Isolation and transcript analysis of gibberellin 20-oxidase genes in pea and bean in relation to fruit development.
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.
García-Martínez
J L
JL
Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Spain.
López-Diaz
I
I
Sánchez-Beltrán
M J
MJ
Phillips
A L
AL
Ward
D A
DA
Gaskin
P
P
Hedden
P
P
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000235:genetics* / Q000254:growth & development; D018628:Gene Dosage; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D017343:Genes, Plant / Q000235:genetics; D005875:Gibberellins / Q000096:biosynthesis; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000235:genetics* / Q000254:growth & development; D010802:Phylogeny; D010946:Plants, Medicinal; D012333:RNA, Messenger / Q000032:analysis; D018749:RNA, Plant / Q000032:analysis; D011994:Recombinant Proteins; D012639:Seeds / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018744:DNA, Plant; D005875:Gibberellins; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C089210:gibberellin, 2-oxoglutarate-oxygen oxidoreductase (20-hydroxylating, oxidizing)