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Molecular Cloning and Organ-Specific Expression of Two Gibberellin 20-Oxidase Genes of Helianthus annuus

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Abstract

The activity of GA 20-oxidase (GA20ox), a 2-oxoglutarate-dependent dioxygenase, is a critical regulatory factor in the gibberellin-(GA)-biosynthetic pathway. Two genes, HaGA20ox1 and HaGA20ox2, along with their corresponding cDNAs exhibiting structural features and homology to GA 20-oxidases of several plant species, have been isolated from vegetative shoots of sunflower (Helianthus annuus). Sequence analysis revealed that both genes consist of three exons and two introns. GA20ox genes from dicot and monocot species showed similar structures. The amino acid sequences deduced from the sunflower cDNA clones showed high level of similarity, with identities of HaGA20ox1/HaGA20ox2 of 84%. The phylogenetic analysis indicated that all monocot and all dicot GA20ox were grouped in two separate clusters. Within the dicot clade, HaGA20ox1 and HaGA20ox2 formed a subclade with GA 20-oxidases from Lactuca sativa (Ls20ox1 and Ls20ox2), Chrysanthemum x morifolium (DgGA20ox1), and Daucus carota (DcGA20ox2). Both HaGA20ox1 and HaGA20ox2 shared the highest similarity with the L. sativa Ls20ox1. The high accumulation of both HaGA20ox transcripts coincided with the period of rapid growth of the sunflower embryo, suggesting a role for GA in the first phase of embryo maturation. HaGA20ox1 mRNA was also detected in all the organs tested but occurred at a higher level in the vegetative shoot and the root, whereas HaGA20ox2 transcripts were preferentially accumulated in inflorescence meristems, vegetative shoots, internodal stem, and roots. Treatment of sunflower plants with an inhibitor of GA biosynthesis showed that HaGA20ox2, but not HaGA20ox1, was subject to feedback regulation by a reduction of bioactive GAs.

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Abbreviations

GA:

gibberellin

GA20ox:

GA 20-oxidase

PAC:

paclobutrazol

RACE:

rapid amplification of cDNA ends

RT-PCR:

reverse transcriptase polymerase chain reaction

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Acknowledgments

This work was supported by a grant from Ministero Italiano dell’Università e Ricerca (PRIN 2006–2008).

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Authors and Affiliations

  1. Sezione di Genetica, Dipartimento di Biologia delle Piante Agrarie, Università di Pisa, Via Matteotti 1B, I-56124, Pisa, Italy

    Filippo G. Carzoli, Vania Michelotti, Marco Fambrini, Mariangela Salvini & Claudio Pugliesi

  2. Scuola Normale Superiore, Università di Pisa, Piazza dei Cavalieri 7, I-56124, Pisa, Italy

    Mariangela Salvini

Authors
  1. Filippo G. Carzoli
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  2. Vania Michelotti
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  3. Marco Fambrini
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  4. Mariangela Salvini
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  5. Claudio Pugliesi
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Corresponding author

Correspondence to Claudio Pugliesi.

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Supplementary Material 1

Amino acid sequence alignment of GA20-oxidases from sunflower, HaGA20ox1 and HaGA20ox2, and several homologous peptides from monocot and dicot species. Asterisks and dots respectively indicate identical and similar amino acid residues. The putative Fe2+-binding motif typical for 2-oxoglutarate-dependent dioxygenases are indicated by boxes. The conserved region NYYPXCXXP thought to be involved in binding the 2-oxoglutarate cosubstrate is indicated in bold italic. The LPWKET residues thought to be involved in binding the GA substrate are underlined. The alignment was obtained by ClustalW version 1.7 (Thompson et al. 1994). The GenBank accession numbers are the same as Fig. 2. (DOC 63.5 KB)

Supplementary Material 2

. Transcript accumulation of HaGA20ox2 in various sunflower organs as determined by RT-PCR analysis. Total RNA was extracted: in a from R, CO, H, VS, ST, YL, ML, and IM; in b from immature embryos (IE) at different days (5, 13, 21, and 28) from DAP and DS. To detect HaGA20ox2 transcript accumulation, PCR amplification was programmed for 35 cycles in a and 32 cycles in b (PPT 121 KB)

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Carzoli, F.G., Michelotti, V., Fambrini, M. et al. Molecular Cloning and Organ-Specific Expression of Two Gibberellin 20-Oxidase Genes of Helianthus annuus . Plant Mol Biol Rep 27, 144–152 (2009). https://doi.org/10.1007/s11105-008-0066-z

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