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. 1995 Jul 3;92(14):6640–6644. doi: 10.1073/pnas.92.14.6640

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

Y L Xu 1, L Li 1, K Wu 1, A J Peeters 1, D A Gage 1, J A Zeevaart 1
PMCID: PMC41574  PMID: 7604047

Abstract

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. De Carolis E., De Luca V. 2-oxoglutarate-dependent dioxygenase and related enzymes: biochemical characterization. Phytochemistry. 1994 Jul;36(5):1093–1107. doi: 10.1016/s0031-9422(00)89621-1. [DOI] [PubMed] [Google Scholar]
  2. Gilmour S. J., Zeevaart J. A., Schwenen L., Graebe J. E. Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod. Plant Physiol. 1986 Sep;82(1):190–195. doi: 10.1104/pp.82.1.190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Goodall G. J., Filipowicz W. Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants. EMBO J. 1991 Sep;10(9):2635–2644. doi: 10.1002/j.1460-2075.1991.tb07806.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Lange T., Hedden P., Graebe J. E. Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8552–8556. doi: 10.1073/pnas.91.18.8552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Lange T. Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm. Planta. 1994;195(1):108–115. doi: 10.1007/BF00206298. [DOI] [PubMed] [Google Scholar]
  6. Leung J., Bouvier-Durand M., Morris P. C., Guerrier D., Chefdor F., Giraudat J. Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase. Science. 1994 Jun 3;264(5164):1448–1452. doi: 10.1126/science.7910981. [DOI] [PubMed] [Google Scholar]
  7. Meyerowitz E. M. Arabidopsis thaliana. Annu Rev Genet. 1987;21:93–111. doi: 10.1146/annurev.ge.21.120187.000521. [DOI] [PubMed] [Google Scholar]
  8. Talon M., Koornneef M., Zeevaart J. A. Endogenous gibberellins in Arabidopsis thaliana and possible steps blocked in the biosynthetic pathways of the semidwarf ga4 and ga5 mutants. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7983–7987. doi: 10.1073/pnas.87.20.7983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Talon M., Zeevaart J. A., Gage D. A. Identification of Gibberellins in Spinach and Effects of Light and Darkness on their Levels. Plant Physiol. 1991 Dec;97(4):1521–1526. doi: 10.1104/pp.97.4.1521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Zeevaart J. A., Gage D. A., Talon M. Gibberellin A1 is required for stem elongation in spinach. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7401–7405. doi: 10.1073/pnas.90.15.7401. [DOI] [PMC free article] [PubMed] [Google Scholar]

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