Mendel’s dwarfing gene: cDNAs from the <i>Le</i> alleles and function of the expressed proteins

<jats:p> The major gibberellin (GA) controlling stem elongation in pea ( <jats:italic>Pisum sativum</jats:italic> L.) is GA <jats:sub>1</jats:sub> , which is formed from GA <jats:sub>20</jats:sub> by 3β-hydroxylation. This step, which limits GA <jats:sub>1</jats:sub> biosynthesis in pea, is controlled by the <jats:italic>Le</jats:italic> 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 <jats:italic>Le</jats:italic> , <jats:italic>le</jats:italic> , <jats:italic>le-3</jats:italic> , and <jats:italic>le</jats:italic> <jats:sup>d</jats:sup> alleles. The cDNA sequences from <jats:italic>le</jats:italic> and <jats:italic>le-3</jats:italic> each contain a base substitution resulting in single amino acid changes relative to the sequence from <jats:italic>Le</jats:italic> . The cDNA sequence from <jats:italic>le</jats:italic> <jats:sup>d</jats:sup> , a mutant derived from an <jats:italic>le</jats:italic> line, contains both the <jats:italic>le</jats:italic> “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 <jats:italic>Escherichia coli</jats:italic> . The expression product for the clone from <jats:italic>Le</jats:italic> converted GA <jats:sub>9</jats:sub> to GA <jats:sub>4</jats:sub> , and GA <jats:sub>20</jats:sub> to GA <jats:sub>1</jats:sub> , with K <jats:sub>m</jats:sub> values of 1.5 μM and 13 μM, respectively. The amino acid substitution in the clone from <jats:italic>le</jats:italic> increased K <jats:sub>m</jats:sub> for GA <jats:sub>9</jats:sub> 100-fold and reduced conversion of GA <jats:sub>20</jats:sub> to almost nil. Expression products from <jats:italic>le</jats:italic> and <jats:italic>le-3</jats:italic> possessed similar levels of 3β-hydroxylase activity, and the expression product from <jats:italic>le</jats:italic> <jats:sup>d</jats:sup> was inactive. Our results suggest that the 3β-hydroxylase cDNA is encoded by <jats:italic>Le. Le</jats:italic> transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds. </jats:p>