Abstract
Phalaenopsis bellina 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 P. bellina. Here, we identified a dual repeat cis-element in the GDPS promoter that is critical for monoterpene biosynthesis in Phalaenopsis orchids. A strong correlation between the dual repeat and the monoterpene production was revealed by examination of the GDPS promoter fragments over 12 Phalaenopsis species. Serial-deletion of the 2-kb GDPS promoter fragments demonstrated that the integrity of the dual repeat was crucial for its promoter activities. By screening the Arabidopsis 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 PbbZIP4 in the transcriptome of P. bellina, showing 83% identity in the DNA binding region with that of AtbZIP18, and the expression level of PbbZIP4 was higher in the scented orchids. In addition, PbbZIP4 transactivated the GDPS promoter fragment containing the dual repeat in dual luciferase assay. Furthermore, transient ectopic expression of PbbZIP4 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 cis-element significant for GDPS gene expression, and thus subsequent monoterpene biosynthesis in the scented Phalaenopsis orchids.
Highlights
Phalaenopsis species are widespread in the tropical Asia regions and includes approximately 56 native species (Christenson, 2001)
We reported that a dual repeat in the upstream promoter fragments of geranyl diphosphate synthase (GDPS) is essential for its transcriptional activation in Phalaenopsis orchids
Further sequence analysis of the PbGDPS promoter showed that a second 75-bp repeat is present downstream from the original 75-bp repeat and formed a dual repeat consisted of the two 75-bp units
Summary
Phalaenopsis species are widespread in the tropical Asia regions and includes approximately 56 native species (Christenson, 2001). The bottlenecks include long generation time (Hsiao et al, 2011a), crossincompatibility due to the differences in genome size and chromosome size among species (Hsiao et al, 2011a,b; Yeh et al, 2014), and negative correlation between floral scent and other favorable traits (Hsiao et al, 2011b), which is occurred in other modern floriculture varieties (Vainstein et al, 2001; Dudareva and Negre, 2005). Alternative approaches to facilitate scented orchid breeding are needed
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