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

1997; National Academy of Sciences; Volume: 94; Issue: 16 Linguagem: Inglês

10.1073/pnas.94.16.8907

1091-6490

David Martin, William M. Proebsting, Peter Hedden,

Legume Nitrogen Fixing Symbiosis

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.