Phytochromes transmit photoperiod information via the evening complex in Brachypodium.

BACKGROUND: Daylength is a key seasonal cue for animals and plants. In cereals, photoperiodic responses are a major adaptive trait, and alleles of clock genes such as PHOTOPERIOD1 (PPD1) and EARLY FLOWERING3 (ELF3) have been selected for in adapting barley and wheat to northern latitudes. How monocot plants sense photoperiod and integrate this information into growth and development is not well understood. RESULTS: We find that phytochrome C (PHYC) is essential for flowering in Brachypodium distachyon. Conversely, ELF3 acts as a floral repressor and elf3 mutants display a constitutive long day phenotype and transcriptome. We find that ELF3 and PHYC occur in a common complex. ELF3 associates with the promoters of a number of conserved regulators of flowering, including PPD1 and VRN1. Consistent with observations in barley, we are able to show that PPD1 overexpression accelerates flowering in short days and is necessary for rapid flowering in response to long days. PHYC is in the active Pfr state at the end of the day, but we observe it undergoes dark reversion over the course of the night. CONCLUSIONS: We propose that PHYC acts as a molecular timer and communicates information on night-length to the circadian clock via ELF3.

Differential phosphorylation of the N-terminal extension regulates phytochrome B signaling.

Phytochrome B (phyB) is an excellent light quality and quantity sensor that can detect subtle changes in the light environment. The relative amounts of the biologically active photoreceptor (phyB Pfr) are determined by the light conditions and light independent thermal relaxation of Pfr into the inactive phyB Pr, termed thermal reversion. Little is known about the regulation of thermal reversion and how it affects plants' light sensitivity. In this study we identified several serine/threonine residues on the N-terminal extension (NTE) of Arabidopsis thaliana phyB that are differentially phosphorylated in response to light and temperature, and examined transgenic plants expressing nonphosphorylatable and phosphomimic phyB mutants. The NTE of phyB is essential for thermal stability of the Pfr form, and phosphorylation of S86 particularly enhances the thermal reversion rate of the phyB Pfr-Pr heterodimer in vivo. We demonstrate that S86 phosphorylation is especially critical for phyB signaling compared with phosphorylation of the more N-terminal residues. Interestingly, S86 phosphorylation is reduced in light, paralleled by a progressive Pfr stabilization under prolonged irradiation. By investigating other phytochromes (phyD and phyE) we provide evidence that acceleration of thermal reversion by phosphorylation represents a general mechanism for attenuating phytochrome signaling.

Thermal Reversion of Plant Phytochromes.

Phytochromes are red/far-red reversible photoreceptors essential for plant growth and development. Phytochrome signaling is mediated by the physiologically active far-red-absorbing Pfr form that can be inactivated to the red-absorbing Pr ground state by light-dependent photoconversion or by light-independent thermal reversion, also termed dark reversion. Although the term "dark reversion" is justified by historical reasons and frequently used in the literature, "thermal reversion" more appropriately describes the process of light-independent but temperature-regulated Pfr relaxation that not only occurs in darkness but also in light and is used throughout the review. Thermal reversion is a critical parameter for the light sensitivity of phytochrome-mediated responses and has been studied for decades, often resulting in contradictory findings. Thermal reversion is an intrinsic property of the phytochrome molecules but can be modulated by intra- and intermolecular interactions, as well as biochemical modifications, such as phosphorylation. In this review, we outline the research history of phytochrome thermal reversion, highlighting important predictions that have been made before knowing the molecular basis. We further summarize and discuss recent findings about the molecular mechanisms regulating phytochrome thermal reversion and its functional roles in light and temperature sensing in plants.

Phytochromes function as thermosensors in Arabidopsis.

Plants are responsive to temperature, and some species can distinguish differences of 1°C. In Arabidopsis, warmer temperature accelerates flowering and increases elongation growth (thermomorphogenesis). However, the mechanisms of temperature perception are largely unknown. We describe a major thermosensory role for the phytochromes (red light receptors) during the night. Phytochrome null plants display a constitutive warm-temperature response, and consistent with this, we show in this background that the warm-temperature transcriptome becomes derepressed at low temperatures. We found that phytochrome B (phyB) directly associates with the promoters of key target genes in a temperature-dependent manner. The rate of phyB inactivation is proportional to temperature in the dark, enabling phytochromes to function as thermal timers that integrate temperature information over the course of the night.

Phytochrome B integrates light and temperature signals in Arabidopsis.

Ambient temperature regulates many aspects of plant growth and development, but its sensors are unknown. Here, we demonstrate that the phytochrome B (phyB) photoreceptor participates in temperature perception through its temperature-dependent reversion from the active Pfr state to the inactive Pr state. Increased rates of thermal reversion upon exposing Arabidopsis seedlings to warm environments reduce both the abundance of the biologically active Pfr-Pfr dimer pool of phyB and the size of the associated nuclear bodies, even in daylight. Mathematical analysis of stem growth for seedlings expressing wild-type phyB or thermally stable variants under various combinations of light and temperature revealed that phyB is physiologically responsive to both signals. We therefore propose that in addition to its photoreceptor functions, phyB is a temperature sensor in plants.

Characterization of photomorphogenic responses and signaling cascades controlled by phytochrome-A expressed in different tissues.

The photoreceptor phytochrome A acts as a light-dependent molecular switch and regulates responses initiated by very low fluences of light (VLFR) and high fluences (HIR) of far-red light. PhyA is expressed ubiquitously, but how phyA signaling is orchestrated to regulate photomorphogenesis is poorly understood. To address this issue, we generated transgenic Arabidopsis thaliana phyA-201 mutant lines expressing the biologically active phyA-YFP photoreceptor in different tissues, and analyzed the expression of several reporter genes, including ProHY5:HY5-GFP and Pro35S:CFP-PIF1, and various FR-HIR-dependent physiological responses. We show that phyA action in one tissue is critical and sufficient to regulate flowering time and root growth; control of cotyledon and hypocotyl growth requires simultaneous phyA activity in different tissues; and changes detected in the expression of reporters are not restricted to phyA-containing cells. We conclude that FR-HIR-controlled morphogenesis in Arabidopsis is mediated partly by tissue-specific and partly by intercellular signaling initiated by phyA. Intercellular signaling is critical for many FR-HIR induced responses, yet it appears that phyA modulates the abundance and activity of key regulatory transcription factors in a tissue-autonomous fashion.

Molecular mechanisms for mediating light-dependent nucleo/cytoplasmic partitioning of phytochrome photoreceptors.

The photoreceptors phytochromes monitor the red/far-red part of the spectrum, exist in the biologically active Pfr (far-red absorbing) or inactive Pr (red absorbing) forms, and function as red/far-red light-regulated molecular switches to modulate plant development and growth. Phytochromes are synthesized in the cytoplasm, and light induces translocation of the Pfr conformer into the nucleus. Nuclear import of phytochromes is a highly regulated process and is fine-tuned by the quality and quantity of light. It appears that phytochrome A (phyA) and phytochrome B (phyB) do not possess active endogenous nuclear import signals (NLSs), thus light-induced translocation of these photoreceptors into the nucleus requires direct protein­protein interactions with their NLS-containing signaling partners. Sub-cellular partitioning of the various phytochrome species is mediated by different molecular machineries. Translocation of phyA into the nucleus is promoted by FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) and FHY1-LIKE (FHL), but the identity of nuclear transport facilitators mediating the import of phyB-E into the nucleus remains elusive. Phytochromes localized in the nucleus are associated with specific protein complexes, termed photobodies. The size and distribution of these structures are regulated by the intensity and duration of irradiation, and circumstantial evidence indicates that they are involved in fine-tuning phytochrome signaling.

Red Light-Regulated Reversible Nuclear Localization of Proteins in Mammalian Cells and Zebrafish.

Protein trafficking in and out of the nucleus represents a key step in controlling cell fate and function. Here we report the development of a red light-inducible and far-red light-reversible synthetic system for controlling nuclear localization of proteins in mammalian cells and zebrafish. First, we synthetically reconstructed and validated the red light-dependent Arabidopsis phytochrome B nuclear import mediated by phytochrome-interacting factor 3 in a nonplant environment and support current hypotheses on the import mechanism in planta. On the basis of this principle we next regulated nuclear import and activity of target proteins by the spatiotemporal projection of light patterns. A synthetic transcription factor was translocated into the nucleus of mammalian cells and zebrafish to drive transgene expression. These data demonstrate the first in vivo application of a plant phytochrome-based optogenetic tool in vertebrates and expand the repertoire of available light-regulated molecular devices.

Systematic analysis of how phytochrome B dimerization determines its specificity.

Phytochromes are red/far-red-light detecting photoreceptors that regulate plant growth and development. They photo-interconvert between an inactive Pr (red-light absorbing) and a physiologically active Pfr (far-red-light absorbing) form, acting as light-controlled molecular switches. Although the two major plant phytochromes A (phyA) and B (phyB) share similar absorption properties, they exhibit dramatic differences in their action spectra. Since both phytochromes antagonistically regulate seedling development under vegetative shade, it is essential for plants to clearly distinguish between phyA and phyB action. This discrimination is not comprehensible solely by the molecular properties of the phytochromes, but is evidently due to the dynamics of the phytochrome system. Using an integrated experimental and mathematical modelling approach we show that phytochrome dimerization is an essential element for phyB function. Our findings reveal that light-independent Pfr to Pr relaxation (dark reversion) and association/dissociation to nuclear bodies (NBs) severely depend on the conformational state of the phyB dimer. We conclude that only Pfr-Pfr homodimers of phyB can be responsible for triggering physiological responses, leading to a suppression of phyB function in the far-red range of the light spectrum.

phyA-GFP is spectroscopically and photochemically similar to phyA and comprises both its native types, phyA' and phyA''.

Low-temperature fluorescence investigations of phyA-GFP used in experiments on its nuclear-cytoplasmic partitioning were carried out. In etiolated hypocotyls of phyA-deficient Arabidopsis thaliana expressing phyA-GFP, it was found that it is similar to phyA in spectroscopic parameters with both its native types, phyA' and phyA'', present and their ratio shifted towards phyA'. In transgenic tobacco hypocotyls, native phyA and rice phyA-GFP were also identical to phyA in the wild type whereas phyA-GFP belonged primarily to the phyA' type. Finally, truncated oat Δ6-12 phyA-GFP expressed in phyA-deficient Arabidopsis was represented by the phyA' type in contrast to full-length oat phyA-GFP with an approximately equal proportion of the two phyA types. This correlates with a previous observation that Δ6-12 phyA-GFP can form only numerous tiny subnuclear speckles while its wild-type counterpart can also localize into bigger and fewer subnuclear protein complexes. Thus, phyA-GFP is spectroscopically and photochemically similar or identical to the native phyA, suggesting that the GFP tag does not affect the chromophore. phyA-GFP comprises phyA'-GFP and phyA''-GFP, suggesting that both of them are potential participants in nuclear-cytoplasmic partitioning, which may contribute to its complexity.

Comparative functional analysis of full-length and N-terminal fragments of phytochrome C, D and E in red light-induced signaling.

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.

Phosphorylation of phytochrome B inhibits light-induced signaling via accelerated dark reversion in Arabidopsis.

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λmax = 730 nm) and inactive Pr (λmax = 660 nm) forms in a red/far-red-dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein-protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB-yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB(Ser86Asp) Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light-induced nuclear import and interaction of phyB(Ser86Asp)-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB-green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.

The mediator complex subunit PFT1 interferes with COP1 and HY5 in the regulation of Arabidopsis light signaling.

Arabidopsis (Arabidopsis thaliana) mutants hypersensitive to far-red light were isolated under a light program of alternating red and far-red light pulses and were named eid (for empfindlicher im dunkelroten Licht). The dominant eid3 mutant carries a missense mutation in a conserved domain of PHYTOCHROME AND FLOWERING TIME1 (PFT1), an important component of the plant mediator coactivator complex, which links promoter-bound transcriptional regulators to RNA polymerase II complexes. Epistatic analyses were performed to obtain information about the coaction between the mutated PFT1(eid3) and positively and negatively acting components of light signaling cascades. The data presented here provide clear evidence that the mutation mainly enhances light sensitivity downstream of phytochrome A (phyA) and modulates phyB function. Our results demonstrate that the Mediator component cooperates with CONSTITUTIVE PHOTORMORPHOGENIC1 in the regulation of light responses and that the hypersensitive phenotype strictly depends on the presence of the ELONGATED HYPOCOTYL5 transcription factor, an important positive regulator of light-dependent gene expression. Expression profile analyses revealed that PFT1(eid3) alters the transcript accumulation of light-regulated genes even in darkness. Our data further indicate that PFT1 regulates the floral transition downstream of phyA. The PFT1 missense mutation seems to create a constitutively active transcription factor by mimicking an early step in light signaling.

Intramolecular uncoupling of chromophore photoconversion from structural signaling determinants drive mutant phytochrome B photoreceptor to far-red light perception.

The phytochrome (phy) photoreceptor family regulates almost all aspects of plant development in a broad range of light environments including seed germination, onset of the photomorphogenic program in seedling stage, the shade avoidance syndrome in competing plant communities, flowering induction and senescence of adult plants. During evolution two clearly distinct classes of phy-s emerged covering these very different physiological tasks. ( 1) PhyA is rapidly degraded in its activated state. PhyA functions in controlling seed germination at very low light intensities (very low fluence response, VLFR) and seedling establishment under photosynthetic shade conditions (high irradiance response, HIR) where the far-red portion of the transmitted light to understorey habitats is substantially enhanced. Arabidopsis phyB together with phyC, D and E belongs to the relatively stable sensor class in comparison to the light labile phyA. PhyB functions at all stages of development including seed germination and seedling establishment, mediates classical red/far-red reversible low fluence responses (LFR) as well as red light high irradiance responses, and it is considered to be the dominating phytochrome sensor of its class.

A short amino-terminal part of Arabidopsis phytochrome A induces constitutive photomorphogenic response.

Phytochrome A (phyA) is the dominant photoreceptor of far-red light sensing in Arabidopsis thaliana. phyA accumulates at high levels in the cytoplasm of etiolated seedlings, and light-induced phyA signaling is mediated by a complex regulatory network. This includes light- and FHY1/FHL protein-dependent translocation of native phyA into the nucleus in vivo. It has also been shown that a short N-terminal fragment of phyA (PHYA406) is sufficient to phenocopy this highly regulated cellular process in vitro. To test the biological activity of this N-terminal fragment of phyA in planta, we produced transgenic phyA-201 plants expressing the PHYA406-YFP (YELLOW FLUORESCENT PROTEIN)-DD, PHYA406-YFP-DD-NLS (nuclear localization signal), and PHYA406-YFP-DD-NES (nuclear export signal) fusion proteins. Here, we report that PHYA406-YFP-DD is imported into the nucleus and this process is partially light-dependent whereas PHYA406-YFP-DD-NLS and PHYA406-YFP-DD-NES display the expected constitutive localization patterns. Our results show that these truncated phyA proteins are light-stable, they trigger a constitutive photomorphogenic-like response when localized in the nuclei, and neither of them induces proper phyA signaling. We demonstrate that in vitro and in vivo PHYA406 Pfr and Pr bind COP1, a general repressor of photomorphogenesis, and co-localize with it in nuclear bodies. Thus, we conclude that, in planta, the truncated PHYA406 proteins inactivate COP1 in the nuclei in a light-independent fashion.

Effects of missense mutation on structure and function of photoreceptor.

Phytochromes (PHYs) are photoreceptors of the red (R ~660 nm) and far-red (FR ~730 nm) light, and they control a wide range of responses affecting crucial aspects of plant life. There are five genes PHYA-PHYE encoding for phytochromes of different but overlapping function. One of these, PHYA has the unique function controlling specific responses in high irradiance far-red, as well as in very weak light. Appropriate PHYA functioning requires not only the photoreversibility of molecule but also the proper nuclear localization and degradation of receptor. Recently, we identified and described a mutant PHYA allele (phyA-5) in Arabidopsis thaliana, which showed reduced binding affinity to FHY1/FHL, the proteins regulating its nuclear transport, resulting in impaired nuclear localization and altered signaling under certain conditions. We present here a hypothesis to explain how the identified amino acid substitution may lead to structural changes manifested as altered signaling and phenotype displayed by the phyA-5 mutant.

Interaction with plant transcription factors can mediate nuclear import of phytochrome B.

Phytochromes (phy) are red/far-red-absorbing photoreceptors that regulate the adaption of plant growth and development to changes in ambient light conditions. The nuclear transport of the phytochromes upon light activation is regarded as a key step in phytochrome signaling. Although nuclear import of phyA is regulated by the transport facilitators far red elongated hypocotyl 1 (FHY1) and fhy1-like, an intrinsic nuclear localization signal was proposed to be involved in the nuclear accumulation of phyB. We recently showed that nuclear import of phytochromes can be analyzed in a cell-free system consisting of isolated nuclei of the unicellular green algae Acetabularia acetabulum. We now show that this system is also versatile to elucidate the mechanism of the nuclear transport of phyB. We tested the nuclear transport characteristics of full-length phyB as well as N- and C-terminal phyB fragments in vitro and showed that the nuclear import of phyB can be facilitated by phytochrome-interacting factor 3 (PIF3). In vivo measurements of phyB nuclear accumulation in the absence of PIF1, -3, -4, and -5 indicate that these PIFs are the major transport facilitators during the first hours of deetiolation. Under prolonged irradiations additional factors might be responsible for phyB nuclear transport in the plant.

Red light-induced phytochrome relocation into the nucleus in Adiantum capillus-veneris.

Phytochromes in seed plants are known to move into nuclei in a red light-dependent manner with or without interacting factors. Here, we show phytochrome relocation to the nuclear region in phytochrome-dependent Adiantum capillus-veneris spore germination by partial spore-irradiation experiments. The nuclear or non-nuclear region of imbibed spores was irradiated with a microbeam of red and/or far-red light and the localization of phytochrome involved in spore germination was estimated from the germination rate. The phytochrome for spore germination existed throughout whole spore under darkness after imbibition, but gradually migrated to the nuclear region following red light irradiation. Intracellular distribution of PHY-GUS fusion proteins expressed in germinated spores by particle bombardment showed the migration of Acphy2, but not Acphy1, into nucleus in a red light-dependent manner, suggesting that Acphy2 is the photoreceptor for fern spore germination.