Biosynthetic regulation of floral scent in phalaenopsis

Floral scent is a key modulating factor in plant—insect interactions and thus plays a central role in successful pollination. Orchidaceae, containing more than 25,000 species, is one of the largest monocotyledon families. Pollinators have played an important role in driving floral diversification in orchids, which has obviously been advantageous for the evolution of this highly successful family. We investigate how flower fragrance is generated in certain Phalaenopsis orchids by identifying floral expressed-sequence-tags (ESTs) and deducing the floral scent biosynthesis pathway of Phalaneopsis bellina using bioinformatics analysis. The major chemical components in the P. bellina flower are found to be monoterpene of geraniol, linalool, and their derivatives, and maximally emitted from day 5 post-anthesis to day 7 postanthesis. The encoded enzymes collection in the biosynthetic pathway from glycerolaldehyde-3-phosphate (G3P) to geraniol and linalool are recognized through data mining of the P. bellina floral EST database (dbEST). Transcripts preferentially expressed in P. bellina include those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase (GDPS). EST filtering results showed that transcripts encoding signal transduction, Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, are detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpoint 66 percent of the biosynthetic steps from G3P to geraniol, linalool and their derivatives. We then focused on the key enzymes in this pathway, the geranyl diphosphate synthase (GDPS) which catalyzes the formation of GDP for further synthesis of geraniol, linalool and their derivatives. The novel PbGDPS enzyme lacks the conserved aspartate-rich motif DD(X)2-4D, possesses a dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and functions as a homodimer. Homology modeling and site-direct mutagenesis of PbGDPS indicate that the Glu-rich motif may provide a binding site for Mg2+ and catalyze the formation of prenyl products. Expression pattern results show that the PbGDPS was flower specific and maximal PbGDPS expression is concomitant with maximal emission of monoterpenes on day 5 post-anthesis, thereby suggesting that PbGDPS plays a crucial role in scent production/emission in orchid flowers. In conclusion, these approaches demonstrate the opportunity to study floral scent in the non-model plants such as orchids, and moreover to accelerate molecular breeding in the orchid industry.