Functional characterization of alkaline phosphatases involved alarm pheromone in the vetch aphid Megoura viciae.

The alkaline phosphatases (ALPs) are highly promiscuous enzymes and have been extensively investigated in mammals for their medical significance, but their functional promiscuity is relatively poorly understood in insects. Here, we first identified four ALP genes (designated as MvALP1-4) in the vetch aphid Megoura viciae that contained one alkaline phosphatase site, three metal-binding sites, and varied other functional sites. Phylogenetic analysis, molecular docking and the spatiotemporal expression profiling of MvALP1-4 were very different, indicating a promiscuous functionality. We also found that MvALP4 involved the biosynthesis of aphid alarm pheromones (EßF) in vitro and in vivo. Finally, transcriptome analysis in the stimulated and unstimulated aphids supported the involvement of MvALPs in the biosynthesis of aphid alarm pheromones. Our study identified a multifunctional ALP involved terpene synthase enzyme activity in the aphid, which contributes to the understanding of the functional plasticity of ALPs in insects.

Farnesyl/geranylgeranyl diphosphate synthases regulate the biosynthesis of alarm pheromone in a unique manner in the vetch aphid Megoura viciae.

Farnesyl/geranylgeranyl diphosphate synthases (FPPS/GGPPS) as the short-chain prenyltransferases catalyse the formation of the acyclic precursors (E)-FPP and (E)-GGPP for isoprenoid biosynthesis. Here, we first cloned the cDNAs encoding FPPS and GGPPS in the vetch aphid Megoura viciae (designated as MvFPPS and MvGGPPS). They had an open reading frame of 1185 and 930 bp in length, encoding 395 and 309 amino acids, with a theoretical isoelectric point of 6.52 and 6.21, respectively. Sequence alignment and phylogenetic analysis showed that MvFPPS and MvGGPPS shared the conserved aspartate-rich motifs characterized by all prenyltransferases identified to date and were clustered with their homologues in two large clades. RNA interference (RNAi) combined with gas chromatography/mass spectrometry (GC-MS) analysis showed that both MvFPPS and MvGGPPS were involved in the biosynthesis of alarm pheromone. Spatiotemporal expression profiling showed that the expression of MvFPPS and MvGGPPS was significantly higher in embryos than in other tissues. RNAi and GC-MS performed specifically in embryos corroborated the function of MvFPPS and MvGGPPS. In vitro, enzymatic activity assay and product analysis demonstrated that MvFPPS could catalysed the formation of (E)-FPP using DMAPP or (E)-GPP as the allylic cosubstrates in the presence of IPP, while MvGGPPS could only use (E)-GPP or (E)-FPP as cosubstrates. Functional interaction analysis using RNAi revealed that MvGGPPS exerts unidirectional functional compensation for MvFPPS. Moreover, it can regulate the biosynthesis of alarm pheromone by imposing a negative feedback regulation on MvFPPS. Our study helps to understand the molecular regulatory mechanism of terpenoid biosynthesis in the aphid.