Search

Plastid-to-nucleus retrograde signals emitted by dysfunctional chloroplasts impact photomorphogenic development, but the molecular link between retrograde- and photosensory-receptor signalling has remained unclear. Here, we show that the phytochrome and retrograde signalling (RS) pathways converge antagonistically to regulate the expression of the nuclear-encoded transcription factor GLK1, a key regulator of a light-induced transcriptional network central to photomorphogenesis. GLK1 gene transcription is directly repressed by PHYTOCHROME-INTERACTING FACTOR (PIF)-class bHLH transcription factors in darkness, but light-activated phytochrome reverses this activity, thereby inducing expression. Conversely, we show that retrograde signals repress this induction by a mechanism independent of PIF mediation. Collectively, our data indicate that light at moderate levels acts through the plant's nuclear-localized sensory-photoreceptor system to induce appropriate photomorphogenic development, but at excessive levels, sensed through the separate plastid-localized RS system, acts to suppress such development, thus providing a mechanism for protection against photo-oxidative damage by minimizing the tissue exposure to deleterious radiation.

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., (© The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.)

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., (Copyright © 2014, American Association for the Advancement of Science.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

The phytochrome (phy)-interacting basic helix-loop-helix transcription factors (PIFs) constitutively sustain the etiolated state of dark-germinated seedlings by actively repressing deetiolation in darkness. This action is rapidly reversed upon light exposure by phy-induced proteolytic degradation of the PIFs. Here, we combined a microarray-based approach with a functional profiling strategy and identified four PIF3-regulated genes misexpressed in the dark (MIDAs) that are novel regulators of seedling deetiolation. We provide evidence that each one of these four MIDA genes regulates a specific facet of etiolation (hook maintenance, cotyledon appression, or hypocotyl elongation), indicating that there is branching in the signaling that PIF3 relays. Furthermore, combining inferred MIDA gene function from mutant analyses with their expression profiles in response to light-induced degradation of PIF3 provides evidence consistent with a model where the action of the PIF3/MIDA regulatory network enables an initial fast response to the light and subsequently prevents an overresponse to the initial light trigger, thus optimizing the seedling deetiolation process. Collectively, the data suggest that at least part of the phy/PIF system acts through these four MIDAs to initiate and optimize seedling deetiolation, and that this mechanism might allow the implementation of spatial (i.e., organ-specific) and temporal responses during the photomorphogenic program.

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.

Contrary to expectations based on the visible phenotypic behavior of seedlings undergoing de-etiolation in response to continuous red light (Rc), previous gene expression profiling showed that one or more of the five-membered phytochrome (phy) family of Arabidopsis, other than phyB, is predominantly responsible for transducing the Rc signals to light-responsive genes. To begin to identify which phys are involved, and to define potential primary targets of phy signaling, we have examined the genome-wide expression profiles of genes responding to Rc within 1 h (early response genes) of initial exposure of dark-grown wild-type, phyA, phyB and phyAphyB double mutant seedlings to the light signal. The data show that phyA has a quantitatively dominant role in Rc-induced expression of these early response genes, that phyB has minimal detectable regulatory activity in the presence of phyA, but assumes a quantitatively larger role in its absence, and that phyA and phyB combined are responsible for the full extent of Rc responsiveness of 96% of these genes. No evidence was obtained of a significant role for the remaining family members, phyC, phyD or phyE, in this process. In striking contrast, Rc-imposed repression of early response gene expression remains quantitatively strong in the phyAphyB double mutant, as well as the monogenic mutants, suggesting a significant role for one or more of the other three phys in this response. Examination of the established or predicted functional roles of the early response genes indicates that genes encoding transcription factors represent the largest single category, at a frequency three times their prevalence genome-wide. This dominance is particularly striking among those genes responding most robustly to the Rc signal, where >50% are classified as involved in transcriptional regulation, suggesting that these may have potentially primary regulatory roles at the interface between phy signaling and the light-responsive transcriptional network. Integration of the present data with those of a previous genome-scale transcriptional analysis of a pif3 mutant, suggests a complex network involving perception and transduction of inductive Rc signals by both phyA and phyB through both PIF3 and other undefined signaling partners to early response genes.

One of the key developmental processes during photomorphogenesis is the differentiation of prolamellar bodies of proplastids into thylakoid membranes containing the photosynthetic pigment-protein complexes of chloroplasts. To study the regulatory events controlling pigment-protein complex assembly, including the biosynthesis of metabolic precursors and pigment end products, etiolated Arabidopsis thaliana seedlings were irradiated with continuous red light (Rc), which led to rapid greening, or continuous far-red light (FRc), which did not result in visible greening, and subjected to analysis by oligonucleotide microarrays and targeted metabolite profiling. An analysis using BioPathAt, a bioinformatic tool that allows the visualization of post-genomic data sets directly on biochemical pathway maps, indicated that in Rc-treated seedlings mRNA expression and metabolite patterns were tightly correlated (e.g., Calvin cycle, biosynthesis of chlorophylls, carotenoids, isoprenoid quinones, thylakoid lipids, sterols, and amino acids). K-means clustering revealed that gene expression patterns across various biochemical pathways were very similar in Rc- and FRc-treated seedlings (despite the visible phenotypic differences), whereas a principal component analysis of metabolite pools allowed a clear distinction between both treatments (in accordance with the visible phenotype). Our results illustrate the general importance of integrative approaches to correlate post-genomic data sets with phenotypic outcomes.

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.

Different Arabidopsis phytochrome (phy) family members (phyA through phyE) display differential photosensory and/or physiological functions in regulating growth and developmental responses to light signals. To identify the genes regulated by phyB in response to continuous monochromatic red light (Rc) during the induction of seedling de-etiolation, we have performed time-course, microarray-based expression profiling of wild type (WT) and phyB null mutants. Comparison of the observed expression patterns with those induced by continuous monochromatic far-red light (FRc; perceived exclusively by phyA) in WT and phyA null-mutant seedlings suggests early convergence of the FRc and Rc photosensory pathways to control a largely common transcriptional network. phyB mutant seedlings retain a surprisingly high level of responsiveness to Rc for the majority of Rc-regulated genes on the microarray, indicating that one or more other phys have a major role in regulating their expression. Combined with the robust visible morphogenic phenotype of the phyB mutant in Rc, these data suggest that different members of the phy family act in organ-specific fashion in regulating seedling de-etiolation. Specifically, phyB appears to be the dominant, if not exclusive, photoreceptor in regulating a minority population of genes involved in suppression of hypocotyl cell elongation in response to Rc signals. By contrast, this sensory function is apparently shared by one or more other phys in regulating the majority Rc-responsive gene set involved in other important facets of the de-etiolation process in the apical region, such as cotyledon cell expansion.

The signaling pathways by which the phytochrome (phy) family of photoreceptors transmits sensory information to light-regulated genes remain to be fully defined. Evidence for a relatively direct pathway has been provided by the binding of one member of the family, phyB, to a promoter-element-bound, basic helix-loop-helix protein, PIF3, specifically upon light-induced conversion of the photoreceptor molecule to its biologically active conformer (Pfr). Here, we show that phyA also binds selectively and reversibly to PIF3 upon photoconversion to Pfr, but that the apparent affinity of PIF3 for phyA is 10-fold lower than for phyB. This result is consistent with previous in vivo data from PIF3-deficient Arabidopsis, indicating that PIF3 has a major role in phyB signaling, but a more minor role in phyA signaling. We also show that phyB binds stoichiometrically to PIF3 at an equimolar ratio, suggesting that the resultant complex is the unit active in transcriptional regulation at target promoters. Deletion mapping suggests that a 37-aa segment present at the N terminus of phyB, but absent from phyA, contributes strongly to the high binding affinity of phyB for PIF3. Conversely, deletion mapping and point mutation analysis of PIF3 for determinants involved in recognition of phyB indicates that the PAS domain of PIF3 is a major contributor to this interaction, but that a second determinant in the C-terminal domain is also necessary.

In a genetic screen of available T-DNA-mutagenized Arabidopsis populations for loci potentially involved in phytochrome (phy) signaling, we identified a mutant that displayed reduced seedling deetiolation under continuous red light, but little if any change in responsiveness to continuous far-red light. This behavior suggests disruption of phyB, but not phyA signaling. We have cloned the mutant locus by using the T-DNA insertion and found that the disrupted gene is identical to the recently described GIGANTEA (GI) gene identified as being involved in control of flowering time. The encoded GI polypeptide has no sequence similarity to any known proteins in the database. However, by using beta-glucuronidase-GI and green fluorescent protein-GI fusion constructs, we have shown that GI is constitutively targeted to the nucleus in transient transfection assays. Optical sectioning by using the green fluorescent protein-GI fusion protein showed green fluorescence throughout the nucleoplasm. Thus, contrary to previous computer-based predictions that GI would be an integral plasma membrane-localized polypeptide, the data here indicate that it is a nucleoplasmically localized protein. This result is consistent with the proposed role in phyB signaling, given recent evidence that early phy signaling events are nuclear localized.

The five members of the phytochrome photoreceptor family of Arabidopsis thaliana control morphogenesis differentially in response to light. Genetic analysis has identified a signaling pathway that is specifically activated by phytochrome A. A component in this pathway, SPA1 (for "suppressor of phyA-105"), functions in repression of photomorphogenesis and is required for normal photosensory specificity of phytochrome A. Molecular cloning of the SPA1 gene indicates that SPA1 is a WD (tryptophan-aspartic acid)-repeat protein that also shares sequence similarity with protein kinases. SPA1 can localize to the nucleus, suggesting a possible function in phytochrome A-specific regulation of gene expression.

Accumulating evidence indicates that individual members of the phytochrome family of photoreceptors have differential but interactive roles in controlling plant responses to light. To investigate possible cross-regulation of these receptors, we have identified monoclonal antibodies that specifically detect each of the five Arabidopsis phytochromes, phyA to phyE (phytochrome A holoprotein; PHYA, phytochrome A apoprotein; PHYA, phytochrome A gene; phyA, mutant allele of phytochrome A gene), on immunoblots and have used them to analyze the effects of phyA and phyB null mutations on the levels of all five family members. In phyB mutants, but not in phyA mutants, a four- to six-fold reduction in the level of phyC is observed in tissues grown either in the dark or in the light. Coordinate expression of phyB and phyC is induced in the phyB mutant background by the presence of a complementing PHYB transgene. However, in transgenic lines that overexpress phyB 15- to 20-fold, phyC is not similarly overexpressed. In these overexpressor lines, the levels of phyA, phyC, and phyD are increased two- to four-fold over normal in light-grown but not dark-grown seedlings. These observations indicate that molecular mechanisms for coordination or cross-regulation of phytochrome levels are active in Arabidopsis and have implications for the interpretation of phytochrome mutants and overexpressor lines.

hy8 long hypocotyl mutants of Arabidopsis defective in responsiveness to prolonged far-red light (the so-called "far-red high-irradiance response") are selectively deficient in functional phytochrome A. To define the molecular lesion in these mutants, we sequenced the phytochrome A gene (phyA) in lines carrying one or other of two classes of hy8 alleles. The hy8-1 and hy8-2 mutants that express no detectable phytochrome A each have a single nucleotide change that inserts a translational stop codon in the protein coding sequence. These results establish that phyA resides at the HY8 locus. The hy8-3 mutant that expresses wild-type levels of photochemically active phytochrome A has a glycine-to-glutamate missense mutation at residue 727 in the C-terminal domain of the phyA sequence. Quantitative fluence rate response analysis showed that the mutant phytochrome A molecule produced by hy8-3 exhibited no detectable regulatory activity above that of the phyA-protein-deficient hy8-2 mutant. This result indicates that glycine-727, which is invariant in all sequenced phytochromes, has a function important to the regulatory activity of phytochrome A but not to photoperception.