Isolation of an elicitor-stimulated 5-epi-aristolochene synthase gene (gPEAS1) from chili pepper (Capsicum annuum)

Phytoalexins play an important role in the inducible defense responses of plants against diseases caused by fungi. Some enzymes, involved in the respective biosynthetic pathways, catalyze key steps. Farnesyl pyrophosphate (FPP) is a key intermediate in the biosynthesis of terpenes. It may be converted into several cyclic and some acyclic sesquiterpenic derivatives, sterol precursors, or to geranylgeranyl pyrophosphate according to the requirements of the plant cells. Specific cyclization reactions of FPP are catalyzed by particular sesquiterpene cyclases. The 5-epi-aristolochene synthase (EAS) enzyme of tobacco (Nicotiana tabacum) and pepper (Capsicum annuum) produce the 5-epi-aristolochene, which is the immediate precursor of the bicyclic phytoalexin capsidiol. Oligonucleotides homologous to 3'-end specific regions of tobacco and Hyoscyamus muticus inducible sesquiterpene cyclase genes were used with the system for rapid amplification of cDNA ends (3'-RACE) technique to prepare pepper EAScDNA fragments (PEAS-cDNA). Three specific PEAScDNAs were isolated (PEAS1, PEAS18, and PEAS55). Northern blots of total RNA samples from pepper plant tissues challenged with cellulase or Phytophthora capsici showed different expression levels of the respective transcripts. PEAS1 was used to identify the corresponding elicitor-stimulated gene (gPEAS1). The nucleotide sequence of the proposed gPEAS1 promoter showed putative stimuli- and tissue-specific responsive elements. A pepper 5-epi-aristolochene synthase gene family of 5-8 members was demonstrated by Southern blot.