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

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.

• Authors: J L García-Martínez, I López-Diaz, M J Sánchez-Beltrán, A L Phillips, D A Ward, P Gaskin, P Hedden
• Journal: Plant molecular biology (Plant Mol Biol) Vol. 33 Issue 6 Pg. 1073-84 (Apr 1997) ISSN: 0167-4412 [Print] Netherlands
• PMID: 9154988 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

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

Topics

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