The LS locus of pea encodes the gibberellin biosynthesis enzyme ent-kaurene synthase A

Abstract

Gibberellins (GAs) are hormones required for several aspects of plant development, including internode elongation and seed development in pea (Pisum sativum L.). The first committed step in the GA biosynthesis pathway is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene via copalyl diphosphate (CDP). These two reactions are catalyzed by the cyclases ent-kaurene synthase A (KSA) and ent-kaurene synthase B (KSB), respectively. Previous genetic and biochemical analysis of the GA-responsive ls-1 mutant of pea suggested that GA levels are reduced in a developmental- and organ-specific manner due to reduced GA biosynthesis. Analysis of cellfree enzyme preparations from WT and ls-1 embryos at contact point reveals that ls-1 reduces the activity of KSA but not KSB. To characterize the ls-1 mutation in more detail, a cDNA coding for a pea KSA was cloned and shown to be encoded by the LS locus. The ls-1 mutation results from an intronic G to A substitution that causes impaired RNA splicing. To determine the activity of the KSAs encoded by the LS and ls-1 alleles, a new in vitro assay for combined KSA and KSB activity has been developed using the KSB gene of pumpkin. Using recombinant WT KSA and KSB fusion proteins, GGDP is converted to ent-kaurene in vitro. Based on the sequence of RT-PCR products, three different truncated KSA proteins are predicted to exist in ls-1 plants. The most abundant mutant KSA protein does not possess detectable activity in vitro. Nevertheless, the ls-1 allele is not null and is able to encode at least a partially functional KSA since a more severe is allele has been identified. The ls-1 mutation has played a key role in identifying a role for GAs in pea seed development in the first few days after fertilization, but not in older seeds. KSA expression in seeds is developmentally regulated and parallels overall GA biosynthesis, suggesting that KSA expression may play an important role in the regulation of GA biosynthesis and seed development.