(3R)-linalool synthase from Artemisia annua L.: cDNA isolation, characterization, and wound induction

Artemisia annua is an annual herb used in traditional Chinese medicine, A c DNA library was constructed from leaves of A. annua seedlings and target se quences were amplified by PCR using degenerate primers derived from a conse nsus sequence of angiosperm terpene synthases, Two clones, QH1 and QH5, wit h high sequence similarity to plant monoterpene synthases were ultimately o btained and expressed in Escherichia coli. These cDNAs encode peptides of 5 67 aa (65.7 kDa) and 583 aa (67.4 kDa), respectively, and display 88% ident ity with each other and 42% identity with Mentha spicata limonene synthase, The two recombinant enzymes yielded no detectable activity with isopenteny l diphosphate, dimethylallyl diphosphate, chrysanthemyl diphosphate, farnes yl diphosphate, (+)-copalyl diphosphate, or geranylgeranyl diphosphate, but were active with geranyl diphosphate in yielding (3R)-linalool as the sole product in the presence of divalent metal cation cofactors. QH1-linalool s ynthase displays a K-m value of 64 mu M for geranyl diphosphate, which is c onsiderably higher than other known monoterpene synthases, and a K-m value of 4.6 mM for Mg+2. Transcripts of QH1 and QH5 could be detected by RT-PCR in the leaves and inflorescence of a annua but not in the stem stele or roo ts; transcripts of QH5 could also be detected in stem epidermis. Linalool c ould not be detected by CC-MS in the essential oil of A. annua, nor in acid or base hydrolysates of aqueous extracts of leaves. RT-PCR demonstrated a wound-inducible increase in QH1 and QH5 transcript abundance in both leaves and stems over a 3-day time course. (C) 1999 Academic Press.