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

Abstract

Artemisia annua is an annual herb used in traditional Chinese medicine. A cDNA library was constructed from leaves of A. annua seedlings and target sequences were amplified by PCR using degenerate primers derived from a consensus sequence of angiosperm terpene synthases. Two clones, QH1 and QH5, with high sequence similarity to plant monoterpene synthases were ultimately obtained and expressed in Escherichia coli. These cDNAs encode peptides of 567 aa (65.7 kDa) and 583 aa (67.4 kDa), respectively, and display 88% identity with each other and 42% identity with Mentha spicata limonene synthase. The two recombinant enzymes yielded no detectable activity with isopentenyl diphosphate, dimethylallyl diphosphate, chrysanthemyl diphosphate, farnesyl 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 synthase displays a K(m) value of 64 microM for geranyl diphosphate, which is considerably 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 roots; transcripts of QH5 could also be detected in stem epidermis. Linalool could not be detected by GC-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.