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

Abstract

The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA1, which is formed from GA20 by 3beta-hydroxylation. This step, which limits GA1 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 3beta-hydroxylase from lines of pea carrying the Le, le, le-3, and led 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 led, 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 GA9 to GA4, and GA20 to GA1, with Km values of 1.5 microM and 13 microM, respectively. The amino acid substitution in the clone from le increased Km for GA9 100-fold and reduced conversion of GA20 to almost nil. Expression products from le and le-3 possessed similar levels of 3beta-hydroxylase activity, and the expression product from led was inactive. Our results suggest that the 3beta-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.

Figure 1

GA 3β-hydroxylation.

Figure 2

Alignment of deduced GA 3β-hydroxylase amino acid sequences from pea (Le; accession no. AF001219) and arabidopsis (accession no. L37126), indicating the substitutions in sequences from le, le-3, and le^d pea lines. Light/dark shading indicates similar/identical residues. Bar, initial PCR product; asterisks, conserved residues that in isopenicillin N synthase bind iron.

Figure 3

Analysis of line I₃ (Le). (A) Southern blot of genomic DNA (30 μg per lane). PstI cut poorly, and signal was not detected in this lane. (B) Northern blot of poly(A)⁺ RNA isolated from different organs (3 μg per lane; overnight exposure). Signal was not detected in flowers and fruits even after 1-week exposure. Arrow indicates direction of electrophoresis.

Figure 4

Variation in expression of the GA 3β-hydroxylase gene among pea lines (1 μg poly(A)⁺ RNA per slot). Average length of internode (i-node) 5–6 is given for reference.

Figure 5

Michaelis–Menten plots for GA 3β-hydroxylation by cell lysates from E. coli expressing cDNA clones from Le (A) or le (B) lines. [¹⁴C]GA₉ or [¹⁴C]GA₂₀ was incubated for 15 min at 30°C with lysate and cofactors. The amount of lysate used in A was 2 μl (19 μg protein) and 20 μl (192 μg protein) with GA₉ and GA₂₀, respectively; in B, 25 μl (240 μg protein) was used with GA₉. Lysate of the clone from le had no activity on GA₂₀.