Mechanistic duality of transcription factor function in phytochrome signaling

Abstract

The phytochrome (phy) family of sensory photoreceptors (phyA-E in Arabidopsis) elicit changes in gene expression after light-induced migration to the nucleus, where they interact with basic helix-loop-helix transcription factors, such as phytochrome-interacting factor 3 (PIF3). The mechanism by which PIF3 relays phy signals, both early after initial light exposure and later during long-term irradiation, is not understood. Using transgenically expressed PIF3 variants, carrying site-specific amino acid substitutions that block the protein from binding either to DNA, phyA, and/or phyB, we examined the involvement of PIF3 in early, phy-induced marker gene expression and in modulating long-term, phy-imposed inhibition of hypocotyl cell elongation under prolonged, continuous irradiation. We describe an unanticipated dual mechanism of PIF3 action that involves the temporal uncoupling of its two most central molecular functions. We find that in early signaling, PIF3 acts positively as a transcription factor, exclusively requiring its DNA-binding capacity. Contrary to previous proposals, PIF3 functions as a constitutive coactivator in this process, without the need for phy binding and subsequent phy-induced modifications. This finding implies that another factor(s) is conditionally activated by phy and functions in concert with PIF3, to induce target gene transcription. In contrast, during long-term irradiations, PIF3 acts exclusively through its phyB-interacting capacity to control hypocotyl cell elongation, independently of its ability to bind DNA. Unexpectedly, PIF3 uses this capacity to regulate phyB protein abundance (and thereby global photosensory sensitivity) to modulate this long-term response rather than participating directly in the transduction chain as a signaling intermediate.

Fig. 1.

PIF3 acts as a constitutive transcription factor in ELIP2 induction independently of phyA or phyB interaction but requiring DNA association. (A) Schematics and terminology for pif3::YFP:PIF3 transgenic pif3-3 rescue constructs. (B) Schematics and terminology for genomic HA:PIF3 transgenic pif3-3 rescue constructs. (C) ELIP2 induction at 1 h requires PIF3 DNA but not phyB binding. Four-day dark-grown seedlings were maintained in the dark (D) or exposed for 1 h to Rc at 9 μmol m⁻² s⁻¹ (R). Total RNA was extracted and probed for ELIP2 and rehybridized for 18S rRNA as a loading control. HA:WT-PIF3 caused overexpression of ELIP2 at 1 h, consistent with the higher PIF3 levels in these lines compared with Col WT seedlings (SI Fig. 7). The numbers 1 and 2 refer to independent transgenic lines 1 and 2. (D) ELIP2 induction at 1 h Rc does not require binding of PIF3 to phyA. Treatments were as in C. (E) ELIP2 induction at 1 h FRc does not require binding of PIF3 to phyA. Treatments were as in C, except that FR seedlings were exposed for 1 h to FRc at 15 μmol m⁻² s⁻¹. (F) ELIP2 induction at 1 h Rc does not require PIF3 binding to phyA or phyB. Treatments of Col, pif3, YFP:WT-PIF3, or YFP:PIF3mAPAmAPB seedlings were as in C. For quantification of blots in C–F see SI Fig. 9A. (G) PIF3 is a transcriptional activator with maximal activity in darkness. Four-day-old dark-grown Col WT seedlings were bombarded with effector constructs expressing a GBD:PIF3 fusion (GBD:PIF3), GBD:AD fusion, or GBD alone. Seedlings were treated for 15 min with FR light and then kept in darkness for further 18 h (D) or treated every 2 h with 5-min R light pulses (R). Each column represents the mean of four biological replicates, error bars denote SE. Fold activation represents fold increase in the photon count of the firefly versus Renilla luciferases of GBD effector alone, which is set to 1. GBD, Gal4 DNA-binding domain; AD, Gal4 activation domain. Schematics of constructs used here can be found in SI Fig. 15.

Fig. 2.

phy-induced degradation of PIF3 acts as a timing gate on PIF3 constitutive transcriptional activity. (A) ELIP2 mRNA persists after induction peak in prolonged Rc in YFP:PIF3mAPAmAPB lines. Four-day-old dark-grown Col, pif3, or YFP:WT-PIF3, YFP:PIF3mbHLH, or YFP:PIF3mAPAmAPB seedlings were maintained in dark (0) or exposed to Rc at 9 μmol m⁻² s⁻¹ for 1, 12, or 16 h. RNA processing was as in Fig. 1 C–F. (B) Quantification of ELIP2 mRNA levels under prolonged Rc. Results shown are from triplicate RNA blot data as in A. Error bars represent SE. (C) YFP:PIF3mAPAmAPB protein levels persist in prolonged Rc. Four-day-old dark-grown pif3, YFP:WT-PIF3, or YFP:PIF3mAPAmAPB seedlings were kept in darkness (0) or exposed to Rc at 9 μmol m⁻² s⁻¹ for 1 or 16 h. Direct protein extracts were probed for YFP:PIF3 fusion proteins by using purified PIF3 antiserum.

Fig. 3.

PIF3 interaction with phyB but not with DNA is required for control of cell elongation responses under long-term Rc irradiation. (A) Visible phenotype of 4-day dark-grown genomic-HA:PIF3 transgenic seedlings. Shown are Col wild-type (Col), parental pif3-3 mutant, and one representative transgenic line for each construct (see Fig. 1B) (additional independent transgenic lines are shown in SI Fig. 12). Col denotes the parental wild type of pif3. (B) Visible seedling phenotypes of the same lines as in A grown for 4 days in Rc at 9 μmol m⁻² s⁻¹. (C) Hypocotyl lengths of genomic-HA:PIF3 transgenic seedlings after 4-day growth in darkness or Rc at 9 μmol m⁻² s⁻¹. Data are for the same genotypes as in A. The horizontal dotted line denotes hypocotyl length of the pif3-3 parental line. Error bars denote SE. (D) Hypocotyl lengths of PIF3::YFP:PIF3 transgenic seedlings. Growth and light treatments were as in C. Data are for the Col wild-type, parental pif3-3 mutant, and one representative transgenic line for each construct (see Fig. 1A) (additional independent transgenic lines are shown in SI Fig. 12). The horizontal dotted line denotes hypocotyl length of the pif3-3 parental line. Error bars denote SE.

Fig. 4.

Under long-term, Rc, PIF3 acts to regulate phyB protein levels in an APB-dependent manner. (A) PIF3 affects phyB levels in 4-day Rc-grown plants through the APB. pif3, Col, HA:WT-PIF3, or HA:PIF3mAPB seedlings were grown either 4 days in darkness (4d Dark) or 4 days under Rc at 9 μmol m⁻² s⁻¹ (4d Rc). Direct extracts were probed for phyB protein or α-tubulin with monoclonal antibodies. (B) Quantification of differences in phyA and phyB levels of 4-day, Rc-grown seedlings. Three independent lots of pif3, Col, HA:WT-PIF3, or HA:PIF3mAPB seedlings were grown, and protein extracts were probed for phyA or phyB and tubulin as a loading control. To ensure linearity of phyA, phyB, and tubulin immunoblot signals, a 2-fold extract dilution curve was run and exposed in parallel to the three replicates. Error bars represent SE. (C) Effect of PIF3 on phyB protein levels occurs late after exposure to Rc. pif3, Col, and HA:WT-PIF3 (WT-PIF3) seedlings grown 4 days in darkness were maintained in the dark (0) or exposed to 3, 6, 12, or 24 h of Rc at 9 μmol m⁻² s⁻¹. Protein extraction and phyB Western blotting were as in A. (D) PIF3 does not affect PHYB transcript levels of 4-day, Rc-grown seedlings. pif3, Col, HA:WT-PIF3, and HA:PIF3mAPB seedlings were grown for 4 days in Rc as above. Total RNA was extracted, blotted, and probed for PHYB or 18S rRNA as a loading control. (Left) One representative blot. (Right) Quantification of three independent replicates.

Fig. 5.

Model for contrasting PIF3 actions in early light-induced gene expression and long-term growth responses. In darkness, PIF3 is either resident together with protein X at a single PIF3-dependent early-response gene (PIF3-DERG) promoter, like ELIP2 (Left) or at the promoter of the PROTEIN X gene (Right). PIF3 and protein X are both required for the induction of PIF3-DERG by light, indicating that they both act positively in this process. PIF3 acts constitutively, requiring its bHLH domain. After light exposure, phys concomitantly both activate protein X transcriptional activity and induce PIF3 phosphorylation and degradation via the PIF3 APA and APB motifs, in the time window when PIF3-DERG is being induced. This action results in a transient PIF3-DERG induction profile. Under long-term irradiation conditions, the low steady-state levels of PIF3 are insufficient for maintaining high PIF3-DERG expression but act now, postranslationally, to induce phyB degradation, in a process requiring the PIF3 APB for direct association with the photoreceptor. This mechanism thus involves indirect global control of phyB output, in this case, hypocotyl growth.