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 GA(1), which is formed from GA(20) by 3β-hydroxylation. This step, which limits GA(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 3β-hydroxylase from lines of pea carrying the Le, le, le-3, and le(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(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 GA(9) to GA(4), and GA(20) to GA(1), with K(m) values of 1.5 μM and 13 μM, respectively. The amino acid substitution in the clone from le increased K(m) for GA(9) 100-fold and reduced conversion of GA(20) to almost nil. Expression products from le and le-3 possessed similar levels of 3β-hydroxylase activity, and the expression product from le(d) was inactive. Our results suggest that the 3β-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.