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

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, GA(19), GA(20) and GA(1) 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 GA(12). 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 BC(1) progeny, the deletion co-segregated with the dwarf phenotype. The deletion was generated near to a breakpoint of a more complex chromosome rearrangement.

Nighttime Sugar Starvation Orchestrates Gibberellin Biosynthesis and Plant Growth in Arabidopsis

“…HPLC analysis was performed with a Kontron instrument equipped with a UV absorbance detector operating at 214 nm, while the gas chromatography-tandem mass spectrometry analysis was performed on a Saturn 2200 quadruple ion trap mass spectrometer coupled to a CP-3800 gas chromatograph (Varian Analytical Instruments) equipped with a MEGA snc (http://www.mega.mi.it) 1MS capillary column (30 m 3 0.25-mm i.d. and 0.25-µm film thickness) as described (Fambrini et al, 2011). GAs were identified by comparing the full mass spectra with those of the authentic compounds.…”

“…HPLC analysis was performed as described above. The fraction corresponding to the elution volume of standard kaurene was analyzed with gas chromatography-tandem mass spectrometry as previously outlined (Fambrini et al, 2011).…”

A transposon-mediate inactivation of a CYCLOIDEA-like gene originates polysymmetric and androgynous ray flowers in Helianthus annuus

“…The GenomeWalker DNA walking approach was used to extend the sequence to 5 0 untranslated region of the HaCYC2c gene (Fambrini et al 2011;Siebert et al 1995). DNA was extracted from leaves of 30-days-old plants of both TURF and mutant plants with the NucleoSpin Ò Plant II DNA extraction and purification protocol for plant tissue, according to the manufacturer's instructions (MachereyNagel GmbH & Co., Düren, Germany).…”

Gibberellin Biosynthesis

“…KAO is encoded by two fully redundant genes in Arabidopsis, although in other species differences in kao expression domains has been reported and loss of function may result in dwarfism (see, e.g. Fambrini et al, 2011). There are multiple ga20ox and ga3ox genes differing considerably in their intensity and pattern of expression and therefore their roles in plant development.…”