Acceleration of flowering during shade avoidance in Arabidopsis alters the balance between FLOWERING LOCUS C-mediated repression and photoperiodic induction of flowering

Abstract

The timing of the floral transition in Arabidopsis (Arabidopsis thaliana) is influenced by a number of environmental signals. Here, we have focused on acceleration of flowering in response to vegetative shade, a condition that is perceived as a decrease in the ratio of red to far-red radiation. We have investigated the contributions of several known flowering-time pathways to this acceleration. The vernalization pathway promotes flowering in response to extended cold via transcriptional repression of the floral inhibitor FLOWERING LOCUS C (FLC); we found that a low red to far-red ratio, unlike cold treatment, lessened the effects of FLC despite continued FLC expression. A low red to far-red ratio required the photoperiod-pathway genes GIGANTEA (GI) and CONSTANS (CO) to fully accelerate flowering in long days and did not promote flowering in short days. Together, these results suggest a model in which far-red enrichment can bypass FLC-mediated late flowering by shifting the balance between FLC-mediated repression and photoperiodic induction of flowering to favor the latter. The extent of this shift was dependent upon environmental parameters, such as the length of far-red exposure. At the molecular level, we found that far-red enrichment generated a phase delay in GI expression and enhanced CO expression and activity at both dawn and dusk. Finally, our analysis of the contribution of PHYTOCHROME AND FLOWERING TIME1 (PFT1) to shade-mediated rapid flowering has led us to suggest a new model for the involvement of PFT1 in light signaling.

Figure 1.

Prolonged exposure to a low R:FR ratio bypasses FLC-mediated late flowering without lowering FLC expression, but FLC blocks acceleration of flowering in response to a transient far-red exposure. A and B, Flowering time in high R:FR conditions (white bars; R:FR approximately 5) and low R:FR conditions (gray bars; R:FR approximately 0.15). Plants were grown in LD cycles (16 h of light/8 h of dark). Error bars represent sd. C, Transcript levels in 7-d-old seedlings assayed by semiquantitative RT-PCR. Seeds were germinated in continuous white light for 5 d and then either left in these high R:FR conditions for 2 d (R:FR approximately 6) or exposed to far-red-enriched light for 2 d (R:FR approximately 0.04) prior to tissue collection. UBQ, UBIQUITIN loading control. D, Flowering time under high R:FR conditions (white bars, W), low R:FR conditions (dark gray bars, FR), or conditions of 5 d of low R:FR exposure followed by a return to high R:FR exposure (light gray bars, 5 d FR). Light sources were as in A and B.

Figure 2.

The triple mutant phyB;D;E suppresses the late flowering of Col-FRI without lowering FLC expression. A, Flowering time in high R:FR conditions (white bars; R:FR approximately 5) and low R:FR conditions (gray bars; R:FR approximately 0.15). Plants were grown in LD cycles (16 h of light/8 h of dark). Error bars represent sd. B, Representative plants from A. All four plants in each panel were photographed at the same time. C, Transcript levels in 9-d-old seedlings assayed by semiquantitative RT-PCR. Seeds were germinated in continuous white light for 5 d and then either left in these high R:FR conditions for 4 d (R:FR approximately 6) or exposed to far-red-enriched light for 4 d (R:FR approximately 0.04) prior to tissue collection. UBQ, UBIQUITIN loading control.

Figure 3.

co-9 and gi-2 mutants have an attenuated flowering response to a low R:FR ratio. A, Average petiole length of the first four true leaves of 8 to 12 soil-grown plants. Seeds were germinated in continuous white light for 3 d (R:FR approximately 6), transplanted to a LD chamber (16 h of light/8 h of dark), and then either maintained in white light (white bars; R:FR approximately 5) or exposed to far-red enriched light (gray bars; R:FR approximately 0.15) for 12 d prior to petiole measurement. Error bars represent se. B, Representative plants from A, photographed on the day of petiole measurement. C and D, Flowering time in high R:FR conditions (white bars; R:FR approximately 5) and low R:FR conditions (gray bars; R:FR approximately 0.15). Plants were grown in LD cycles (16 h of light/8 h of dark). Error bars represent sd.

Figure 4.

Expression patterns of GI, CO, and FT are altered during far-red exposure. Relative transcript levels in seedlings were assayed by quantitative RT-PCR. Wild-type Col seeds were germinated in white light for 5 d (A–E; 3 d of continuous light plus 2 d of LD cycles of 16 h of light/8 h of dark) or 6 d (F; 6 d of SD cycles of 8 h of light/16 h of dark) and then either left in high R:FR cycles (bottom of schematics, gray lines; R:FR approximately 5) or shifted to low R:FR cycles (top of schematics, black lines; R:FR approximately 0.15). Tissue was collected at 4-h intervals starting at dawn on the relevant day of far-red exposure. cDNA from each time point was used to amplify both UBIQUITIN (as a loading control) and the gene of interest, and the amount of the latter was calculated relative to the former. All values were then normalized so that peak expression of the gene of interest on the 1st d of white light conditions was equal to 1. Dotted versus solid lines represent independent biological replicates. Error bars show se for three technical replicates. A, Time course of FT mRNA abundance. See text and schematic for details of light regime. B, Time course of FT mRNA abundance on the 2nd and 5th d of far-red exposure. C, Time course of FT mRNA abundance on the 3rd d of far-red exposure in Col seedlings (triangles) versus co-9 seedlings (circles). D, As in B (day 5), but for CO transcript levels. The dawn time point is repeated at the end of the time course for comparative purposes. E, As in A, but for GI transcript levels. F, Time course of GI mRNA abundance on the 2nd d of far-red exposure during SD conditions. An additional time point taken 6 h after dawn is not shown for the sake of clarity, but it also showed no difference between low and high R:FR conditions.

Figure 5.

A low R:FR ratio that is maximally effective in long days does not accelerate flowering in SD conditions. Flowering time in high R:FR conditions (white bars; R:FR approximately 5) and low R:FR conditions (gray bars; R:FR approximately 0.15). Plants were grown in SD cycles (8 h of light/16 h of dark). Error bars represent sd. Ws, Wassilewskija.

Figure 6.

pft1 suppresses the rapid flowering of phyB but only weakly suppresses the rapid flowering of phyB;D;E and does not inhibit rapid flowering in response to a low R:FR ratio. A, Flowering time in high R:FR conditions (white bars; R:FR approximately 5) and low R:FR conditions (gray bars; R:FR approximately 0.15). Plants were grown in LD cycles (16 h of light/8 h of dark). Error bars represent sd. B, Representative plants from white light-grown plants in A. All plants were photographed on the same day.

Figure 7.

Model for the acceleration of flowering in response to the removal of PHY Pfr during far-red enrichment. Reduction in Pfr levels relieves repression of the photoperiod pathway at several stages (light gray lines). Regulation of CO transcription might involve modification of the phase of GI expression. Removal of Pfr may also release a photoperiod-independent floral promotion pathway (dashed line) under certain conditions (e.g. total loss of PHYB Pfr). PFT1 negatively regulates Pfr signaling pathways, so loss of PFT1 in the pft1 mutant leads to more effective floral repression and later flowering only when there is at least some Pfr present (e.g. in the phyB mutant, which still has high PHYD and PHYE Pfr levels). Derepression of the photoperiod pathway (black lines) during far-red exposure shifts the balance toward floral promotion even in the presence of an active FRI allele and elevated FLC levels; FLC-mediated repression of flowering (dark gray lines) buffers the acceleration but is eventually bypassed.