Cloning and Bacterial Expression of Sesquiterpene Cyclase, a Key Branch Point Enzyme for the Synthesis of Sesquiterpenoid Phytoalexin Capsidiol in UV-Challenged Leaves of Capsicum annuum

Abstract

Sesquiterpene cyclase, a branch point enzyme in the general isoprenoid pathway for the synthesis of phytoalexin capsidiol, was induced in detached leaves of Capsicum annuum (pepper) by UV treatment. The inducibility of cyclase enzyme activities paralleled the absolute amount of cyclase protein(s) of pepper immunodetected by monoclonal antibodies raised against tobacco sesquiterpene cyclase. A cDNA library was constructed with poly(A)+ RNA isolated from 24 h UV-challenged leaves of pepper. A cDNA clone for sesquiterpene cyclase in pepper was isolated by using a tobacco 5-epi aristolochene synthase gene as a heterologous probe. The predicted protein encoded by this cDNA was comprised of 559 amino acids and had a relative molecular mass of 65,095. The primary structural information from the cDNA clone revealed that it shared 77%, 72% and 49% identity with 5-epi aristolochene, vetispiradiene, and cadinene synthase, respectively. The enzymatic product catalyzed by the cDNA clone in bacteria was identified as 5-epi aristolochene, as judged by argentation TLC. RNA blot hybridization demonstrated the induction of an mRNA consistent with the induction of cyclase enzyme activity in UV-treated pepper.

Expression pattern of (+)-δ-cadinene synthase genes and biosynthesis of sesquiterpene aldehydes in plants of Gossypium arboreum L.

“…In G. hirsutum foliar tissues inoculated with a phytopathogenic bacterium Xanthomonas campestris, an elevated d-cadinene synthase activity was observed (Davis et al 1996). Similar activation of sesquiterpene cyclase genes by elicitation has been reported for tobacco and Hyoscyamus muticus (Facchini and Chappell 1992;Back and Chappell 1995). However, in contrast to tobacco plants that accumulate secondary sesquiterpenes only after induction (VoÈ geli and Chappell 1990;Yin et al 1997), cotton plants constitutively accumulate sesquiterpenoids in their pigmented glands.…”

“…Because of this central function, this enzyme is highly regulated (Chappell 1995;McGarvey and Croteau 1995;Starks et al 1997;Bohlmann et al 1998b). Investigations of plant terpene cyclases have drawn much attention recently, and cDNAs encoding various sesquiterpene cyclases have been isolated from a number of plant species (Facchini and Chappell 1992;Back and Chappell 1995;Back et al 1998;Bohlmann et al 1998a;Colby et al 1998;Steele et al 1998a), including two (+)-d-cadinene synthases (CADs) from Gossypium arboreum L. (Chen et al 1995(Chen et al , 1996. However, up to now only a few studies have been conducted on plant cyclase genes and their expression patterns.…”

Volatile sesquiterpenes from fungi: what are they good for?

“…The resulting metabolites are modulated by other enzymes which are induced by environmental stress factors. Little is known about the specific mechanisms in fungi but from plants several environmental factors, including UV-radiation (Back et al 1998), infection (Townsend et al 2005) and herbivore attack (Yuan et al 2008), have been identified which lead to the enhanced production of volatile sesquiterpenes.…”

Metabolic engineering of biosynthetic pathways leading to isoprenoids: Mono- and sesquiterpenes in plastids and cytosol

“…5-epi-aristolochene synthases form Nicotiana tabacum (Facchini & Chappell 1992) and from Capsicum annum (Back et al 1998), a vetispiradiene synthase from Hyoscyamus muticus (Back & Chappell 1995), a (E)-b-farnesene synthase from Mentha piperita and Citrus junos (Crock et al 1997, Maruyama et al 2001), a 6-selinene synthase and a gammahumulene synthase from Abies grandis (Steele 1998), 6-cadinene synthases from Gossypium arboretum (Chen et al 1995(Chen et al , 1996, a E-.alpha.-bisabolene synthase from Abies grandis (Bohlmann et al 1998), a germacrene C synthase from Lycopersicon esculentum (Colby et al 1998), an epi-cedrol synthase and an amorpha-4,11-diene synthase from Artemisia annua (Mercke et al 1999(Mercke et al , 2000, a germacrene D synthase from Lycopersicon esculentum (van der Hoeven et al 2000) and germacrene A synthases from Lactuca sativa (Bennett et al 2002), from Cichorium intybus (Bouwmeester et al 2002) and from Solidago Canadensis (Prosser et al 2002). Exhaustive reviews have been made on the subject covering some latest findings (Chang et al 2007, Cheng et al 2007).…”