Transcription factor PIF4 controls the thermosensory activation of flowering

Abstract

Plant growth and development are strongly affected by small differences in temperature. Current climate change has already altered global plant phenology and distribution, and projected increases in temperature pose a significant challenge to agriculture. Despite the important role of temperature on plant development, the underlying pathways are unknown. It has previously been shown that thermal acceleration of flowering is dependent on the florigen, FLOWERING LOCUS T (FT). How this occurs is, however, not understood, because the major pathway known to upregulate FT, the photoperiod pathway, is not required for thermal acceleration of flowering. Here we demonstrate a direct mechanism by which increasing temperature causes the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) to activate FT. Our findings provide a new understanding of how plants control their timing of reproduction in response to temperature. Flowering time is an important trait in crops as well as affecting the life cycles of pollinator species. A molecular understanding of how temperature affects flowering will be important for mitigating the effects of climate change.

Fig. 1. PIF4 is necessary for the thermal induction of flowering in short photoperiods

(a) pif4-101 plants do not show acceleration of flowering at 27 °C compared to Col-0. Inset shows a 16 week old pif4-101 plant grown at 27 °C. (b) rosette leaf numbers at flowering for Col-0 and pif4-101 grown at 22 °C and 27 °C in short photoperiod conditions (error bars are +/− SD, n=6). (c) FT expression as measured by Q-PCR in 4 week old plants at 22 °C and 27 °C under short photoperiods in a PIF4 dependent manner. (data from 3 biological replicates, Error bars are +/− SD) (d) 35S::PIF4 overexpression triggers very early flowering. (e) Leaf numbers at flowering for Col-0 and 35S::PIF4 in long photoperiods (error bars are +/− SD, n=5). (f) CO and FT gene expression data measured by Q-PCR in Col-0 and 35S::PIF4 at 21 °C in long photoperiods (Samples taken 2 weeks after sowing; data from 3 biological replicates. All error bars are +/− SD). (g) FT is required for the early flowering phenotype of 35S::PIF4 plants. When crossed into the ft-3 background, the early flowering of 35S::PIF4 is completely suppressed. Inset is a top view of the 35S::PIF4 ft-3 plant showing that petiole elongation growth is retained. (h) Flowering time data for Col-0, Ler, 35S::PIF4, ft-3, and 35S::PIF4 ft-3 plants (error bars are +/− SD, n=5).

Fig. 2. Regulation of PIF4 by temperature

(a) Trancriptional regulation of PIF4 by temperature. 10 day-old Col-0 seedlings grown at 12, 17, 22 and 27 °C under short photoperiods were analyzed for PIF4 expression by Q-PCR. Data shown is from three biological replicates. Error bars are +/− SD. (b) Flowering phenotype of 35S::PIF4 is temperature dependent. While 35S::PIF4 plants (right) flower very early at 22 °C (upper panel) as compared to Col-0 (left), this phenotype is largely suppressed by growth at 12 °C (lower panel). (c) PIF4 protein levels in 35S::PIF4 plants are not affected by growth temperature. Seven day old 35S::PIF4:HA seedlings grown at 17 °C were transferred to 12, 17, 22 and 27 °C under short photoperiods for 2 days and samples were collected at the end of night (EON) before light and after 4 hours under illumination. While PIF4:HA protein levels are independent of growth temperatures, the protein is robustly degraded in presence of light. PIF4:HA protein was detected by HRP conjugated anti-HA antibody. Stained lower half of the gel used for immunoblot is shown as loading control.

Fig. 3. PIF4 directly binds the FT promoter in a temperature dependent manner

(a) GUS histochemical analysis of the expression domains of FT and PIF4 in rosette leaves. (b) ChIP analysis shows PIF4 binding to the FT locus in vivo in seedlings. The At5g45280 promoter is a positive control for PIF4 binding activity, the HSP70 promoter has been used as a negative control. (c) Schematic summarising structure of the FT promoter and positioning of Q-PCR amplicons for ChIP analysis. (d) ChIP analysis of 35S::PIF4:HA at 12 °C and 27 °C (2 week old seedlings, short photoperiods). (e) ChIP analysis of PIF4::PIF4:ProA at 17, 22 and 27 °C (4 week old soil-grown plants, short photoperiods). (f) Analysis of H2A.Z occupancy at the FT locus at 17 °C and 27 °C (3 week old plate grown plants, short photoperiods). (g) ChIP analysis of PIF4 binding to FT in Col-0 and arp6-1 (3 week old soil-grown plants, 22 °C short photoperiod). (For all ChIP experiments, plant materials were collected at the end of dark period before lights come ON and were protected from light until frozen. All data presented are from two independent ChIP experiments; all error bars are +/− SD).

Fig. 4. PIF4 integrates environmental signals

(a) Suppression of flowering at 12 °C is significantly repressed in the absence of DELLA mediated repression. (b) Leaf number at flowering for della global is reduced compared to Ler at 12 °C (error bars are +/− SD, n=6). (c) A schematic representation of temperature dependent FT regulation by PIF4. Temperature induced H2A.Z nucleosome dynamics can regulate PIF4 binding to target loci for transcriptional activation.