The mechanism by which the phytochrome (phy) photoreceptor family transduces informational light signals to photoresponsive genes is unknown. Using a yeast two-hybrid screen, we have identified a phytochrome-interacting factor, PIF3, a basic helix-loop-helix protein containing a PAS domain. PIF3 binds to wild-type C-terminal domains of both phyA and phyB, but less strongly to signaling-defective, missense mutant-containing domains. Expression of sense or antisense PIF3 sequences in transgenic Arabidopsis perturbs photoresponsiveness in a manner indicating that PIF3 functions in both phyA and phyB signaling pathways in vivo. PIF3 localized to the nucleus in transient transfection experiments, indicating a potential role in controlling gene expression. Together, the data suggest that phytochrome signaling to photoregulated genes includes a direct pathway involving physical interaction between the photoreceptor and a transcriptional regulator.

No abstract

Regulatable transgene systems providing easily controlled, conditional induction or repression of expression are indispensable tools in biomedical and agricultural research and biotechnology. Several such systems have been developed for eukaryotes. Most of these rely on the administration of either exogenous chemicals or heat shock. Despite the general success of many of these systems, the potential for problems, such as toxic, unintended, or pleiotropic effects of the inducing chemical or treatment, can impose limitations on their use. We have developed a promoter system that can be induced, rapidly and reversibly, by short pulses of light. This system is based on the known red light-induced binding of the plant photoreceptor phytochrome to the protein PIF3 and the reversal of this binding by far-red light. We show here that yeast cells expressing two chimeric proteins, a phytochrome-GAL4-DNA-binding-domain fusion and a PIF3-GAL4-activation-domain fusion, are induced by red light to express selectable or "scorable" marker genes containing promoters with a GAL4 DNA-binding site, and that this induction is rapidly abrogated by subsequent far-red light. We further show that the extent of induction can be controlled precisely by titration of the number of photons delivered to the cells by the light pulse. Thus, this system has the potential to provide rapid, noninvasive, switchable control of the expression of a desired gene to a preselected level in any suitable cell by simple exposure to a light signal.

Dark-grown seedlings exhibit skotomorphogenic development. Genetic and molecular evidence indicates that a quartet of Arabidopsis Phytochrome (phy)-Interacting bHLH Factors (PIF1, 3, 4, and 5) are critically necessary to maintaining this developmental state and that light activation of phy induces a switch to photomorphogenic development by inducing rapid degradation of the PIFs. Here, using integrated ChIP–seq and RNA–seq analyses, we have identified genes that are direct targets of PIF3 transcriptional regulation, exerted by sequence-specific binding to G-box (CACGTG) or PBE-box (CACATG) motifs in the target promoters genome-wide. In addition, expression analysis of selected genes in this set, in all triple pif-mutant combinations, provides evidence that the PIF quartet members collaborate to generate an expression pattern that is the product of a mosaic of differential transcriptional responsiveness of individual genes to the different PIFs and of differential regulatory activity of individual PIFs toward the different genes. Together with prior evidence that all four PIFs can bind to G-boxes, the data suggest that this collective activity may be exerted via shared occupancy of binding sites in target promoters.

Light signals perceived by the phytochromes induce the transition from skotomorphogenic to photomorphogenic development (deetiolation) in dark-germinated seedlings. Evidence that a quadruple mutant (pifq) lacking four phytochromeinteracting bHLH transcription factors (PIF1, 3, 4, and 5) is constitutively photomorphogenic in darkness establishes that these factors sustain the skotomorphogenic state. Moreover, photoactivated phytochromes bind to and induce rapid degradation of the PIFs, indicating that the photoreceptor reverses their constitutive activity upon light exposure, initiating photomorphogenesis. Here, to define the modes of transcriptional regulation and cellular development imposed by the PIFs, we performed expression profile and cytological analyses of pifq mutant and wild-type seedlings. Dark-grown mutant seedlings display cellular development that extensively phenocopies wild-type seedlings grown in light. Similarly, 80% of the gene expression changes elicited by the absence of the PIFs in dark-grown pifq seedlings are normally induced by prolonged light in wild-type seedlings. By comparing rapidly light-responsive genes in wild-type seedlings with those responding in darkness in the pifq mutant, we identified a subset, enriched in transcription factor-encoding genes, that are potential primary targets of PIF transcriptional regulation. Collectively, these data suggest that the transcriptional response elicited by light-induced PIF proteolysis is a major component of the mechanism by which the phytochromes pleiotropically regulate deetiolation and that at least some of the rapidly light-responsive genes may comprise a transcriptional network directly regulated by the PIF proteins.

The phytochrome (phy) family of sensory photoreceptors transduce informational light signals to selected nuclear genes, inducing plant growth and developmental responses appropriate to the environment. Existing data suggest that one signaling pathway by which this occurs involves direct, intranuclear interaction of the photoactivated phy molecule with PIF3, a basic helix-loop-helix transcription factor. Here, we provide evidence from recently identified pif3 mutant alleles that PIF3 is necessary for early chloroplast greening and rapid phy-induced expression of nuclear genes encoding chloroplast components upon first exposure of seedlings to light. Therefore, these data indicate that PIF3 functions to transduce phy signals to genes involved in a critical facet of the early seedling deetiolation process, the generation of a functional photosynthetic apparatus. When transgenically expressed GUS:PIF3 fusion protein constructs were used, we found that PIF3 protein levels are rapidly and reversibly modulated by the photoreceptor over diurnal cycles in Arabidopsis seedlings. The PIF3 protein declines rapidly to a basal steady-state level upon initial light exposure, but reaccumulates to preirradiation levels in darkness during the subsequent night period. These data suggest that PIF3 may function in early phy signaling at the dark-to-light transition, not only during initial seedling deetiolation, but daily at dawn under diurnal light-dark cycles.

The phytochrome photoreceptor family directs plant gene expression by switching between biologically inactive and active conformers in response to the sequential absorption of red and farred photons. Several intermediates that act late in the phytochrome signalling pathway have been identified, but fewer have been identified that act early in the pathway. We have cloned a nuclear basic helix-loop-helix protein, PIF3, which can bind to non-photoactive carboxy-terminal fragments of phytochromes A and B and functions in phytochrome signalling in vivo. Here we show that full-length photoactive phytochrome B binds PIF3 in vitro only upon light-induced conversion to its active form, and that photoconversion back to its inactive form causes dissociation from PIF3. We conclude that photosensory signalling by phytochrome B involves light-induced, conformer-specific recognition of the putative transcriptional regulator PIF3, providing a potential mechanism for direct photoregulation of gene expression.

Following light-induced nuclear translocation, phytochrome photoreceptors interact with and induce rapid phosphorylation and degradation of bHLH transcription factors, such as PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), to regulate gene expression. Concomitantly this interaction triggers feedback reduction of phytochrome B (phyB) levels. Light-induced phosphorylation of PIF3 is necessary for the degradation of both proteins. We report that this PIF3 phosphorylation induces, and is necessary for, recruitment of LRB (Light-Response Bric-a-Brack/Tramtrack/Broad (BTB)) E3 ubiquitin ligases to the PIF3-phyB complex. The recruited LRBs promote concurrent polyubiqutination and degradation of both PIF3 and phyB in vivo. These data reveal a linked signal-transmission and attenuation mechanism involving mutually assured destruction of the receptor and its immediate signaling partner.