Molecular cloning and functional characterization of four monoterpene synthase genes from Citrus unshiu Marc

We isolated four novel cDNA clones for monoterpene synthase genes, CitMTSE1, CitMTS3, CitMTS61, and CitMTS62, from Satsuma mandarin (Citrus unshiu Marc.). Functional tests of in vitro translation demonstrated that CitMTSE1, CitMTS3, CitMTS61, and CitMTS62 coded d-limonene synthase, gamma-terpinene synthase, gamma-terpinene synthase, and beta-pinene synthase, respectively. Their predicted proteins possessed typical characteristics for monoterpene synthases, such as transit peptides in the N-terminus, RR sequences, and DDXXD motifs. In citrus monoterpene synthase genes, the enzymatic product-specific interval length variations among conservative domains were conserved among interspecies, and they were conspicuous between limonene synthase genes and other cyclic monoterpene synthase genes, although the total amino acid length of each clone was similar. A phylogenetic tree indicated that differentiation in citrus monoterpene synthases might occur before the divergence of Citrus species. Besides, the mRNA expressions of most clones were active in the peel at an early stage of fruit development. CitMTSE1 and CitMTS3 were also powerfully expressed in the flower at anthesis, suggesting a different regulation mechanism.