James M. Tepperman

PIF3, a Phytochrome-Interacting Factor Necessary for Normal Photoinduced Signal Transduction, Is a Novel Basic Helix-Loop-Helix Protein

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.

Multiple transcription-factor genes are early targets of phytochrome A signaling

The phytochrome family of sensory photoreceptors directs adaptational changes in gene expression in response to environmental light signals. Using oligonucleotide microarrays to measure expression profiles in wild-type and phytochrome A (phyA) null-mutant Arabidopsis seedlings, we have shown that 10% of the genes represented on the array are regulated by phyA in response to a continuous far-red light signal. Strikingly, 44% of the genes responding to the signal within 1 h are predicted to encode multiple classes of transcriptional regulators. Together with previous data, this observation suggests that phyA may regulate seedling photomorphogenesis by direct targeting of light signals to the promoters of genes encoding a master set of diverse transcriptional regulators, responsible in turn for orchestrating the expression of multiple downstream target genes in various branches of a phyA-regulated transcriptional network.

A light-switchable gene promoter system

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.

Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light

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 far-red 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.

Definition of Early Transcriptional Circuitry Involved in Light-Induced Reversal of PIF-Imposed Repression of Photomorphogenesis in Young Arabidopsis Seedlings

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 phytochrome-interacting 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.

A Quartet of PIF bHLH Factors Provides a Transcriptionally Centered Signaling Hub That Regulates Seedling Morphogenesis through Differential Expression-Patterning of Shared Target Genes in Arabidopsis

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.

Dynamic Antagonism between Phytochromes and PIF Family Basic Helix-Loop-Helix Factors Induces Selective Reciprocal Responses to Light and Shade in a Rapidly Responsive Transcriptional Network in Arabidopsis

Genome-wide expression profiling identifies core elements of a transcriptional network that is regulated rapidly and reciprocally by light and vegetational shade signals, via a phytochrome transcription factor transduction interface. This signaling hub functions continuously to control early seedling and juvenile plant growth and development in response to the prevailing light environment. Plants respond to shade-modulated light signals via phytochrome (phy)-induced adaptive changes, termed shade avoidance. To examine the roles of Phytochrome-Interacting basic helix-loop-helix Factors, PIF1, 3, 4, and 5, in relaying such signals to the transcriptional network, we compared the shade-responsive transcriptome profiles of wild-type and quadruple pif (pifq) mutants. We identify a subset of genes, enriched in transcription factor–encoding loci, that respond rapidly to shade, in a PIF-dependent manner, and contain promoter G-box motifs, known to bind PIFs. These genes are potential direct targets of phy-PIF signaling that regulate the primary downstream transcriptional circuitry. A second subset of PIF-dependent, early response genes, lacking G-box motifs, are enriched for auxin-responsive loci, and are thus potentially indirect targets of phy-PIF signaling, mediating the rapid cell expansion induced by shade. Comparing deetiolation- and shade-responsive transcriptomes identifies another subset of G-box–containing genes that reciprocally display rapid repression and induction in response to light and shade signals. These data define a core set of transcriptional and hormonal processes that appear to be dynamically poised to react rapidly to light-environment changes via perturbations in the mutually antagonistic actions of the phys and PIFs. Comparing the responsiveness of the pifq and triple pif mutants to light and shade confirms that the PIFs act with overlapping redundancy on seedling morphogenesis and transcriptional regulation but that each PIF contributes differentially to these responses.

phyB is evolutionarily conserved and constitutively expressed in rice seedling shoots.

SummarySouthern blot analysis indicates that the rice genome contains single copies of genes encoding type A (phyA) and type B (phyB) phytochromes. We have isolated overlapping cDNA and genomic clones encoding the entire phyB polypeptide. This monocot sequence is more closely related to phyB from the dicot, Arabidopsis (73% amino acid sequence identity), than it is to the phyA gene in the rice genome (50% identity). These data support the proposal that phyA and phyB subfamilies diverged early in plant evolution and that subsequent divergence accompanied the evolution of monocots and dicots. Moreover, since rice and Arabidopsis phyB polypeptides are more closely related to one another (73% identity) than are monocot and dicot phyA sequences (63–65% identity), it appears that phyB has evolved more slowly than phyA. Sequence conservation between phyA and phyB is greatest in a central core region surrounding the chromophore attachment site, and least toward the amino-terminal and carboxy-terminal ends of the polypeptides, although hydropathy analysis suggests that the overall structure of the two phytochromes has been conserved. Gene-specific Northern blot analysis indicates that, whereas phyA is negatively regulated by phytochrome in rice seedling shoots in the manner typical of monocots, phyB is constitutively expressed irrespective of light treatment. In consequence, phyA and phyB transcripts are equally abundant in fully green tissue. Since Arabidopsis phyB mRNA levels are also unaffected by light, the present results suggest that this mode of regulation is evolutionarily conserved among phyB genes, perhaps reflecting differences in the functional roles of the different phytochrome subfamilies.

A mutually assured destruction mechanism attenuates light signaling in Arabidopsis

Emerging from the shade into the light As a growing seedling emerges into the light, it needs to shift its developmental program to grow toward the light. Signaling components that flip the switch from growth in the shade to growth in the light include phytochromes, which are sensitive to red light, and transcription factors that drive the shade-adapted pattern of development. Ni et al. now show how phosphorylation sets these signaling partners up for destruction. The signaling established by red light invokes photomorphogenesis by promoting the destruction of the photoreceptor and its signaling partner. Science, this issue p. 1160 Phytochrome and its partner transcription factor go to their doom together in response to a change in light quality. After light-induced nuclear translocation, phytochrome photoreceptors interact with and induce rapid phosphorylation and degradation of basic helix-loop-helix 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.

Combinatorial Complexity in a Transcriptionally Centered Signaling Hub in Arabidopsis

A subfamily of four Phytochrome (phy)-Interacting bHLH transcription Factors (PIFs) collectively promote skotomorphogenic development in dark-grown seedlings. This activity is reversed upon exposure to light, by photoactivated phy molecules that induce degradation of the PIFs, thereby triggering the transcriptional changes that drive a transition to photomorphogenesis. The PIFs function both redundantly and partially differentially at the morphogenic level in this process. To identify the direct targets of PIF transcriptional regulation genome-wide, we analyzed the DNA-binding sites for all four PIFs by ChIP-seq analysis, and defined the genes transcriptionally regulated by each PIF, using RNA-seq analysis of pif mutants. Despite the absence of detectable differences in DNA-binding-motif recognition between the PIFs, the data show a spectrum of regulatory patterns, ranging from single PIF dominance to equal contributions by all four. Similarly, a broad array of promoter architectures was found, ranging from single PIF-binding sites, containing single sequence motifs, through multiple PIF-binding sites, each containing one or more motifs, with each site occupied preferentially by one to multiple PIFs. Quantitative analysis of the promoter occupancy and expression level induced by each PIF revealed an intriguing pattern. Although there is no robust correlation broadly across the target-gene population, examination of individual genes that are shared targets of multiple PIFs shows a gradation in correlation from strongly positive, through uncorrelated, to negative. This finding suggests a dual-layered mechanism of transcriptional regulation, comprising both a continuum of binding-site occupancy by each PIF and a superimposed layer of local regulation that acts differentially on each PIF, to modulate its intrinsic transcriptional activation capacity at each site, in a quantitative pattern that varies between the individual PIFs from gene to gene. These findings provide a framework for probing the mechanisms by which transcription factors with overlapping direct-target genes integrate and selectively transduce signals to their target networks.