Gibberellins regulate the transcription of the continuous flowering regulator, RoKSN, a rose TFL1 homologue

Abstract

The role of gibberellins (GAs) during floral induction has been widely studied in the annual plant Arabidopsis thaliana. Less is known about this control in perennials. It is thought that GA is a major regulator of flowering in rose. In spring, low GA content may be necessary for floral initiation. GA inhibited flowering in once-flowering roses, whereas GA did not block blooming in continuous-flowering roses. Recently, RoKSN, a homologue of TFL1, was shown to control continuous flowering. The loss of RoKSN function led to continuous flowering behaviour. The objective of this study was to understand the molecular control of flowering by GA and the involvement of RoKSN in this inhibition. In once-flowering rose, the exogenous application of GA(3) in spring inhibited floral initiation. Application of GA(3) during a short period of 1 month, corresponding to the floral transition, was sufficient to inhibit flowering. At the molecular level, RoKSN transcripts were accumulated after GA(3) treatment. In spring, this accumulation is correlated with floral inhibition. Other floral genes such as RoFT, RoSOC1, and RoAP1 were repressed in a RoKSN-dependent pathway, whereas RoLFY and RoFD repression was RoKSN independent. The RoKSN promoter contained GA-responsive cis-elements, whose deletion suppressed the response to GA in a heterologous system. In summer, once-flowering roses did not flower even after exogenous application of a GA synthesis inhibitor that failed to repress RoKSN. A model is presented for the GA inhibition of flowering in spring mediated by the induction of RoKSN. In summer, factors other than GA may control RoKSN.

Fig. 1.

Management of R.×wichurana (RW) after GA₃ and PCB treatments. (A) In December, vegetative shoots were pruned at six nodes. Plants remained outside for vernalization. At the end of January, plants were transferred to a tunnel. New shoots developed from axillary buds of the previous years. These shoots were mainly terminated by an inflorescence. After floral development, new shoots grew from the base. Since RW is an OF rose, the shoots remained vegetative. In summer, these shoots were pruned at six nodes and the fate of new emerging shoots from axillary buds was studied. Under controlled conditions, these shoots remained vegetative. RW plants were treated with GA₃ (0, 30, 70, or 140 µM) during vegetative growth and floral initiation between January and March (hatched boxes). Another set of RWs was treated with GA₃ (30 µM) and PCB (14, 56, and 112 µM) during vegetative growth between August and September. Arrows and asterisks indicate the sampling carried out for RNA extraction and histological analysis, respectively. (B) Experiments with different durations and periods of GA₃ (30 µM) treatment. In the ‘Late treatment’, the experiment started at different times and ended at the same time, whereas in the ‘Early treatment’, the experiment started at the same time and ended at different times. Numbers indicate the days after the beginning of treatment (DAB). Broken lines represent the pruned shoots of the previous years, large arrows represent new indeterminate growing shoots, and black circles represent axillary buds. Open circles represent flowers. (This figure is available in colour at JXB online.)

Fig. 2.

Effect of GA₃ treatment on average internode size in centimetres (A) and percentage of shoots that become floral (B) on R.×wichurana (RW). RW plants were treated with GA₃ (0, 30, 70, or 140 µM) for 70 d, and with GA₃ (30 µM) for different durations and periods. Data were the means of 10 plant observations. There was a statistically significant difference (Waerden’s test, P < 0.05; and the χ² test, P < 0.0033, for the comparison of percentages) between means with different letters.

Fig. 3.

Longitudinal cross-section through apices treated with GA₃ (30 µM; B, D, F, and H) or not (A, C, E, and G). In the untreated plant, the vegetative meristem (A: 42 DAB) progressively changed into a floral meristem (C, E, and G; 56, 63, and 70 DAB, respectively), whereas in the GA₃ (30µM)-treated plants, the meristem remained vegetative for the same stage of development (B, D, F, and H, 42, 56, 63, and 70 DAB, respectively). VM, vegetative meristem; LP, leaf primordia; FM, floral meristem; S, sepal; FB, floral bud; P, petal; St, stamen; Pi, pistil. Scale bar=100 µm. (This figure is available in colour at JXB online.)

Fig. 4.

Transcript accumulation of floral genes in spring and later in summer in GA₃-treated (squares), PCB-treated (triangles), or untreated RW (diamonds). Transcript accumulation of floral genes was followed by qPCR in axillary shoots for (A) RoKSN, (B) RoFD, (C) RoFT, (D) RoLFY, (E) RoSOC1, and (F) RoAP1. The x-axis indicates the number of days at which apices were harvested after the beginning of the GA₃ or PCB treatment. The transcript accumulation levels are expressed in relation to the first sample, harvested in January, for each gene according to the Pfaffl ratio (Pfaffl, 2001) (base value=1);. The grey box represents the floral initiation determined according to the histological analysis (Fig. 2). Kruskall–Wallis test on two biological replicates (P < 0.05) was realized, and asterisks represent statistical differences between GA₃-treated and untreated plants; triangles represent statistical differences between PCB-treated and untreated plants. (G) Transcript accumulation of floral genes in GA₃-treated (grey box), and untreated (black box) LWP. The transcript accumulation levels are expressed in relation to the untreated plants. Data with different letters indicate a significant difference (Kruskall–Wallis test P < 0.05). nd, not detected

Fig. 5.

Analysis of GA-responsive elements in the RoKSN promoter. (A) Alignment of the four TFL1 homologue promoters using MVISTA. Conserved regions (>70% similarity on a 100bp window) are in red and surrounded by dotted lines (blocks A and B). Alignment was performed with TFL1 (Arabidopis), FvKSN (F. vesca), MdTFL1 (M. domestica), MdTFL1a (M. domestica), and PpTFL1 (P. persica). (B) Successive promoter deletions fused with a GFP::GUS gene. (C) GFP fluorescence and GUS activity. GFP fluorescence observed under a confocal microscope for the four different promoter deletions. Untreated and treated plants are in the left and right panel, respectively. GUS activity for the different deletions in the presence (grey) or absence (black) of GA₃ (10 µM) by transient expression in tobacco leaves after Agrobacterium infiltration. Data are expressed relative to the minimal untreated promoter (p200). Data with different letters indicate a significant difference (Kruskall–Wallis test P < 0.05). (This figure is available in colour at JXB online.)