Functional analysis of Pogostemon cablin farnesyl pyrophosphate synthase gene and its binding transcription factor PcWRKY44 in regulating biosynthesis of patchouli alcohol

Abstract

Farnesyl pyrophosphate synthase (FPPS) plays an important role in the synthesis of plant secondary metabolites, but its function and molecular regulation mechanism remain unclear in <i>Pogostemon cablin</i>. In this study, the full-length cDNA of the FPP synthase gene from <i>P. cablin</i> (<i>PcFPPS</i>) was cloned and characterized. The expressions of <i>PcFPPS</i> are different among different tissues (highly in <i>P. cablin</i> flowers). Subcellular localization analysis in protoplasts indicated that PcFPPS was located in the cytoplasm. PcFPPS functionally complemented the lethal <i>FPPS</i> deletion mutation in yeast CC25. Transient overexpression of <i>PcFPPS</i> in <i>P. cablin</i> leaves accelerated terpene biosynthesis, with an ~47% increase in patchouli alcohol. Heterologous overexpression of <i>PcFPPS</i> in tobacco plants was achieved, and it was found that the FPP enzyme activity was significantly up-regulated in transgenic tobacco by ELISA analysis. In addition, more terpenoid metabolites, including stigmasterol, phytol, and neophytadiene were detected compared with control by GC-MS analysis. Furthermore, with dual-LUC assay and yeast one-hybrid screening, we found 220 bp promoter of <i>PcFPPS</i> can be bound by the nuclear-localized transcription factor PcWRKY44. Overexpression of <i>PcWRKY44</i> in <i>P. cablin</i> upregulated the expression levels of <i>PcFPPS</i> and patchoulol synthase gene (<i>PcPTS</i>), and then promote the biosynthesis of patchouli alcohol. Taken together, these results strongly suggest the <i>PcFPPS</i> and its binding transcription factor PcWRKY44 play an essential role in regulating the biosynthesis of patchouli alcohol.