Stamen-derived bioactive gibberellin is essential for male flower development of Cucurbita maxima L

Abstract

Gibberellin (GA) signalling during pumpkin male flower development is highly regulated, including biosynthetic, perception, and transduction pathways. GA 20-oxidases, 3-oxidases, and 2-oxidases catalyse the final part of GA synthesis. Additionally, 7-oxidase initiates this part of the pathway in some cucurbits including Cucurbita maxima L. (pumpkin). Expression patterns for these GA-oxidase-encoding genes were examined by competitive reverse transcription-PCR (RT-PCR) and endogenous GA levels were determined during pumpkin male flower development. In young flowers, GA20ox3 transcript levels are high in stamens, followed by high levels of the GA precursor GA(9). Later, just before flower opening, transcript levels for GA3ox3 and GA3ox4 increase in the hypanthium and stamens, respectively. In the stamen, following GA3ox4 expression, bioactive GA(4) levels rise dramatically. Accordingly, catabolic GA2ox2 and GA2ox3 transcript levels are low in developing flowers, and increase in mature flowers. Putative GA receptor GID1b and DELLA repressor GAIPb transcript levels do not change in developing flowers, but increase sharply in mature flowers. Emasculation arrests floral development completely and leads to abscission of premature flowers. Application of GA(4) (but not of its precursors GA(12)-aldehyde or GA(9)) restores normal growth of emasculated flowers. These results indicate that de novo GA(4) synthesis in the stamen is under control of GA20ox3 and GA3ox4 genes just before the rapid flower growth phase. Stamen-derived bioactive GA is essential and sufficient for male flower development, including the petal and the pedicel growth.

Fig. 1.

Gibberellin biosynthesis in pumpkin plants. This simplified pathway only shows the final part for the synthesis of the non-13-hydroxylated GAs. Reactions are catalysed by recombinant GA20ox4 (a), GA3ox4 (b), GA2ox2 (c), and GA2ox3 (d).

Fig. 2.

Phylogenetic relationship of pumpkin (Cm) and cucumber (Csa) GA-related proteins (Huang et al., 2009) using the program Geneious Pro 5.4. For this study newly isolated genes from pumpkin are in bold. CmGAIP and CmGAIPb were isolated originally by Haywood et al. (2005). For the other CmGA-oxidases see Lange et al. (2005). For Cucumis sativus: Csa613 (ACHR01000613; ORF, bp 29 702–30 198 and 30 355–30 982), Csa614 (ACHR01000614; ORF, bp 8355–8848 and 8929–9559); for other Csa genes see the Cucurbit Genomics Database (www.icugi.org/cgi-bin/ICuGI/genome/cuke.cgi).

Fig. 3.

Stages of flower development. Values for day after appearance (DAA) of flower buds shown are ±SEM (n = 12). Tissues are as indicated: hypanthium (H), sepals (S), petals (P), and stamens (St). Bar=1 cm.

Fig. 4.

Transcript levels of gibberellin oxidase genes in different floral organs during male flower development. Developmental stages and floral organs used for analysis by competitive RT-PCR were as described in Fig. 3. For gene abbreviations, see Fig. 2. HSP, H, S, and P were analysed together.

Fig. 5.

Emasculation arrests flower growth that is rescued by bioactive GA₄ application. Male flower buds of developmental stage II (Fig. 3) were either left intact (A) or were emasculated (B). Eleven days after the treatment flowers are illustrated that developed from intact buds (C) or from emasculated buds treated either with water (D) or with an aqueous solution of GA₄ as described in the Materials and methods (E). Bar=1 cm.

Fig. 6.

Effect of the gibberellin precursors GA₁₂-aldehyde and GA₉, and of bioactive GA₄ on growth of emasculated pumpkin flowers. Male flower buds of developmental stage II (Fig. 3) were either kept intact or emasculated. The latter ones were treated either with water, or with an aqueous solution of GA₁₂-aldehyde, GA₉, or GA₄ as described in the Materials and methods. The length of the flower (A) and of the pedicel (B) was measured at day 6, day 9, and day 12 after the treatments. Values shown are ±SEM (n=10). Asterisks indicate values that are statistically different from those of intact flowers (Student’s t test, P < 0.05).