Isolation and transcript analysis of gibberellin 20-oxidase genes in pea and bean in relation to fruit development

Plant Mol Biol. 1997 Apr;33(6):1073-84. doi: 10.1023/a:1005715722193.

Abstract

PCR was used with degenerate primers based on conserved amino acid sequences in gibberellin (GA) 20-oxidases to isolate cDNA clones for these enzymes from young seeds of pea (Pisum sativum) and developing embryos of French bean (Phaseolus vulgaris). One GA 20-oxidase cDNA (Ps27-12) was obtained from pea and three (Pv 15-11, Pv73-1 and Pv85-26) from bean. Their identities were confirmed by demonstrating that fusion proteins expressed in Escherichia coli exhibited GA 20-oxidase activity, converting [14C]GA12 to [14C]GA9. The intermediates in this three-step reaction, GA15 and GA24, were also identified as products. The expression proteins from three of the clones (Ps27-12, Pv15-11 and Pv73-1) were also shown to convert GA53 to GA20, as effectively as they did GA12. On the basis of transcript levels measured by northern blot analysis, the pea GA 20-oxidase gene is most highly expressed in young leaves, fully expanded internodes, very young seeds (until 4 days after anthesis) and expanding pods (from 3 days after anthesis at least until day 6). Expression in pods from 3-day-old unpollinated ovaries is higher than in those from pollinated ovaries. Treatment of unpollinated ovaries with GA3 to induce parthenocarpic fruit-set severely reduced the amount of GA 20-oxidase mRNA, whereas treatment with 2,4-D, although inducing fruit-set, did not reduce the levels of these transcripts. Plant decapitation above an unpollinated ovary resulted in very high levels of GA 20-oxidase mRNA in the pod. The three GA 20-oxidase genes from French bean showed very different patterns of expression: Pv 15-1 was expressed in the roots, young leaves, and developing seeds, but most highly in immature cotyledons, while Pv73-1 has a similar expression pattern to Ps27-12, with transcripts found only in young seeds and young leaves, where it was particularly abundant. Transcripts corresponding to Pv85-26 were detected in developing seeds, and just traces in the young leaves. Southern blot analysis indicated that the bean GA 20-oxidases are each encoded by single-copy genes, whereas one more gene, homologous to Ps27-12, could also exist in pea.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cloning, Molecular
  • DNA, Plant / analysis
  • Escherichia coli / genetics
  • Fabaceae / genetics*
  • Fabaceae / growth & development
  • Gene Dosage
  • Gene Expression Regulation, Plant / physiology*
  • Genes, Plant / genetics
  • Gibberellins / biosynthesis
  • Mixed Function Oxygenases / genetics*
  • Molecular Sequence Data
  • Peas / genetics*
  • Peas / growth & development
  • Phylogeny
  • Plants, Medicinal*
  • RNA, Messenger / analysis
  • RNA, Plant / analysis
  • Recombinant Proteins
  • Seeds / chemistry
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid

Substances

  • DNA, Plant
  • Gibberellins
  • RNA, Messenger
  • RNA, Plant
  • Recombinant Proteins
  • Mixed Function Oxygenases
  • gibberellin, 2-oxoglutarate-oxygen oxidoreductase (20-hydroxylating, oxidizing)

Associated data

  • GENBANK/U70471
  • GENBANK/U70530
  • GENBANK/U70531
  • GENBANK/U70532