Mendel's dwarfing gene: cDNAs from the Le alleles and function of the expressed proteins

The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is G[A.sub.1], which is formed from G[A.sub.20] by 3[Beta]-hydroxylation. This step, which limits G[A.sub.1] biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3[Beta]-hydroxylase from lines of pea carrying the Le, le, le-3, and [le.sup.d] alleles. The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le. The cDNA sequence from [le.sup.d], a mutant derived from an le line, contains both the le 'mutation' and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in Escherichia coli. The expression product for the clone from Le converted G[A.sub.9] to G[A.sub.4], and G[A.sub.20] to G[A.sub.1], with gm values of 1.5 [[micro]molar] and 13 [[micro]molar], respectively. The amino acid substitution in the clone from le increased [K.sub.m] for G[A.sub.9] 100-fold and reduced conversion of G[A.sub.20] to almost nil. Expression products from le and le-3 possessed similar levels of 3[Beta]-hydroxylase activity, and the expression product from [le.sup.d] was inactive. Our results suggest that the 3[Beta]-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in inter-nodes and leaves of established seedlings, and in developing seeds.