AUTHOR=Chuang Yu-Chen , Hung Yi-Chu , Hsu Chi-Yu , Yeh Chuan-Ming , Mitsuda Nobutaka , Ohme-Takagi Masaru , Tsai Wen-Chieh , Chen Wen-Huei , Chen Hong-Hwa 

TITLE=A Dual Repeat Cis-Element Determines Expression of GERANYL DIPHOSPHATE SYNTHASE for Monoterpene Production in Phalaenopsis Orchids

JOURNAL=Frontiers in Plant Science

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2018.00765

DOI=10.3389/fpls.2018.00765

ISSN=1664-462X

ABSTRACT=<p><italic>Phalaenopsis bellina</italic> is a scented orchid emitting large amount of monoterpenes. GERANYL DIPHOSPHATE SYNTHASE (PbGDPS) is the key enzyme for monoterpene biosynthesis, and shows concomitant expression with the emission of monoterpenes during flower development in <italic>P. bellina</italic>. Here, we identified a dual repeat <italic>cis</italic>-element in the <italic>GDPS</italic> promoter that is critical for monoterpene biosynthesis in <italic>Phalaenopsis</italic> orchids. A strong correlation between the dual repeat and the monoterpene production was revealed by examination of the <italic>GDPS</italic> promoter fragments over 12 <italic>Phalaenopsis</italic> species. Serial-deletion of the 2-kb <italic>GDPS</italic> promoter fragments demonstrated that the integrity of the dual repeat was crucial for its promoter activities. By screening the <italic>Arabidopsis</italic> transcription factors (TFs) cDNA library using yeast one-hybrid assay, AtbZIP18, a member of group I of bZIP TFs, was identified to be able to bind the dual repeat. We then identified <italic>PbbZIP4</italic> in the transcriptome of <italic>P. bellina</italic>, showing 83% identity in the DNA binding region with that of AtbZIP18, and the expression level of <italic>PbbZIP4</italic> was higher in the scented orchids. In addition, PbbZIP4 transactivated the <italic>GDPS</italic> promoter fragment containing the dual repeat in dual luciferase assay. Furthermore, transient ectopic expression of <italic>PbbZIP4</italic> induced a 10-fold production of monoterpenoids in the scentless orchid. In conclusion, these results indicate that the dual repeat is a real TF-bound <italic>cis</italic>-element significant for <italic>GDPS</italic> gene expression, and thus subsequent monoterpene biosynthesis in the scented <italic>Phalaenopsis</italic> orchids.</p>