AUTHOR=Huang Hsin , Kuo Yi-Wei , Chuang Yu-Chen , Yang Ya-Ping , Huang Li-Min , Jeng Mei-Fen , Chen Wen-Huei , Chen Hong-Hwa 

TITLE=Terpene Synthase-b and Terpene Synthase-e/f Genes Produce Monoterpenes for Phalaenopsis bellina Floral Scent

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.700958

DOI=10.3389/fpls.2021.700958

ISSN=1664-462X

ABSTRACT=<p>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. <italic>Phalaenopsis bellina</italic> is a scented orchid with monoterpenes as major VOCs, comprising linalool, geraniol, and their derivatives. Comparative transcriptomics analysis identified four <italic>terpene synthase-b</italic> (<italic>TPS-b</italic>) genes and two <italic>TPS-e/f</italic> genes with differential gene expression between scented and scentless <italic>Phalaenopsis</italic> species. Here, we confirmed their differential expression between scented and scentless <italic>Phalaenopsis</italic> orchids and excluded one <italic>TPS-b</italic> candidate. We analyzed the temporal and spatial expression and functionally characterized these <italic>TPSs</italic>. Both <italic>TPS-b</italic> and <italic>TPS-e/f</italic> 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 <italic>PbTPS5</italic> and <italic>PbTPS10</italic>. The <italic>TPS-b</italic> genes are expressed exclusively in reproductive organs, whereas the <italic>TPS-e/f</italic> genes are expressed in both reproductive and vegetative organs. <italic>In planta</italic> functional characterization of both <italic>PbTPS5</italic> and <italic>PbTPS10</italic> in tobacco and scentless <italic>Phalaenopsis</italic> plants did not produce terpenoids. Further ectopic expression in scented <italic>Phalaenopsis</italic> cultivar <italic>P.</italic> I-Hsin Venus showed that linalool was the main product, with <italic>PbTPS10</italic> displaying 3-fold higher activity than <italic>PbTPS5.</italic> On <italic>in vitro</italic> enzyme assay with purified recombinant TPS-b proteins ectopically expressed in <italic>Escherichia coli</italic>, geraniol was the product catalyzed by <italic>PbTPS5</italic> and <italic>PbTPS9</italic>. <italic>PbTPS3</italic> was a linalool/(β)-cis-ocimene synthase and <italic>PbTPS4</italic> a linalool synthase. In conclusion, both <italic>TPS-b</italic> and <italic>TPS-e/f</italic> enzymes orchestrated floral monoterpene biosynthesis in <italic>P. bellina</italic>.</p>