MENDELS DWARFING GENE - CDNAS FROM THE LE ALLELES AND FUNCTION OF THEEXPRESSED PROTEINS

The major gibberellin (GA) controlling stem elongation in pea (Pisum s ativum L.) is GA(1), which is formed from GA(20) by 3 beta-hydroxylati on. This step, which limits GA(1) biosynthesis in pea, is controlled b y 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(d) all eles, The cDNA sequences from le and le-3 each contain a base substitu tion resulting in single amino acid changes relative to the sequence f rom 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 prod uct for the clone from Le converted GA(9) to GA(4), and GA(20) to GA(1 ), with K-m values of 1.5 mu M and 13 mu M, respectively. The amino ac id 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 fr om le and le-3 possessed similar levels of 3 beta-hydroxylase activity , and the expression product from le(d) was inactive. Our results sugg est that the 3 beta-hydroxylase cDNA is encoded by Le, Le transcript i s expressed in roots, shoots, and cotyledons of germinating pea seedli ngs, in internodes and leaves of established seedlings, and in develop ing seeds.