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The extreme dwarf phenotype of the GA-sensitive mutant of sunflower, dwarf2, is generated by a deletion in the ent-kaurenoic acid oxidase1 (HaKAO1) gene sequence

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Abstract

A dwarf mutant, dw arf 2 (dw2), was isolated from sunflower (Helianthus annuus). The most obvious alterations of dw2 plants were the lack of stem growth, reduced size of leaves, petioles and flower organs, retarded flower development. Pollen and ovules were produced but the filaments failed to extrude the anthers from the corolla. The dw2 phenotype was mainly because of reduced cell size. In dw2 leaves, the dark-green color was not so much due to higher pigment content, but was correlated with a changed leaf morphology. The mutant responded to the application of bioactive gibberellins (GAs). The levels of ent-7α-hydroxykaurenoic acid, GA19, GA20 and GA1 in dw2 seedlings were severely decreased relative to those in its wild type (WT). ent-Kaurenoic acid was actively converted to ent-7α-hydroxykaurenoic acid in WT plants but quite poorly in dw2 plants. All together these data suggested that the dw2 mutation severely reduced the flux through the biosynthetic pathway leading to active GAs by hampering the conversion of ent-kaurenoic acid to GA12. Two ent-kaurenoic acid oxidase (KAO) genes were identified. HaKAO1 was expressed everywhere in sunflower organs, while HaKAO2 was mainly expressed in roots. We demonstrated that a DNA deletion in HaKAO1 of dw2 generated aberrant mRNA-splicing, causing a premature stop codon in the amino acid sequence. In dw2 calli, Agrobacterium-mediated transfer of WT HaKAO1 cDNA restored the WT endogenous levels of GAs. In segregating BC1 progeny, the deletion co-segregated with the dwarf phenotype. The deletion was generated near to a breakpoint of a more complex chromosome rearrangement.

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

    Marco Fambrini, Mariangela Salvini & Claudio Pugliesi

  2. Dipartimento di Biologia delle Piante Agrarie, Sezione di Fisiologia Vegetale, Università di Pisa, Via Mariscoglio 34, 56124, Pisa, Italy

    Lorenzo Mariotti, Sandro Parlanti, Piero Picciarelli & Nello Ceccarelli

  3. Scuola Normale Superiore, Piazza dei Cavalieri 7, 56126, Pisa, Italy

    Mariangela Salvini

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  1. Marco Fambrini
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  2. Lorenzo Mariotti
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  3. Sandro Parlanti
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  4. Piero Picciarelli
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Correspondence to Claudio Pugliesi.

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Marco Fambrini and Lorenzo Mariotti contributed equally to this work.

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Suppl. Fig. 6

Pictures of untransformed (a, b) and transformed (c, d) calli obtained from dwarf2 (dw2) mutant leaves of sunflower (Helianthus annuus L.). Genetic transformation was performed with an Agrobacterium strain harbouring the wild type (WT) HaKAO1 cDNA fused in a vector pK7WGF2 (35S::GFP) (Karimi et al. 2002). Pictures were taken using bright field (a, c) and GFP (b, d) filters. Scale bars = 100 μm. (TIFF 25490 kb)

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Suppl. Fig. 10 a

Schematic diagram explanations of PCR conduced on genomic DNA of wild type (DW2) and dwarf2 (dw2) mutant of sunflower (Helianthus annuus L.) to test the chromosomal rearrangement occurring in the dw2 genome. The 6th intron of the HaKAO1 gene and the position of primers are indicated; the 403-bp deletion (403) is depicted as gray bar; the 7th exon of the HaKAO1 gene is depicted as black box. The 20 nucleotides adjacent to the primer FIN2 displayed 100% identity between the two genotypes and are showed. The region adjacent to these nucleotides displayed a low identity (see also Suppl. Fig. 9) and is depicted as black bar in DW2 and gray bar in dw2. The arrows indicate the 5’- 3’-primer direction. b The genomic PCR products of HaKAO1 from wild type (DW2) and mutant (dw2) using four different primer combinations. The products were resolved on ethidium bromide-stained 1.5% TAE-agarose gel and visualized under UV light. The size of the PCR products are shown. (TIFF 8297 kb)

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Fambrini, M., Mariotti, L., Parlanti, S. et al. The extreme dwarf phenotype of the GA-sensitive mutant of sunflower, dwarf2, is generated by a deletion in the ent-kaurenoic acid oxidase1 (HaKAO1) gene sequence. Plant Mol Biol 75, 431–450 (2011). https://doi.org/10.1007/s11103-011-9740-x

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