Abstract
Changes in light quality indicative of competition for this essential resource influence plant growth and developmental transitions. Little is known about neighbor proximity-induced acceleration of reproduction. phytochrome B (phyB) senses light cues from plant competitors ultimately leading to the expression of the floral inducers FLOWERING LOCUS (FT) and TWIN SISTER of FT (TSF). Here we show that three PHYTOCHROME INTERACTING FACTOR (PIF) transcriptional regulators act directly downstream of phyB to promote expression of FT and TSF. Neighbor proximity enhances PIF accumulation towards the end of the day coinciding with enhanced floral inducer expression. We present evidence for direct PIF-mediated TSF expression. The relevance of our findings is illustrated by the prior identification of FT, TSF and PIF4 as loci underlying flowering time regulation in nature.
One Sentence Summary PIF transcription factors mediate reproductive transition in response to neighbor proximity light cues in Arabidopsis.
Plants depend on sunlight to fuel photosynthesis. Therefore, growing with potentially reduced light availability, as encountered in dense plant communities, constitutes a threat for plant growth and development. Plants perceive potential competitors because of the reflected far-red (FR) light from neighbors, resulting in reduced red (R)/FR ratio (R/FR), which leads to the conversion phytochromes (phy) photoreceptors to their inactive Pr form. In shade-intolerant plants, this triggers organ elongation to outgrowth competitors and precocious flowering (1). Accelerated flowering results from the transcriptional induction of the florigen FLOWERING LOCUS T (FT) and its close homologue TWIN SYSTER OF FT (TSF) in the vasculature (2-5), followed by their transport to the shoot apical meristem. In Arabidopsis, low R/FR ratio promotes floral transition in a photoperiod-dependent manner (6), in agreement with the attenuated low R/FR response of the photoperiodic mutant constans (co) (2, 6). PHYTOCHROME AND FLOWERING TIME 1 (PFT1) was proposed to control flowering in response to simulated shade (4), but was later shown to respond normally to low R/FR (6). Here we investigate how phytochromes mediate early flowering in response to low R/FR.
The bHLH transcription factors PHYTOCHROME-INTERACTING FACTORS (PIF) play major roles in neighbor detection responses downstream of phyB (7, 8). Enhanced PIF expression induces precocious flowering through FT and TSF in the phloem (9-11). Moreover, plants with impaired HFR1 function, a repressor of PIF activity (12), display an increased FT expression in response to low R/FR (13). Therefore, we hypothesized that PIFs might control flowering time in response to low R/FR. We scored the flowering transition of PIF loss-offunction mutants under simulated neighbor detection (fig. S1; hereafter referred to as low R/FR) and showed that PIF7 plays a prominent function in flowering transition under low R/FR (Table 1, experiment 1). In addition, mutations in PIF4 and PIF5 further enhanced the pif7 phenotype, indicating that these genes also contribute to the response (Fig. 1A; Table 1, experiment 1, significant interaction between genotype and condition p < 0.001; Fig. S2). Moreover, while pif3pif4pif5 and pif4pif5pif7 both flower slightly late in high R/FR, specifically pif4pif5pif7 flowered later than the wild type in low R/FR (Fig. S2). Next, we checked whether PIFs mediate precocious flowering of the constitutive shade-avoidance mutant phyB. Consistent with our data in low R/FR, mutations in PIF4, PIF5 and PIF7 were required to fully suppress early flowering in phyB, including in non-inductive photoperiods (Fig. 1B; Table 1, experiment 2; Fig. S3). We conclude that PIF4, PIF5 and PIF7 act genetically downstream of phyB to control low R/FR-induced flowering.
Because flowering in low R/FR depends on the growth condition and genetic background (2, 6, 14), we tested the flowering response of ft, tsf, fttsf and co. In our conditions ft and tsf mutants responded strongly to low R/FR (2), and phyBtsf flowered as phyB indicating that neither FT nor TSF alone accounted for early flowering (Fig. S4 and S5). In contrast, fttsf double mutant presented a reduced low R/FR response, similar to co (Fig. S4), confirming that FT and TSF together are required to accelerate flowering in low R/FR (2). Next, we determined whether PIFs contribute to FT and TSF transcriptional regulation in low R/FR (2, 4-6). Transcriptome data (15) showed that FT mRNA levels increased in cotyledons within 90 minutes after transfer to low R/FR, while such a rapid induction was not observed for TSF (Fig. S6). We therefore monitored FT and TSF expression in the wild type and pif4pif5pif7 for several days after transfer from high to low R/FR at ZT16, as FT and TSF expression peak at dusk (6, 16). FT and TSF expression were similar in pif4pif5pif7 and wild-type plants in high R/FR. In contrast FT and TSF up-regulation by low R/FR were strongly impaired in pif4pif5pif7 (Fig. 1C). Consistent with the importance of PIF-dependent TSF up-regulation, ftpif4pif5pif7 quadruple mutants flowered later compared to ft and similar to fttsf under low R/FR (Table 1, experiment 3; Fig. S7). In contrast, CO mRNA expression was only marginally increased by light treatments in both genotypes (Fig. 1C). These results identify PIFs as important mediators of FT and TSF-induced early flowering in response to low R/FR.
To better understand how PIFs control FT and TSF expression we investigated their temporal and spatial expression pattern. Consistent with the vascular expression of FT and TSF during floral transition (2, 17), promoter-GUS fusions showed broad PIF4 and PIF5 expression, including the leaf vasculature in seedlings and adult plants (Fig. 2A; Fig. S8). This is consistent with tissue-specific expression analysis of PIF4, PIF5 and PIF7 (18) indicating that PIF4, PIF5, PIF7, FT and TSF are all expressed in the vasculature. FT mRNA expression in the wild type displayed two strong peaks in response to low R/FR, the first early in the light period and the highest peak around dusk (Fig. S9A) (6). In contrast, there was no induction of FT expression by low R/FR in pif4pif5pif7 (Fig. S9A). TSF expression and its regulation by low R/FR and the PIFs were very similar to FT (Figure S9B). PIF7 expression showed a strong diel oscillation with a peak in the morning as previously observed for PIF4 and PIF5 (19) (Fig. S9C). However, low R/FR ratio did not affect significantly PIF7 mRNA expression throughout the day (fig. S9C) (15), suggesting that transcription regulation of PIFs alone cannot account for increased FT and TSF expression in low R/FR. Given that phyB inactivation under low R/FR stabilizes PIF4 and PIF5 proteins (7) we decided to investigate PIF protein accumulation. Using genomic HA-tagged lines driven by their own promoters (Fig. S10A-D), we observed diel protein oscillation of HA-tagged PIF4, PIF5 and PIF7 matching mRNA levels (Fig 2B, C; Fig. S11). Interestingly, PIF4 and PIF5, proteins accumulated to higher levels in low R/FR specifically toward the end of the day, correlating with FT and TSF expression (Fig 2B; Fig. S9 and S11). Such a regulation was less apparent for PIF7 (Fig 2C), however PIF7 nuclear import is induced by low R/FR (20), indicating a different mode of low R/FR regulation for this PIF.
Because TSF was shown to integrate environmental signals to influence flowering time in nature (21), we focused our analysis on PIF regulation of TSF expression. PIF4 and PIF5 preferentially bind to G-boxes (CACGTG) and PBE-boxes (CATGTG) (22, 23). Because PIF7 plays a central role in low R/FR-induced flowering and little is known about its DNA binding preference, we tested its DNA binding specificity using protein-binding microarrays. In agreement with recent DAP-seq data (24) and similar to other PIFs (22, 23), we found that PIF7 binds with high affinity to a G-box (Fig 3A). Moreover, as observed for other PIFs, among E-boxes it showed the highest affinity for the PBE-box (Fig. 3A). We identified 2 PBE-boxes in the TSF promoter located 990 and 437 bases upstream of the ATG (Fig. 3B). Interestingly, the analysis of previously published ChIP-seq data (25) revealed a high-confidence PIF4 binding peak overlapping the first PBE-box (-437) (Fig. 3B). To test whether these PBE-boxes are biologically relevant for PIF-mediated TSF expression we fused its promoter with luciferase and performed transient expression assays in Nicotiana benthamiana. Consistent with PIFs directly regulating TSF expression, PIF4 and PIF7 led to TSF expression that was almost completely abolished by a single nucleotide mutation of either 1 (-437) or both PBE-boxes (Fig. 3C; fig S12). Taken together, our data suggest that PIF4 and PIF7 directly bind to PBE-boxes at TSF promoter to induce its expression.
Our experiments identify PIF4, PIF5 and PIF7 as transcription factors acting downstream of phyB to induce flowering response to neighbor proximity through the floral inducers FT and TSF. However, our genetic data indicate that additional mechanisms contribute to this regulation. Indeed, similar to co and fttsf (fig S4), copif4pif5pif7 are still responsive to low R/FR (Table 1, experiment 4). Therefore, we identify one important flowering-time control mechanism operating in low R/FR and provide evidences for direct regulation of TSF by the PIFs. Interestingly, as previously shown for CO (2), low R/FR leads to increased PIF4 and PIF5 proteins levels (Fig. 2B-C; Fig. S11), consistent with the proposed coordinated regulation of FT and TSF expression by PIFs and CO (11) (Fig. 1, 2, 3C, 3D, table 1). The regulation we uncovered here is likely to be significant in natural environments as “florigen” genes FT and TSF as well as PIF4 were identified as genes underlying regulation of flowering time in nature (21, 26). Understanding this regulatory mechanism may also be relevant to increase yields on restricted agricultural land.
Acknowledgments
We thank Koji Goto for pFT::GUS line and co-101 seeds, Andrea Maran and Séverine Lorrain for genomic pPIF5::PIF5-3HA and pPIF4/5::GUS lines, Markus Schmid for ft-10 mutant, Prof. Hongtao Liu for pGREENII-0800 vector, Rodrigo S. Reis dual-luciferase assistance, Genomic Technologies Facility (GTF) for qPCR assistance, Adriana Arongaus and Martina Legris for critical reading of the manuscript. Work in the Fankhauser lab is funded by the University of Lausanne and grants from the Swiss National Science Foundation (n° 310030B_179558 and CRSII3_154438). VCG was supported by EMBO long-term fellowship (ALTF 293-2013).