The GA2 Locus of Arabidopsis thalianaEncodes ent-Kaurene Synthase of Gibberellin Biosynthesis

Abstract

The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. GAs are a group of diterpene compounds, some of which are plant-growth regulators that control many aspects of plant development, such as seed germination, shoot elongation, and flower development. A number of GAresponsive dwarf mutants that are deficient in the biosynthesis of active GAs have been characterized in various plant species (for a recent review, see Hedden and Kamiya, 1997;Ross et al., 1997). Biochemical characterization of these mutants has contributed to the elucidation of GA biosynthetic pathways. ent-Kaurene is an early intermediate in GA biosynthesis and is synthesized by the cyclization of GGDP via CDP. In plants, this two-step cyclization involves two distinct enzymes: CPS catalyzes the cyclization of GGDP to CDP, which is then converted to entkaurene by KS. CPS and KS were formally called entkaurene synthases A and B, respectively (Hedden and Kamiya, 1997). Biochemical studies suggested that CPS and KS may interact with each other (Duncan and West, 1981) and that these enzymes are localized in plastids (Railton et al., 1984;Sun and Kamiya, 1994; Aach et al., 1995). There is some evidence that ent-kaurene biosynthesis is controlled by environmental conditions such as photoperiod (Zeevaart and Gage, 1993) and temperature (Moore and Moore, 1991), as well as during plant development (Chung and Coolbaugh, 1986;Silverstone et al., 1997).From Arabidopsis thaliana six GA-responsive dwarf mutants (ga1 through ga6) have been isolated and characterized (Koornneef and van der Veen, 1980;Sponsel et al., 1997). Each mutant is blocked in a specific step in GA biosynthesis. The GA1 locus has been cloned (Sun et al., 1992) and shown to encode CPS (Sun and Kamiya, 1994). The GA4 and GA5 genes code for dioxygenases involved in later steps of the GA biosynthetic pathway (Chiang et al., 1995;Xu et al., 1995). The GA2, GA3, and GA6 loci may encode enzymes catalyzing steps in the GA biosynthetic pathway or regulators of these enzymes.The ga2-1 mutant is a nongerminating, extreme dwarf, which is phenotypically similar to strong alleles of the ga1 and ga3 mutants. (Koornneef and van der Veen, 1980). The biochemical characterization of the ga2 mutant was described by Zeevaart and Talon (1992). The ga2 mutant is responsive to exogenous ent-kaurene and is lacking in the ability to accumulate ent-kaurene when treated with an inhibitor of ent-kaurene metabolism. A cell-free extract prepared from siliques of the...

Identification of Syn-Pimara-7,15-Diene Synthase Reveals Functional Clustering of Terpene Synthases Involved in Rice Phytoalexin/Allelochemical Biosynthesis

“…Slightly higher similarity (approximately 30% identity) was found with the identified gymnosperm bifunctional class II/I diterpene synthases, which are involved in labdane-related biosynthesis (Stofer Vogel et al, 1996;Schepmann et al, 2001;Martin et al, 2004). Further, the sequence is moderately similar (39%-42% identity) to the known ent-kaurene synthases involved in gibberellin biosynthesis (Yamaguchi et al, 1996(Yamaguchi et al, , 1998Richman et al, 1999). Finally, comparison with the recently identified ent-CPP specific cassa-12,15-diene synthase (Cho et al, 2004) revealed significant homology (approximately 51% identity).…”

“…A putative class I terpene synthase involved in labdane-related biosynthesis was identified by a BLAST search of the GenBank database (www.ncbi.nlm.nih.gov:80/BLAST/) with the amino acid sequence of ent-kaurene synthase from Arabidopsis (Yamaguchi et al, 1998). RT-PCR reactions were performed to verify expression of the predicted gene in UVirradiated leaves by generating a fragment of the corresponding sequence.…”

Stemar‐13‐ene synthase, a diterpene cyclase involved in the biosynthesis of the phytoalexin oryzalexin S in rice

“…Similarity was found with OsDTC1 (ent-cassa-12,15-diene synthase) (48% identity), ent-KS (39-41% identity) [22][23][24], abietadiene synthase (AS) [25] (32% identity), and ent-CPS [26,27] (29% identity). AS has two active sites responsible for KS-type and CPS-type activities.…”

A genomic approach to characterization of the Citrus terpene synthase gene family

“…These are the AtTPSGA1 gene for copalyl diphosphate synthase (Sun and Kamiya, 1994) and the AtTPSGA2 transcript which encodes kaurene synthase (Yamaguchi et al, 1998), both of which are involved in the formation of gibberellins, and the AtTPS10 transcript for myrcene/ocimene synthase (Bohlmann et al, 2000) required for the formation of acyclic monoterpenes. In an initial attempt to identify the members of the Citrus terpene synthase gene family, we have performed an in silico screen of the CitEST database for putative terpene synthase sequences.…”

“…Class I terpene synthases include AtTPS GA1 (Sun and Kamiya, 1994), which is a member of the TPS-c group of angiosperm copalyl diphosphate synthases. This class also includes the AtTPS GA2 enzyme (Yamaguchi et al, 1998), which is a diterpene synthase of the TPS-e subfamily of kaurene synthases. These terpene synthases are involved in the biosynthesis of gibberellic acid and putative Citrus ortologues were found for both of them.…”