Search

The phytochrome (phy)-PHYTOCHROME-INTERACTING FACTOR (PIF) sensory module perceives and transduces light signals to direct target genes (DTGs), which then drive the adaptational responses in plant growth and development appropriate to the prevailing environment. These signals include the first exposure of etiolated seedlings to sunlight upon emergence from subterranean darkness and the change in color of the light that is filtered through, or reflected from, neighboring vegetation (“shade”). Previously, we identified three broad categories of rapidly signal-responsive genes: those repressed by light and conversely induced by shade; those repressed by light, but subsequently unresponsive to shade; and those responsive to shade only. Here, we investigate the potential role of epigenetic chromatin modifications in regulating these contrasting patterns of phy-PIF module-induced expression of DTGs in Arabidopsis (Arabidopsis thaliana). Using RNA-seq and ChIP-seq to determine time-resolved profiling of transcript and histone 3 lysine 4 trimethylation (H3K4me3) levels, respectively, we show that, whereas the initial dark-to-light transition triggers a rapid, apparently temporally coincident decline of both parameters, the light-to-shade transition induces similarly rapid increases in transcript levels that precede increases in H3K4me3 levels. Together with other recent findings, these data raise the possibility that, rather than being causal in the shade-induced expression changes, H3K4me3 may function to buffer the rapidly fluctuating shade/light switching that is intrinsic to vegetational canopies under natural sunlight conditions.

O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUS’s O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing., AtACINUS is an Arabidopsis homolog of a mammalian splicing regulator and previously found to be O-GlcNAcyated. Here Bi et al. characterize the interactors and targets of AtACINUS, show it is required for development and stress responses and provide evidence that its O-glycosylation affects alternative splicing.

As sessile organisms, plants must adapt to a changing environment, sensing variations in resource availability and modifying their development in response. Light is one of the most important resources for plants, and its perception by sensory photoreceptors (e.g. phytochromes) and subsequent transduction into long-term transcriptional reprogramming have been well characterized. Chromatin changes have been shown to be involved in photomorphogenesis. However, the initial short-term transcriptional changes produced by light and what factors enable these rapid changes are not well studied. Here, we identify rapidly light-responsive, PIF (Phytochrome Interacting Factor) direct-target genes (LRP-DTGs). We found that a majority of these genes also show rapid changes in Histone 3 Lysine-9 acetylation (H3K9ac) in response to the light signal. Detailed time-course analysis of transcriptional and chromatin changes showed that, for light-repressed genes, H3K9 deacetylation parallels light-triggered transcriptional repression, while for light-induced genes, H3K9 acetylation appeared to somewhat precede light-activated transcription. However, real-time imaging of transcription elongation revealed that, in fact, H3K9 acetylation also parallels transcriptional induction. Collectively, the data raise the possibility that light-induced transcriptional and chromatin-remodeling processes are mechanistically intertwined. Histone modifying proteins involved in long term light responses do not seem to have a role in this fast response, indicating that different factors might act at different stages of the light response. This work not only advances our understanding of plant responses to light, but also unveils a system in which rapid chromatin changes in reaction to an external signal can be studied under natural conditions.

Light-environment signals, sensed by plant phytochrome photoreceptors, are transduced to target genes through direct regulation of PHYTOCHROME-INTERACTING FACTOR (PIF) transcription factor abundance and activity. Previous genome-wide DNA-binding and expression analysis has identified a set of genes that are direct targets of PIF transcriptional regulation. However, quantitative analysis of promoter occupancy versus expression level has suggested that unknown “trans factors” modulate the intrinsic transcriptional activation activity of DNA-bound PIF proteins. Here, using computational analysis of published data, we have identified PSEUDO-RESPONSE REGULATORS (PRR5 and PRR7) as displaying a high frequency of colocalization with the PIF proteins at their binding sites in the promoters of PIF Direct Target Genes (DTGs). We show that the PRRs function to suppress PIF-stimulated growth in the light and vegetative shade and that they repress the rapid PIF-induced expression of PIF-DTGs triggered by exposure to shade. The repressive action of the PRRs on both growth and DTG expression requires the PIFs, indicating direct action on PIF activity, rather than a parallel antagonistic pathway. Protein interaction assays indicate that the PRRs exert their repressive activity by binding directly to the PIF proteins in the nucleus. These findings support the conclusion that the PRRs function as direct outputs from the core circadian oscillator to regulate the expression of PIF-DTGs through modulation of PIF transcriptional activation activity, thus expanding the roles of the multifunctional PIF-signaling hub.

Light-induced ‘speckles’, or photobodies, have been long known in plants, but the mechanistic basis of their formation along with their cellular function have remained a mystery. Answers to both questions have now been provided.

O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential inArabidopsisbut have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressorFLCand mediates AS ofABH1andHAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUS’s O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in thesecandspymutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing.

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., This work was supported by Marie Curie International Reintegration Grant PIRG06‐GA‐2009‐256420 and by funds from Universitat Ramon Llull/Obra Social la Caixa (2016‐URL‐Internac‐019, 2018‐LC‐05 and 2018‐URL‐IR2nQ‐019) to P.L., by NIH (5R01GM047475‐24) and USDA ARS Current Research Information System (2030‐21000‐051‐00D) grants to P.H.Q., and by grants from the Spanish ‘Ministerio de Economía y Competitividad’ (MINECO) (BIO2012‐31672 and BIO2015‐68460‐P), by FEDER / Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación (Project Reference BIO2015‐68460‐P) and from the CERCA Programme/Generalitat de Catalunya (Project References 2014‐SGR‐1406 and 2017SGR‐718) to E.M. We acknowledge financial support by the CERCA Programme/Generalitat de Catalunya and from MINECO through the ‘Severo Ochoa Programme for Centers of Excellence in R&D’ 2016‐2019 (SEV‐2015‐0533).

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

Transcriptional activation domains (TADs) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5'-phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negative selection that can be used for both reverse-1- and 2-hybrid screens. With this method we were able to identify two distinct regions in PIF3 with transcriptional activation activity, both of which are functionally conserved in PIF1, PIF4, and PIF5. Both are collectively necessary for full PIF3 transcriptional activity, but neither is sufficient to induce transcription autonomously. We also found that the TAD appear to overlap physically with other PIF3 functions, such as phyB binding activity and consequent phosphorylation. Our protocol should provide a valuable tool for identifying, analyzing and characterizing novel TADs in eukaryotic transcription factors, and thus potentially contribute to the unraveling of the mechanism underlying transcriptional activation.

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., This work was supported by Spanish Ministerio de Ciencia e Innovación Grant BIO2009-07675, Ministerio de Economía Grant BIO2012-31672, and Generalitat de Catalunya Grant 2009-SGR-206 (to E.M.); by a Comissionat per a Universitats i Recerca del Departament d’Innovació, Universitats i Empresa Fellowship from the Generalitat de Catalunya (Beatriu de Pinós program) and by Marie Curie International Reintegration Grant PIRG06-GA-2009-256420 and Generalitat de Catalunya Grant 2014-SGR-00268 (to P.L.); by Marie Curie Career Integration Grant 10-GA-2011-304008 (to C.D.); and by NIH Grant 2R01 GM-047475 and US Department of Agriculture Agricultural Research Service Current Research Information System Grant 5335-21000-032-00D (to P.H.Q.). J.S. was supported by a Junta para la Ampliación de Estudios Predoctoral Fellowship, and C.D. was supported by a Postdoctoral Fellowship from the Center for Research in Agricultural Genomics.

SummaryEnvironmental changes strongly affect plant growth and development. Phytohormones, endogenous plant-made small molecules such as ethylene, regulate a wide range of processes throughout the lifetime of plants [1, 2]. The ability of plants to integrate external signals with endogenous regulatory pathways is vital for their survival [3, 4]. Ethylene has been found to suppress hypocotyl elongation in darkness [5] while promoting it in light [6, 7]. How ethylene regulates hypocotyl elongation in such opposite ways is largely unknown. In particular, how light modulates and even reverses the function of ethylene has yet to be characterized. Here we show that the basic-helix-loop-helix transcription factor phytochrome-interacting factor 3 (PIF3) is directly activated by ETHYLENE-INSENSITIVE 3 (EIN3) and is indispensible for ethylene-induced hypocotyl elongation in light. Ethylene via EIN3 concomitantly activates two contrasting pathways: the PIF3-dependent growth-promoting pathway and an ethylene response factor 1 (ERF1)-mediated growth-inhibiting pathway. In the light, growth-promoting PIFs are limiting due to light-dependent destabilization, and thus ethylene stimulates growth under these conditions. In contrast, ERF1 is destabilized, and thus limiting, under dark conditions, explaining why ethylene inhibits growth in the dark. Our findings provide a mechanistic insight into how light modulates internal hormone-regulated plant growth.

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., This work was supported by the “Comissionat per a Universitats i Recerca del Departament d’Innovació, Universitats i Empresa” fellowship of the Generalitat de Catalunya (Beatriu de Pinós Program) and Marie Curie International Reintegration Grant PIRG06-GA-2009-256420 to P.L., by grants from Marie Curie IRG-046568, Spanish “Ministerio de Ciencia e Innovación” BIO2006-09254 and BIO2009-07675, and Generalitat de Catalunya 2009-SGR-206 to E.M., and by National Institutes of Health Grant GM-47475, Department of Energy Grant DEFG03-87ER13742, and USDA Agricultural Research Service Current Research Information System Grant 5335-21000-027-00D to P.H.Q.

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., This work was supported by a Junta para la Ampliación de Estudios predoctoral fellowship from Consejo Superior de Investigaciones Científicas to J.S. (JaePre_08_01049), by a “Comissionat per a Universitats i Recerca del Departament d’Innovació, Universitats i Empresa” fellowship of the Generalitat de Catalunya (Beatriu de Pinós program) and Marie Curie IRG PIRG06-GA-2009-256420 grant to P.L., and by Marie Curie IRG-046568, Spanish “Ministerio de Ciencia e Innovación” BIO2006-09254 and BIO2009-07675, and Generalitat de Catalunya 2009-SGR-206 grants to E.M.

A small subset of basic helix–loop–helix transcription factors called PIFs (phytochrome-interacting factors) act to repress seed germination, promote seedling skotomorphogenesis and promote shade-avoidance through regulated expression of over a thousand genes. Light-activated phytochrome molecules directly reverse these activities by inducing rapid degradation of the PIF proteins. Here, we review recent advances in dissecting this signaling pathway and examine emerging evidence that indicates that other pathways also converge to regulate PIF activity, including the gibberellin pathway, the circadian clock and high temperature. Thus PIFs have broader roles than previously appreciated, functioning as a cellular signaling hub that integrates multiple signals to orchestrate regulation of the transcriptional network that drives multiple facets of downstream morphogenesis. The relative contributions of the individual PIFs to this spectrum of regulatory functions ranges from quantitatively redundant to qualitatively distinct., This work was supported by the ‘Comissionat per a Universitats i Recerca del Departament d’Innovació, Universitats i Empresa’, of the Generalitat de Catalunya (Beatriu de Pinós program) and by Marie Curie International Reintegration Grant PIRG06-GA-2009-256420 to P. Leivar, and by National Institutes of Health Grant GM-47475, Department of Energy Grant DEFG03-87ER13742, and USDA Agricultural Research Service Current Research Information System Grant 5335-21000-027-00D to P.H. Quail.

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., This work was supported by a postdoctoral fellowship from the Spanish Ministry of Education and Science to P.L. and by National Institutes of Health Grant GM-47475, Department of Energy Grant DEFG03-87ER13742, and USDA Agricultural Research Service Current Research Information System Grant 5335-21000-027-00D to P.H.Q.

Light signals are fundamental to the growth and development of plants. Red and far-red light are sensed using the phytochrome family of plant photoreceptors. Individual phytochromes display both unique and overlapping roles throughout the life cycle of plants, regulating a range of developmental processes from seed germination to the timing of reproductive development. The evolution of multiple phytochrome photoreceptors has enhanced plant sensitivity to fluctuating light environments, diversifying phytochrome function, and facilitating conditional cross-talk with other signalling systems. The isolation of null mutants, deficient in all individual phytochromes, has greatly advanced understanding of phytochrome functions in the model species, Arabidopsis thaliana. The creation of mutants null for multiple phytochrome combinations has enabled the dissection of redundant interactions between family members, revealing novel regulatory roles for this important photoreceptor family. In this review, current knowledge of phytochrome functions in the light-regulated development of Arabidopsis is summarised.

We define the photoresponsiveness, during seedling de-etiolation, of PHYTOCHROME-INTERACTING FACTOR 3-LIKE 1 (PIL1), initially identified by microarray analysis as an early-response gene that is robustly repressed by first exposure to light. We show that PIL1 mRNA abundance declines rapidly, with a half-time of 15 min, to a new steady-state level, 10-fold below the initial dark level, within 45 min of first exposure to red light. Analysis of phy-null mutants indicates that multiple phytochromes, including phyA and phyB, impose this repression. Conversely, PIL1 expression is rapidly derepressed by subsequent far-red irradiation of previously red light-exposed seedlings. However, the magnitude of this derepression is modulated over time, in a biphasic manner, in response to increasing duration of pre-exposure to continuous red light: (i) an early phase (up to about 6 h) of relatively rapidly increasing effectiveness of far-red reversal of repression, as declining phyA levels relieve initial very low fluence suppression of this response; and (ii) a second phase (beyond 6 h) of gradually declining effectiveness of far-red reversal, to only 20% of maximal derepression, within 36 h of continuous red light exposure, with no evidence of circadian modulation of this responsiveness, an observation in striking contrast to a previous report for entrained, green seedlings exposed to vegetative shade. These data, together with analysis of phytochrome signaling mutants and overexpressors with aberrant de-etiolation phenotypes, suggest that the second-phase decline in robustness of PIL1 derepression is an indirect consequence of the global developmental transition from the etiolated to the de-etiolated state, and that circadian coupling of derepression requires entrainment.

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 (phyA–phyE in Arabidopsis thaliana) induces changes in target-gene expression upon light-induced translocation to the nucleus, where certain members interact with selected members of the constitutively nuclear basic helix-loop-helix transcription factor family, such as PHYTOCHROME-INTERACTING FACTOR3 (PIF3). Previous evidence indicates that the binding of the photoactivated photoreceptor molecule to PIF3 induces rapid phosphorylation of the transcription factor in the cell prior to its degradation via the ubiqitin-proteosome system. To investigate whether this apparent primary signaling mechanism can be generalized to other phy-interacting partners, we have examined the molecular behavior of a second related phy-interacting member of the basic helix-loop-helix family, PIF5, during early deetiolation, immediately following initial exposure of dark-grown seedlings to light. The data show that red light induces very rapid phosphorylation and subsequent degradation (t 1/2 < 5 min) of PIF5 via the proteosome system upon irradiation. Photobiological and genetic evidence indicates that the photoactivated phy molecule acts within 60 s to induce this phosphorylation of PIF5, and that phyA and phyB redundantly dominate this process, with phyD playing an apparently minor role. Collectively, the data support the proposal that the rapid phy-induced phosphorylation of PIF3 and PIF5 may represent the biochemical mechanism of primary signal transfer from photoactivated photoreceptor to binding partner, and that phyA and phyB (and possibly phyD) may signal to multiple, shared partners utilizing this common mechanism.

Identification of all genes involved in the phytochrome (phy)-mediated responses of plants to their light environment is an important goal in providing an overall understanding of light-regulated growth and development. This article highlights and integrates the central findings of two recent comprehensive studies in Arabidopsis that have identified the genome-wide set of phy-regulated genes that respond rapidly to red-light signals upon first exposure of dark-grown seedlings, and have tested the functional relevance to normal seedling photomorphogenesis of an initial subset of these genes. The data: (a) reveal considerable complexity in the channeling of the light signals through the different phy-family members (phyA to phyE) to responsive genes; (b) identify a diversity of transcription-factor-encoding genes as major early, if not primary, targets of phy signaling, and, therefore, as potentially important regulators in the transcriptional-network hierarchy; and (c) identify auxin-related genes as the dominant class among rapidly-regulated, hormone-related genes. However, reverse-genetic functional profiling of a selected subset of these genes reveals that only a limited fraction are necessary for optimal phy-induced seedling deetiolation.

DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a yeast one-hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bind to hexameric E-box (CANNTG) or N-box (CACG(A/C)G) motifs, depending on transcriptional activity. We have shown that OsPIF14 binds to the OsDREB1B promoter through two N-boxes and that the flanking regions of the hexameric core are essential for protein-DNA interaction and stability. We also showed that OsPIF14 down-regulates OsDREB1B gene expression in rice protoplasts, corroborating the OsPIF14 repressor activity observed in the transactivation assays using Arabidopsis protoplasts. In addition, we showed that OsPIF14 is indeed a phytochrome interacting factor, which preferentially binds to the active form (Pfr) of rice phytochrome B. This raises the possibility that OsPIF14 activity might be modulated by light. However, we did not observe any regulation of the OsDREB1B gene expression by light under control conditions. Moreover, OsPIF14 gene expression was shown to be modulated by different treatments, such as drought, salt, cold and ABA. Interestingly, OsPIF14 showed also a specific cold-induced alternative splicing. All together, these results suggest the possibility that OsPIF14 is involved in cross-talk between light and stress signaling through interaction with the OsDREB1B promoter. Although in the absence of stress, OsDREB1B gene expression was not regulated by light, given previous reports, it remains possible that OsPIF14 has a role in light modulation of stress responses.

In previous time-resolved microarray-based expression profiling, we identified 32 genes encoding putative transcription factors, signaling components, and unknown proteins that are rapidly and robustly induced by phytochrome (phy)-mediated light signals. Postulating that they are the most likely to be direct targets of phy signaling and to function in the primary phy regulatory circuitry, we examined the impact of targeted mutations in these genes on the phy-induced seedling deetiolation process in Arabidopsis thaliana. Using light-imposed concomitant inhibition of hypocotyl and stimulation of cotyledon growth as diagnostic criteria for normal deetiolation, we identified three major mutant response categories. Seven (22%) lines displayed statistically significant, reciprocal, aberrant photoresponsiveness in the two organs, suggesting disruption of normal deetiolation; 13 (41%) lines displayed significant defects either unidirectionally in both organs or in hypocotyls only, suggesting global effects not directly related to photomorphogenic signaling; and 12 (37%) lines displayed no significant difference in photoresponsiveness from the wild type. Potential reasons for the high proportion of rapidly light-responsive genes apparently unnecessary for the deetiolation phenotype are discussed. One of the seven disrupted genes displaying a significant mutant phenotype, the basic helix-loop-helix factor–encoding PHYTOCHROME-INTERACTING FACTOR3-LIKE1 gene, was found to be necessary for rapid light-induced expression of the photomorphogenesis- and circadian-related PSEUDO-RESPONSE REGULATOR9 gene, indicating a regulatory function in the early phy-induced transcriptional network.

*Summary Evidence has been presented that a negative transcriptional feedback loop formed by the genes CIRCADIAN CLOCK ASSOCIATED (CCA1), LATE ELONGATED HYPOCOTYL (LHY) and TIMING OF CAB (TOC1) constitutes the core of the central oscillator of the circadian clock in Arabidopsis. Here we show that these genes are expressed at constant, basal levels in dark-grown seedlings. Transfer to constant red light (Rc) rapidly induces a biphasic pattern of CCA1 and LHY expression, and a reciprocal TOC1 expression pattern over the first 24 h, consistent with initial induction of this synchronous oscillation by the light signal. We have used this assay with wild-type and mutant seedlings to examine the role of these oscillator components, and to determine the function of ELF3 and ELF4 in their light-regulated expression. The data show that whereas TOC1 is necessary for light-induced CCA1/LHY expression, the combined absence of CCA1 and LHY has little effect on the pattern of light-induced TOC1 expression, indicating that the negative regulatory arm of the proposed oscillator is not fully functional during initial seedling de-etiolation. By contrast, ELF4 is necessary for light-induced expression of both CCA1 and LHY, and conversely, CCA1 and LHY act negatively on light-induced ELF4 expression. Together with the observation that the temporal light-induced expression profile of ELF4 is counter-phased to that of CCA1 and LHY and parallels that of TOC1, these data are consistent with a previously unrecognized negative-feedback loop formed by CCA1/LHY and ELF4 in a manner analogous to the proposed CCA1/LHY/ TOC1 oscillator. ELF3 is also necessary for light-induced CCA1/LHY expression, but it is neither light-induced nor clock-regulated during de-etiolation. Taken together, the data suggest (a) that ELF3, ELF4, and TOC1 all function in the primary, phytochrome-mediated light-input pathway to the circadian oscillator in Arabidopsis; and (b) that this oscillator consists of two or more interlocking transcriptional feedback loops that may be differentially operative during initial light induction and under steady-state circadian conditions in entrained green plants.

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.

Several hundred Arabidopsis genes, transcriptionally regulated by phytochrome A (phyA), were previously identified using an oligonucleotide microarray. We have now identified, in silico, conserved sequence motifs in the promoters of these genes by comparing the promoter sequences to those of all the genes present on the microarray from which they were sampled. This was done using a Perl script (called Sift) that identifies over-represented motifs using an enumerative approach. The utility of Sift was verified by analysis of circadian-regulated promoters known to contain a biologically significant motif. Several elements were then identified in phyA-responsive promoters by their over-representation. Five previously undescribed motifs were detected in the promoters of phyA-induced genes. Four novel motifs were found in phyA-repressed promoters, plus a motif that strongly resembles the DE1 element. The G-box, CACGTG, was a prominent hit in both induced and repressed phyA-responsive promoters. Intriguingly, two distinct flanking consensus sequences were observed adjacent to the G-box core sequence: one predominating in phyA-induced promoters, the other in phyA-repressed promoters. Such different conserved flanking nucleotides around the core motif in these two sets of promoters may indicate that different members of the same family of DNA-binding proteins mediate phyA induction and repression. An increased abundance of G-box sequences was observed in the most rapidly phyA-responsive genes and in the promoters of phyA-regulated transcription factors, indicating that G-box-binding transcription factors are upstream components in a transcriptional cascade that mediates phyA-regulated development.

The basic/helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA target sites and that have been well characterized in nonplant eukaryotes as important regulatory components in diverse biological processes. Based on evidence that the bHLH protein PIF3 is a direct phytochrome reaction partner in the photoreceptor's signaling network, we have undertaken a comprehensive computational analysis of the Arabidopsis genome sequence databases to define the scope and features of the bHLH family. Using a set of criteria derived from a previously defined consensus motif, we identified 147 bHLH protein-encoding genes, making this one of the largest transcription factor families in Arabidopsis. Phylogenetic analysis of the bHLH domain sequences permits classification of these genes into 21 subfamilies. The evolutionary and potential functional relationships implied by this analysis are supported by other criteria, including the chromosomal distribution of these genes relative to duplicated genome segments, the conservation of variant exon/intron structural patterns, and the predicted DNA binding activities within subfamilies. Considerable diversity in DNA binding site specificity among family members is predicted, and marked divergence in protein sequence outside of the conserved bHLH domain is observed. Together with the established propensity of bHLH factors to engage in varying degrees of homodimerization and heterodimerization, these observations suggest that the Arabidopsis bHLH proteins have the potential to participate in an extensive set of combinatorial interactions, endowing them with the capacity to be involved in the regulation of a multiplicity of transcriptional programs. We provide evidence from yeast two-hybrid and in vitro binding assays that two related phytochrome-interacting members in the Arabidopsis family, PIF3 and PIF4, can form both homodimers and heterodimers and that all three dimeric configurations can bind specifically to the G-box DNA sequence motif CACGTG. These data are consistent, in principle, with the operation of this combinatorial mechanism in Arabidopsis.

To identify new components in the phytochrome (phy) signaling network in Arabidopsis, we used a sensitized genetic screen for deetiolation-defective seedlings. Two allelic mutants were isolated that exhibited reduced sensitivity to both continuous red and far-red light, suggesting involvement in both phyA and phyB signaling. The molecular lesions responsible for the phenotype were shown to be mutations in the Arabidopsis PSEUDO-RESPONSE REGULATOR7 (PRR7) gene. PRR7 is a member of a small gene family in Arabidopsis previously suggested to be involved in circadian rhythms. A PRR7-beta-glucuronidase fusion protein localized to the nucleus, implying a possible function in the regulation of photoresponsive gene expression. Consistent with this suggestion, prr7 seedlings were partially defective in the regulation of the rapidly light-induced genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), observable as a premature increase in expression level during the second peak of the biphasic induction profile that is elicited upon initial exposure of dark-grown seedlings to light. A similar 3- to 6-h coordinated advance in peak free-running expression of CCA1, LHY, and TIMING-OF-CAB1, which are considered to encode the molecular components of the circadian oscillator in Arabidopsis, was observed in entrained fully green prr7 seedlings compared with wild-type seedlings. Collectively, these data suggest that PRR7 functions as a signaling intermediate in the phytochrome-regulated gene expression responsible for both seedling deetiolation and phasing of the circadian clock in response to light.

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.

The phytochrome (phy) family of sensory photoreceptors (phyA to phyE in Arabidopsis) enables plants to optimize their growth and development under natural light environments. Subcellular localization studies have shown that the photoreceptor molecule is induced to translocate from cytosol to nucleus by light, but direct evidence of the functional relevance of this translocation has been lacking. Here, using a glucocorticoid receptor-based fusion protein system, we demonstrate that both photoactivation and nuclear translocation combined are necessary and sufficient for the biological function of phyB. Conversely, neither artificial nuclear translocation of non-photoactivated phyB nor artificial retention of photoactivated phyB in the cytosol provides detectable biological activity. Together these data indicate that signal transfer from photoactivated phyB to its primary signaling partner(s) is localized in the nucleus, and conversely suggest the absence of a cytosolic pathway from photoactivated phyB to light-responsive genes.

The basic/helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA target sites and that have been well characterized in nonplant eukaryotes as important regulatory components in diverse biological processes. Based on evidence that the bHLH protein PIF3 is a direct phytochrome reaction partner in the photoreceptor's signaling network, we have undertaken a comprehensive computational analysis of the Arabidopsis genome sequence databases to define the scope and features of the bHLH family. Using a set of criteria derived from a previously defined consensus motif, we identified 147 bHLH protein–encoding genes, making this one of the largest transcription factor families in Arabidopsis. Phylogenetic analysis of the bHLH domain sequences permits classification of these genes into 21 subfamilies. The evolutionary and potential functional relationships implied by this analysis are supported by other criteria, including the chromosomal distribution of these genes relative to duplicated genome segments, the conservation of variant exon/intron structural patterns, and the predicted DNA binding activities within subfamilies. Considerable diversity in DNA binding site specificity among family members is predicted, and marked divergence in protein sequence outside of the conserved bHLH domain is observed. Together with the established propensity of bHLH factors to engage in varying degrees of homodimerization and heterodimerization, these observations suggest that the Arabidopsis bHLH proteins have the potential to participate in an extensive set of combinatorial interactions, endowing them with the capacity to be involved in the regulation of a multiplicity of transcriptional programs. We provide evidence from yeast two-hybrid and in vitro binding assays that two related phytochrome-interacting members in the Arabidopsis family, PIF3 and PIF4, can form both homodimers and heterodimers and that all three dimeric configurations can bind specifically to the G-box DNA sequence motif CACGTG. These data are consistent, in principle, with the operation of this combinatorial mechanism in Arabidopsis.

Summary The Arabidopsis mutants far1 and fhy3 display a phenotype of reduced inhibition of hypocotyl elongation, which is specific to far-red light and therefore specific to the phytochrome A (phyA)-signaling pathway. We report that the proteins encoded by the FAR1 and FHY3 genes are both related to the transposases of type II MuDR family transposons. We demonstrate that the FAR1 protein is capable of activating transcription in Arabidopsis, indicating that it may define a type of transcriptional regulator. Using microarray expression analysis, we show that of 293 mRNAs twofold induced in wild-type Col-0 plants by continuous far-red light, 85% show reduced responsiveness in the fhy3 mutant. Notable alterations were observed in the responses of genes encoding certain transcription factors, proteins involved in cell wall extension, and proteins related to redox balance control. We also found genes, including some involved in transcriptional control, which showed altered transcriptional behavior in the dark-grown mutant plants. Taken together, our data suggest that FAR1 and FHY3 may function 'permissively' outside the signal transduction pathway of light- regulated development, yet be required for the expression of transcriptional regulatory components. An alternative possibility is that their role includes both light-signal transduction and transcriptional regulation of other genes not responsive to light. We propose that FAR1 and FHY3 control the expression of their target genes by a mechanism that has evolved directly from the way that an ancestral, MuDRA-like trans- posase bound to the TIRs of mobile elements.

The phytochrome family of sensory photoreceptors transduces environmental light signals to responsive nuclear genes by poorly defined pathways. The recent application of yeast two-hybrid library screens to the identification of components that physically interact with members of the phytochrome family has dramatically altered previous views of the likely intracellular signaling pathways. The evidence indicates that one pathway involves light-triggered translocation of the photoreceptor molecule from cytoplasm to nucleus where it binds specifically in its biologically active form to a promoter-bound basic helix-loop-helix protein. The phytochrome molecules are proposed to function as integral, light-switchable components of transcriptional regulator complexes targeting environmental light signals directly and instantly to specific gene promoters.

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 β−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 plasmamembrane-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.

As part of an effort to isolate new Arabidopsis mutants specifically defective in responsiveness to red light, we identified srl1 (short hypocotyl in red light) by screening an EMS-mutagenized M2 population derived from a phytochrome B (phyB)-overexpressor line (ABO). The srl1 mutant shows enhanced responsiveness to continuous red but not far-red light, in both wild-type and ABO backgrounds, consistent with involvement in the phyB-signaling pathway but not that of phyA. The hypersensitive phenotype of srl1 is not due to overexpression of endogenous phyA or phyB, and the locus maps to the center of chromosome 2, distinct from any other known photomorphogenic mutants. srl1 seedlings display enhancement of several phyB-mediated responses, including shorter hypocotyls, more expanded cotyledons, shorter petioles and modestly higher levels of CAB gene expression under red light than the wild type. Double mutant analyses show that the hypersensitive phenotype of srl1 is completely phyB-dependent. The data suggest, therefore, that SRL1 may encode a negatively acting component specific to the phyB-signaling pathway.

Although sequences representing members of the phytochrome (phy) family of photoreceptors have been reported in numerous species across the phylogenetic spectrum, relatively few phytochrome genes (PHY) have been fully characterized. Using rice, we have cloned and characterized the first PHYC gene from a monocot. Comparison of genomic and cDNA PHYC sequences shows that the rice PHYC gene contains three introns in the protein-coding region typical of most angiosperm PHY genes, in contrast to Arabidopsis PHYC, which lacks the third intron. Mapping of the transcription start site and 5′-untranslated region of the rice PHYC transcript indicates that it contains an unusually long, intronless, 5′-untranslated leader sequence of 715 bp. PHYC mRNA levels are relatively low compared to PHYA and PHYB mRNAs in rice seedlings, and are similar in dark- and light-treated seedlings, suggesting relatively low constitutive expression. Genomic mapping shows that the PHYA, PHYB, and PHYC genes are all located on chromosome 3 of rice, in synteny with these genes in linkage group C (sometimes referred to as linkage group A) of sorghum. Phylogenetic analysis indicates that rice phyC is closely related to sorghum phyC, but relatively strongly divergent from Arabidopsis phyC, the only full-length dicot phyC sequence available.

The negatively photoregulatedPHYA gene has a complex promoter structure in Arabidopsis, with three active transcription start sites. To identify the photoreceptors responsible for regulation of this gene, and to assess the relative roles of the three transcription start sites, we analyzed the changes in PHYA transcript levels in wild-type and photoreceptor mutant seedlings under various irradiation conditions. Continuous far-red or red light exposures each induced a significant decline in transcript levels in wild-type etiolated seedlings. Analysis of mutants specifically lacking either phyA or phyB protein demonstrated that these phytochromes are required for the negative regulation induced by far-red and red light, respectively. Ribonuclease protection experiments showed further that this negative regulation is confined almost exclusively to the shortest, most abundant PHYA transcript, and occurs predominantly in shoots. By contrast, both of the other minor transcripts in shoots, and all three transcripts in roots, exhibit near constitutive expression. This complex expression pattern indicates that the PHYAgene is subject to regulation by multiple signals, including environmental, developmental, and organ-specific signals.

Although Arabidopsis has numerous well documented advantages for genetic and molecular analyses, its small size can be a limitation for biochemical and immunochemical assays requiring protein extraction. We have developed a rapid method to extract total protein from small amounts of Arabidopsis tissue that can be used for quantitative immunoblot analysis. The procedure involves direct extraction of tissue into SDS-containing buffer under conditions permitting immediate protein quantification in the extract, using commercially available kits without prior fractionation. This approach provides maximal extraction and quantitative recovery of total cellular protein, together with accurate evaluation of target protein levels as a proportion of the total. We have examined the utility and sensitivity of the procedure using monoclonal antibodies to phytochromes A and C (phyA and phyC), which are high- and low-abundance members, respectively, of the phytochrome family in Arabidopsis. Both phytochromes could be rapidly and readily quantified in the tissues examined, with phyC being detectable in extracts representing as few as five dark-grown seedlings, two light-grown seedlings, or half a single leaf from 3-week-old adult plants. The data indicate that the procedure may have broad utility for the detection and quantitative analysis of many proteins, including those of low abundance, in a variety of applications in Arabidopsis. In one such application, we used transgenic Arabidopsis phyC-overexpressor seedlings to demonstrate that the procedure can be used to detect transgene-encoded protein early at the segregating T2 generation, thereby offering the capacity for accelerated screening and selection of lines engineered to overexpress target proteins.

The phytochrome family of photoreceptors has a well-defined role in regulating gene expression in response to informational light signals. Little is known, however, of the early steps of phytochrome signal transduction. Here we describe a new Arabidopsis mutant, far1 (far-red-impaired response), which has reduced responsiveness to continuous far-red light, but responds normally to other light wavelengths. This phenotype implies a specific requirement for FAR1 in phyA signal transduction. The far1 locus maps to the south arm of chromosome 4, and is not allelic to photomorphogenic loci identified previously. All five far1 alleles isolated have single nucleotide substitutions that introduce stop codons in a single ORF. The FAR1 gene encodes a protein with no significant sequence similarity to any proteins of known function. The FAR1 protein contains a predicted nuclear localization signal and is targeted to the nucleus in transient transfection assays. This result supports an emerging view that early steps in phytochrome signaling may be centered in the nucleus. The FAR1 gene defines a new multigene family, which consists of at least four genes in Arabidopsis. This observation raises the possibility of redundancy in the phyA-signaling pathway, which could account for the incomplete block of phyA signaling observed in the far1 mutant.

Transgenic potato (Solanum tuberosum) plants expressing Arabidopsis phytochrome B were characterized morphologically and physiologically under white light in a greenhouse to explore their potential for improved photosynthesis and higher tuber yields. As expected, overexpression of functional phytochrome B caused pleiotropic effects such as semidwarfism, decreased apical dominance, a higher number of smaller but thicker leaves, and increased pigmentation. Because of increased numbers of chloroplasts in elongated palisade cells, photosynthesis per leaf area and in each individual plant increased. In addition, photosynthesis was less sensitive to photoinactivation under prolonged light stress. The beginning of senescence was not delayed, but deceleration of chlorophyll degradation extended the lifetime of photosynthetically active plants. Both the higher photosynthetic performance and the longer lifespan of the transgenic plants allowed greater biomass production, resulting in extended underground organs with increased tuber yields.

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.

Summary The DNA-binding proteins PF1 and GT-2 are factors that bind to different functionally defined, positively acting cis-elements in the PHYA genes of oat and rice, respectively. PF1 is an HMG-I/Y protein, with its cognate cis-element being an AT-rich sequence, designated PE1, whereas GT-2 is a transcriptional activator with twin DNA binding domains that recognize a triplet of GTboxes in a complex motif designated GTE. To further define the DNA-binding activity of PF1 and to explore potential inter-relationships between the two factors, we have performed a series of in vitro DNA-binding experiments with both PE1 and GTE target sites. The data show that, consistent with its membership of the HMG-I/Y protein family, PF1 can bend DNA when bound to PE1. In addition, PF1 can bind promiscuously, with varying affinity, to other AT-containing motifs, including GTE. When co-incubated with GT-2, PF1 enhances the specific DNA-binding activity of GT-2 toward GTE, the first report of such activity for a plant HMG-I/Y protein. This enhancement takes place without demonstrable physical contact between the two proteins, suggesting the possibility of a novel, indirect mechanism of recruitment involving DNA target-site pre-conditioning. The evidence indicates therefore that PF1 and GT-2 do not perform functionally equivalent roles in positively regulating oat and rice PHYA gene expression. However, the data suggest the possibility that PF1 may act as an architectural factor, promiscuously recognizing a spectrum of AT-containing elements in plant promoters, with the general function of catalyzing enhanced binding of conventional cognate transcriptional regulators to these elements via DNA bending.