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Stomata govern the gaseous exchange between the leaf and the external atmosphere, and their function is essential for photosynthesis and the global carbon and oxygen cycles. Rhythmic stomata movements in daily dark/light cycles prevent water loss at night and allow CO 2 uptake during the day. How the actors involved are transcriptionally regulated and how this might contribute to rhythmicity is largely unknown. Here, we show that morning stomata opening depends on the previous night period. The transcription factors PHYTOCHROME-INTERACTING FACTORS (PIFs) accumulate at the end of the night and directly induce the guard cell-specific K + channel KAT1. Remarkably, PIFs and KAT1 are required for blue light-induced stomata opening. Together, our data establish a molecular framework for daily rhythmic stomatal movements under well-watered conditions, whereby PIFs are required for accumulation of KAT1 at night, which upon activation by blue light in the morning leads to the K + intake driving stomata opening., (© 2024. The Author(s).)

Plastid-to-nucleus retrograde signalling (RS) initiated by dysfunctional chloroplasts impact photomorphogenic development. We have previously shown that the transcription factor GLK1 acts downstream of the RS regulator GUN1 in photodamaging conditions to regulate not only the well established expression of photosynthesis-associated nuclear genes (PhANGs) but also to regulate seedling morphogenesis. Specifically, the GUN1/GLK1 module inhibits the light-induced phytochrome-interacting factor (PIF)-repressed transcriptional network to suppress cotyledon development when chloroplast integrity is compromised, modulating the area exposed to potentially damaging high light. However, how the GUN1/GLK1 module inhibits photomorphogenesis upon chloroplast damage remained undefined. Here, we report the identification of BBX16 as a novel direct target of GLK1. BBX16 is induced and promotes photomorphogenesis in moderate light and is repressed via GUN1/GLK1 after chloroplast damage. Additionally, we showed that BBX16 represents a regulatory branching point downstream of GUN1/GLK1 in the regulation of PhANG expression and seedling development upon RS activation. The gun1 phenotype in lincomycin and the gun1-like phenotype of GLK1OX are markedly suppressed in gun1bbx16 and GLK1OXbbx16. This study identified BBX16 as the first member of the BBX family involved in RS, and defines a molecular bifurcation mechanism operated by GLK1/BBX16 to optimise seedling de-etiolation, and to ensure photoprotection in unfavourable light conditions., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Hypocotyl elongation relies on directional cell expansion, a process under light and circadian clock control. Under short photoperiods (SD), hypocotyl elongation in Arabidopsis thaliana follows a rhythmic pattern, a process in which circadian morning-to-midnight waves of the transcriptional repressors PSEUDO-RESPONSE REGULATORS (PRRs) jointly gate PHYTOCHROME-INTERACTING FACTOR (PIF) activity to dawn. Previously, we described CYCLING DOF FACTOR 5 (CDF5) as a target of this antagonistic PRR/PIF dynamic interplay. Under SD, PIFs induce CDF5 accumulation specifically at dawn, when it promotes the expression of positive cell elongation regulators such as YUCCA8 to induce growth. In contrast to SD, hypocotyl elongation under long days (LD) is largely reduced. Here, we examine whether CDF5 is an actor in this photoperiod specific regulation. We report that transcription of CDF5 is robustly induced in SD compared to LD, in accordance with PIFs accumulating to higher levels in SD, and in contrast to other members of the CDF family, whose expression is mainly clock regulated and have similar waveforms in SD and LD. Notably, when CDF5 was constitutively expressed under LD, CDF5 protein accumulated to levels comparable to SD but was inactive in promoting cell elongation. Similar results were observed for CDF1. Our findings indicate that both CDFs can promote cell elongation specifically in shorter photoperiods, however, their activity in LD is inhibited at the post-translational level. These data not only expand our understanding of the biological role of CDF transcription factors, but also identify a previously unrecognized regulatory layer in the photoperiodic response of hypocotyl elongation., (© 2020 Scandinavian Plant Physiology Society.)

Under photoperiodic conditions, Arabidopsis thaliana seedling growth is inhibited in long days (LDs), but promoted under the extended nights of short days (SDs). This behavior is partly implemented by phytochrome (phy)-imposed oscillations in the abundance of the growth-promoting, phy-interacting bHLH transcription factors PHY-INTERACTING FACTOR 1 (PIF1), PIF3, PIF4 and PIF5 (PIF quartet or PIFq). However, the observation that a pifq mutant is still stimulated to elongate when given a phy-inactivating end-of-day far-red pulse (EODFR), suggests that additional factors are involved in the phy-mediated suppression of growth during the subsequent dark period. Here, by combining growth-analysis of pif7 single- and higher-order mutants with gene expression analysis under SD, LD, SD-EODFR, and LD-EODFR, we show that PIF7 promotes growth during the dark hours of SD, by regulating growth-related gene expression. Interestingly, the relative contribution of PIF7 in promoting growth is stronger under EODFR, whereas PIF3 role is more important under SD, suggesting that PIF7 is a prominent target of phy-suppression. Indeed, we show that phy imposes phosphorylation and inactivation of PIF7 during the light hours in SD, and prevents full dephosphorylation during the night. This repression can be lifted with an EODFR, which correlates with increased PIF7-mediated gene expression and elongation. In addition, our results suggest that PIF7 function might involve heterodimerization with PIF3. Furthermore, our data indicate that a pifqpif7 quintuple mutant is largely insensitive to photoperiod for hypocotyl elongation. Collectively, the data suggest that PIF7, together with the PIFq, is required for the photoperiodic regulation of seasonal growth., (© 2020 Scandinavian Plant Physiology Society.)

How do plants respond to abiotic stresses such as drought, salt or cold? A new study in Arabidopsis reveals that the stress-responsive AHLs antagonize the function of the PIF transcription factors to restrict rosette growth and allow resource reallocation for stress-adaptive responses., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chloroplast biogenesis is indispensable for proper plant development and environmental acclimation. In a screen for mutants affected in photosynthesis, we identified the protein phosphatase7 - like ( pp7l ) mutant, which displayed delayed chloroplast development in cotyledons and young leaves. PP7L, PP7, and PP7-long constitute a subfamily of phosphoprotein phosphatases. PP7 is thought to transduce a blue-light signal perceived by crys and phy a that induces expression of SIGMA FACTOR5 (SIG5). We observed that, like PP7, PP7L was predominantly localized to the nucleus in Arabidopsis ( Arabidopsis thaliana ), and the pp7l phenotype was similar to that of the sig6 mutant. However, SIG6 expression was unaltered in pp7l mutants. Instead, loss of PP7L compromised translation and ribosomal RNA (rRNA) maturation in chloroplasts, pointing to a distinct mechanism influencing chloroplast development. Promoters of genes deregulated in pp7l - 1 were enriched in PHYTOCHROME-INTERACTING FACTOR (PIF)-binding motifs and the transcriptome of pp7l - 1 resembled those of pif and CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) signalosome complex ( csn ) mutants. However, pif and csn mutants, as well as cop1 , cryptochromes (cry)1 cry2 , and phytochromes (phy)A phyB mutants, do not share the pp7l photosynthesis phenotype. PhyB protein levels were elevated in pp7l mutants, but phyB overexpression plants did not resemble pp7l These results indicate that PP7L operates through a different pathway and that the control of greening and photosystem biogenesis can be separated. The lack of PP7L increased susceptibility to salt and high-light stress, whereas PP7L overexpression conferred resistance to high-light stress. Strikingly, PP7L was specifically recruited to Brassicales for the regulation of chloroplast development. This study adds another player involved in chloroplast biogenesis., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Plants coordinate their growth and development with the environment through integration of circadian clock and photosensory pathways. In Arabidopsis thaliana, rhythmic hypocotyl elongation in short days (SD) is enhanced at dawn by the basic-helix-loop-helix (bHLH) transcription factors PHYTOCHROME-INTERACTING FACTORS (PIFs) directly inducing expression of growth-related genes [1-6]. PIFs accumulate progressively during the night and are targeted for degradation by active phytochromes in the light, when growth is reduced. Although PIF proteins are also detected during the day hours [7-10], their growth-promoting activity is inhibited through unknown mechanisms. Recently, the core clock components and transcriptional repressors PSEUDO-RESPONSE REGULATORS PRR9/7/5 [11, 12], negative regulators of hypocotyl elongation [13, 14], were described to associate to G boxes [15], the DNA motifs recognized by the PIFs [16, 17], suggesting that PRR and PIF function might converge antagonistically to regulate growth. Here we report that PRR9/7/5 and PIFs physically interact and bind to the same promoter region of pre-dawn-phased, growth-related genes, and we identify the transcription factor CDF5 [18, 19] as target of this interplay. In SD, CDF5 expression is sequentially repressed from morning to dusk by PRRs and induced pre-dawn by PIFs. Consequently, CDF5 accumulates specifically at dawn, when it induces cell elongation. Our findings provide a framework for recent TIMING OF CAB EXPRESSION 1 (TOC1/PRR1) data [5, 20] and reveal that the long described circadian morning-to-midnight waves of the PRR transcriptional repressors (PRR9, PRR7, PRR5, and TOC1) [21] jointly gate PIF activity to dawn to prevent overgrowth through sequential regulation of common PIF-PRR target genes such as CDF5., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A mechanism for integrating light perception and the endogenous circadian clock is central to a plant's capacity to coordinate its growth and development with the prevailing daily light/dark cycles. Under short-day (SD) photocycles, hypocotyl elongation is maximal at dawn, being promoted by the collective activity of a quartet of transcription factors, called PIF1, PIF3, PIF4, and PIF5 (phytochrome-interacting factors). PIF protein abundance in SDs oscillates as a balance between synthesis and photoactivated-phytochrome-imposed degradation, with maximum levels accumulating at the end of the long night. Previous evidence shows that elongation under diurnal conditions (as well as in shade) is also subjected to circadian gating. However, the mechanism underlying these phenomena is incompletely understood. Here we show that the PIFs and the core clock component Timing of CAB expression 1 (TOC1) display coincident cobinding to the promoters of predawn-phased, growth-related genes under SD conditions. TOC1 interacts with the PIFs and represses their transcriptional activation activity, antagonizing PIF-induced growth. Given the dynamics of TOC1 abundance (displaying high postdusk levels that progressively decline during the long night), our data suggest that TOC1 functions to provide a direct output from the core clock that transiently constrains the growth-promoting activity of the accumulating PIFs early postdusk, thereby gating growth to predawn, when conditions for cell elongation are optimal. These findings unveil a previously unrecognized mechanism whereby a core circadian clock output signal converges immediately with the phytochrome photosensory pathway to coregulate directly the activity of the PIF transcription factors positioned at the apex of a transcriptional network that regulates a diversity of downstream morphogenic responses.

Seedlings growing under diurnal conditions display maximal growth at the end of the night in short-day (SD) photoperiods. Current evidence indicates that this behaviour involves the action of PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) together with PIF4 and PIF5, through direct regulation of growth-related genes at dawn coinciding with a PIF3 accumulation peak generated by phytochrome-imposed oscillations in protein abundance. Here, to assess how alterations in PIF3 levels impact seedling growth, the night-specific accumulation of PIF3 was modulated by releasing SD-grown seedlings into continuous light, or by exposing them to a phytochrome-inactivating end-of-day far-red pulse (EOD-FRp). The data show a strong direct correlation between PIF3 accumulation, PIF3-regulated induction of growth-related genes, and hypocotyl elongation, and suggest that growth promotion in SD conditions involves factors other than PIF3, PIF4, and PIF5. Using a pif1 mutant, evidence is provided that PIF1 also contributes to inducing hypocotyl elongation during the dark period under diurnal conditions. PIF1 displayed constitutive transcript levels in SD conditions, suggesting that phytochrome-imposed oscillations in PIF1 protein abundance determine its accumulation and action during the night, similar to PIF3 and in contrast to PIF4 and PIF5, which oscillate diurnally due to a combination of circadian clock-regulated transcription and light control of protein accumulation. Furthermore, using single and higher order pif mutants, the relative contribution of each member of the PIF quartet to the regulation of morphogenesis and the expression of selected growth marker genes under SD conditions, or under SD conditions supplemented with an EOD-FRp, is defined. Collectively, the data indicate that PIF1, PIF3, PIF4, and PIF5 act together to promote and optimize growth under photoperiodic conditions., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Phytochrome-interacting factors (PIFs) are members of the Arabidopsis thaliana basic helix-loop-helix family of transcriptional regulators that interact specifically with the active Pfr conformer of phytochrome (phy) photoreceptors. PIFs are central regulators of photomorphogenic development that act to promote stem growth, and this activity is reversed upon interaction with phy in response to light. Recently, significant progress has been made in defining the transcriptional networks directly regulated by PIFs, as well as the convergence of other signaling pathways on the PIFs to modulate growth. Here, we summarize and highlight these findings in the context of PIFs acting as integrators of light and other signals. We discuss progress in our understanding of the transcriptional and posttranslational regulation of PIFs that illustrates the integration of light with hormonal pathways and the circadian clock, and we review seedling hypocotyl growth as a paradigm of PIFs acting at the interface of these signals. Based on these advances, PIFs are emerging as required factors for growth, acting as central components of a regulatory node that integrates multiple internal and external signals to optimize plant development.

Phytochromes (phy) C, D and E are involved in the regulation of red/far-red light-induced photomorphogenesis of Arabidopsis thaliana, but only limited data are available on the mode of action and biological function of these lesser studied phytochrome species. We fused N-terminal fragments or full-length PHYC, D and E to YELLOW FLUORESCENT PROTEIN (YFP), and analyzed the function, stability and intracellular distribution of these fusion proteins in planta. The activity of the constitutively nuclear-localized homodimers of N-terminal fragments was comparable with that of full-length PHYC, D, E-YFP, and resulted in the regulation of various red light-induced photomorphogenic responses in the studied genetic backgrounds. PHYE-YFP was active in the absence of phyB and phyD, and PHYE-YFP controlled responses, as well as accumulation, of the fusion protein in the nuclei, was saturated at low fluence rates of red light and did not require functional FAR-RED ELONGATED HYPOCOTYL1 (FHY-1) and FHY-1-like proteins. Our data suggest that PHYC-YFP, PHYD-YFP and PHYE-YFP fusion proteins, as well as their truncated N-terminal derivatives, are biologically active in the modulation of red light-regulated photomorphogenesis. We propose that PHYE-YFP can function as a homodimer and that low-fluence red light-induced translocation of phyE and phyA into the nuclei is mediated by different molecular mechanisms., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Arabidopsis seedlings display rhythmic growth when grown under diurnal conditions, with maximal elongation rates occurring at the end of the night under short-day photoperiods. Current evidence indicates that this behavior involves the action of the growth-promoting bHLH factors PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) at the end of the night, through a coincidence mechanism that combines their transcriptional regulation by the circadian clock with control of protein accumulation by light. To assess the possible role of PIF3 in this process, we have analyzed hypocotyl responses and marker gene expression in pif single- and higher-order mutants. The data show that PIF3 plays a prominent role as a promoter of seedling growth under diurnal light/dark conditions, in conjunction with PIF4 and PIF5. In addition, we provide evidence that PIF3 functions in this process through its intrinsic transcriptional regulatory activity, at least in part by directly targeting growth-related genes, and independently of its ability to regulate phytochrome B (phyB) levels. Furthermore, in sharp contrast to PIF4 and PIF5, our data show that the PIF3 gene is not subject to transcriptional regulation by the clock, but that PIF3 protein abundance oscillates under diurnal conditions as a result of a progressive decline in PIF3 protein degradation mediated by photoactivated phyB, and consequent accumulation of the bHLH factor during the dark period. Collectively, the data suggest that phyB-mediated, post-translational regulation allows PIF3 accumulation to peak just before dawn, at which time it accelerates hypocotyl growth, together with PIF4 and PIF5, by directly regulating the induction of growth-related genes., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

The reversibly red (R)/far-red (FR)-light-responsive phytochrome (phy) photosensory system initiates both the deetiolation process in dark-germinated seedlings upon first exposure to light, and the shade-avoidance process in fully deetiolated seedlings upon exposure to vegetational shade. The intracellular signaling pathway from the light-activated photoreceptor conformer (Pfr) to the transcriptional network that drives these responses involves direct, physical interaction of Pfr with a small subfamily of bHLH transcription factors, termed Phy-Interacting Factors (PIFs), which induces rapid PIF proteolytic degradation. In addition, there is evidence of further complexity in light-grown seedlings, whereby phyB-PIF interaction reciprocally induces phyB degradation, in a mutually-negative, feedback-loop configuration. Here, to assess the relative contributions of these antagonistic activities to the net phenotypic readout in light-grown seedlings, we have examined the magnitude of the light- and simulated-shade-induced responses of a pentuple phyBpif1pif3pif4pif5 (phyBpifq) mutant and various multiple pif-mutant combinations. The data (1) reaffirm that phyB is the predominant, if not exclusive, photoreceptor imposing the inhibition of hypocotyl elongation in deetiolating seedlings in response to prolonged continuous R irradiation and (2) show that the PIF quartet (PIF1, PIF3, PIF4, and PIF5) retain and exert a dual capacity to modulate hypocotyl elongation under these conditions, by concomitantly promoting cell elongation through intrinsic transcriptional-regulatory activity, and reducing phyB-inhibitory capacity through feedback-loop-induced phyB degradation. In shade-exposed seedlings, immunoblot analysis shows that the shade-imposed reduction in Pfr levels induces increases in the abundance of PIF3, and mutant analysis indicates that PIF3 acts, in conjunction with PIF4 and PIF5, to promote the known shade-induced acceleration of hypocotyl elongation. Conversely, although the quadruple pifq mutant displays clearly reduced hypocotyl elongation compared to wild-type in response to prolonged shade, immunoblot analysis detects no elevation in phyB levels in the mutant seedlings compared to the wild-type during the majority of the shade-induced growth period, and phyB levels are not robustly correlated with the growth phenotype across the pif-mutant combinations compared. These results suggest that PIF feedback modulation of phyB abundance does not play a dominant role in modulating the magnitude of the PIF-promoted, shade-responsive phenotype under these conditions. In seedlings grown under diurnal light-dark cycles, the data show that FR-pulse-induced removal of Pfr at the beginning of the dark period (End-of-Day-FR (EOD-FR) treatment) results in longer hypocotyls relative to no EOD-FR treatment and that this effect is attenuated in the pif-mutant combinations tested. This result similarly indicates that the PIF quartet members are capable of intrinsically promoting hypocotyl cell elongation in light-grown plants, independently of the effects of PIF feedback modulation of photoactivated-phyB abundance.

Plants need to accurately adjust their development after germination in the underground darkness to ensure survival of the seedling, both in the dark and in the light upon reaching the soil surface. Recent studies have established that the photoreceptors phytochromes and the bHLH phytochrome interacting factors PIFs regulate seedling development to adjust it to the prevailing light environment during post-germinative growth. However, complete understanding of the downstream regulatory network implementing these developmental responses is still lacking. In a recent work, published in The Plant Cell, we report a subset of PIF3-regulated genes in dark-grown seedlings that we have named MIDAs (MISREGULATED IN DARK). Analysis of their functional relevance using mutants showed that four of them present phenotypic alterations in the dark, and that each affected a particular facet of seedling development, suggesting organ-specific branching in the signal that PIF3 relays downstream. Furthermore, our results also showed an altered response to light in seedlings with an impaired PIF3/MIDA regulatory network, indicating that these factors might also be essential to initiate and optimize the developmental adjustment of the seedling to the 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.

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.

Background: An important contributing factor to the success of terrestrial flowering plants in colonizing the land was the evolution of a developmental strategy, termed skotomorphogenesis, whereby postgerminative seedlings emerging from buried seed grow vigorously upward in the subterranean darkness toward the soil surface., Results: Here we provide genetic evidence that a central component of the mechanism underlying this strategy is the collective repression of premature photomorphogenic development in dark-grown seedlings by several members of the phytochrome (phy)-interacting factor (PIF) subfamily of bHLH transcription factors (PIF1, PIF3, PIF4, and PIF5). Conversely, evidence presented here and elsewhere collectively indicates that a significant component of the mechanism by which light initiates photomorphogenesis upon first exposure of dark-grown seedlings to irradiation involves reversal of this repression by rapid reduction in the abundance of these PIF proteins, through degradation induced by direct interaction of the photoactivated phy molecule with the transcription factors., Conclusions: We conclude that bHLH transcription factors PIF1, PIF3, PIF4, and PIF5 act as constitutive repressors of photomorphogenesis in the dark, action that is rapidly abrogated upon light exposure by phy-induced proteolytic degradation of these PIFs, allowing the initiation of photomorphogenesis to occur.

We show that a previously uncharacterized Arabidopsis thaliana basic helix-loop-helix (bHLH) phytochrome interacting factor (PIF), designated PIF7, interacts specifically with the far-red light-absorbing Pfr form of phyB through a conserved domain called the active phyB binding motif. Similar to PIF3, upon light exposure, PIF7 rapidly migrates to intranuclear speckles, where it colocalizes with phyB. However, in striking contrast to PIF3, this process is not accompanied by detectable light-induced phosphorylation or degradation of PIF7, suggesting that the consequences of interaction with photoactivated phyB may differ among PIFs. Nevertheless, PIF7 acts similarly to PIF3 in prolonged red light as a weak negative regulator of phyB-mediated seedling deetiolation. Examination of pif3, pif4, and pif7 double mutant combinations shows that their moderate hypersensitivity to extended red light is additive. We provide evidence that the mechanism by which these PIFs operate on the phyB signaling pathway under prolonged red light is through maintaining low phyB protein levels, in an additive or synergistic manner, via a process likely involving the proteasome pathway. These data suggest that the role of these phyB-interacting bHLH factors in modulating seedling deetiolation in prolonged red light may not be as phy-activated signaling intermediates, as proposed previously, but as direct modulators of the abundance of the photoreceptor.

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.

Studies with mutants in four members of the five-membered Arabidopsis phytochrome (phy) family (phyA, phyB, phyD, and phyE) have revealed differential photosensory and/or physiological functions among them, but identification of a phyC mutant has proven elusive. We now report the isolation of multiple phyC mutant alleles using reverse-genetics strategies. Molecular analysis shows that these mutants have undetectable levels of phyC protein, suggesting that they are null for the photoreceptor. phyC mutant seedlings were indistinguishable from wild-type seedlings under constant far-red light (FRc), and phyC deficiency had no effect in the phyA mutant background under FRc, suggesting that phyC does not participate in the control of seedling deetiolation under FRc. However, when grown under constant red light (Rc), phyC seedlings exhibited a partial loss of sensitivity, observable as longer hypocotyls and smaller cotyledons than those seen in the wild type. Although less severe, this phenotype resembles the effect of phyB mutations on photoresponsiveness, indicating that both photoreceptors function in regulating seedling deetiolation in response to Rc. On the other hand, phyB phyC double mutants did not show any apparent decrease in sensitivity to Rc compared with phyB seedlings, indicating that the phyC mutation in the phyB-deficient background does not have an additive effect. These results suggest that phyB is necessary for phyC function. This functional dependence correlates with constitutively lower levels of phyC observed in the phyB mutant compared with the wild type, a decrease that seems to be regulated post-transcriptionally. phyC mutants flowered early when grown in short-day photoperiods, indicating that phyC plays a role in the perception of daylength. phyB phyC double mutant plants flowered similarly to phyB plants, indicating that in the phyB background, phyC deficiency does not further accelerate flowering. Under long-day photoperiods, phyA phyC double mutant plants flowered later than phyA plants, suggesting that phyC is able to promote flowering in the absence of phyA. Together, these results suggest that phyC is involved in photomorphogenesis throughout the life cycle of the plant, with a photosensory specificity similar to that of phyB/D/E and with a complex pattern of differential crosstalk with phyA and phyB in the photoregulation of multiple developmental processes.

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.