS-e/f genes showed an increased expression on blooming day or 3 days post-anthesis (D + 3) before the optimal emission of floral scent on D + 5, with especially high expression of PbTPS5 and PbTPS10. The TPS-b genes are expressed exclusively in reproductive organs, whereas the TPS-e/f genes are expressed in both reproductive and vegetative organs. In planta functional characterization of both PbTPS5 and PbTPS10 in tobacco and scentless Phalaenopsis plants did not produce terpenoids. Further ectopic expression in scented Phalaenopsis cultivar P. I-Hsin Venus showed that linalool was the main product, with PbTPS10 displaying 3-fold higher activity than PbTPS5. On in vitro enzyme assay with purified recombinant TPS-b proteins ectopically expressed in Escherichia coli, geraniol was the product catalyzed by PbTPS5 and PbTPS9. PbTPS3 was a linalool/(β)-cis-ocimene synthase and PbTPS4 a linalool synthase. In conclusion, both TPS-b and TPS-e/f enzymes orchestrated floral monoterpene biosynthesis in P. bellina.

兰科植物是物种多样性最为丰富的植物类群，可通过视觉或嗅觉信号与传粉者建立高度特异性的互作关系。挥发性有机化合物（volatile organic compounds, VOCs）的生物合成与向大气中的释放，是介导嗅觉信号、保障成功传粉的关键过程。贝丽娜蝴蝶兰（Phalaenopsis bellina）是一种芳香型兰科植物，其主要挥发性有机成分为单萜类物质，涵盖芳樟醇、香叶醇及其衍生物。通过比较转录组学分析，研究人员在芳香与无香蝴蝶兰物种间鉴定出4个萜合酶-b（terpene synthase-b, TPS-b）基因与2个TPS-e/f基因存在差异表达。本研究验证了上述基因在芳香与无香蝴蝶兰间的差异表达特征，并排除了1个TPS-b候选基因。我们对这些萜合酶基因的时空表达模式进行了分析，并完成了功能鉴定。TPS-b与TPS-e/f两类基因均在开花当日或开花后3天（D+3）出现表达上调，这一时间节点早于花香物质在开花后5天（D+5）达到最优释放量的时刻，其中PbTPS5与PbTPS10的表达水平尤为显著升高。TPS-b基因仅在生殖器官中特异性表达，而TPS-e/f基因则可在生殖器官与营养器官中均有表达。在烟草与无香蝴蝶兰植株中开展的体内（in planta）功能验证实验中，PbTPS5与PbTPS10均未催化生成萜类物质。进一步在芳香型蝴蝶兰品种‘P. I-Hsin Venus’中开展异位表达实验，结果显示芳樟醇为主要催化产物，且PbTPS10的酶活性是PbTPS5的3倍。以在大肠杆菌（Escherichia coli）中异位表达纯化得到的重组TPS-b蛋白开展体外酶活实验，结果显示：PbTPS5与PbTPS9可催化生成香叶醇；PbTPS3为芳樟醇/(β)-顺式罗勒烯合酶，而PbTPS4为芳樟醇合酶。综上，TPS-b与TPS-e/f两类酶协同调控了贝丽娜蝴蝶兰的花部单萜生物合成过程。