Phy tunes: phosphorylation status and phytochrome-mediated signaling

Phytochromes are photoreceptors that regulate various aspects of plant growth and development. In this issue of Cell, Ryu et al. (2005) show that PAPP5, a type 5 protein phosphatase, acts on a biologically active phytochrome, increasing its stability and affinity for a downstream signal transducer and thus enhancing plant photoresponses.     

Autophosphorylation desensitizes phytochrome signal transduction

Plant red/far-red photoreceptor phytochromes are known as autophosphorylating serine/threonine kinases. However, the functional roles of autophosphorylation and kinase activity of phytochromes are largely unknown. We recently reported that the autophosphorylation of phytochrome A (phyA) plays an important role in regulating plant phytochrome signaling by controlling phyA protein stability. Two serine residues in the N-terminal extension (NTE) region were identified as autophosphorylation sites, and phyA mutant proteins with serine-to-alanine mutations were degraded in plants at a significantly slower rate than the wild-type under light conditions, resulting in transgenic plants with hypersensitive light responses. In addition, the autophosphorylation site phyA mutants had normal protein kinase activities. Collectively, our results suggest that phytochrome autophosphorylation provides a mechanism for signal desensitization in phytochrome-mediated light signaling by accelerating the degradation of phytochrome A.Key words: phytochrome, autophosphorylation, phosphorylation, protein kinase, protein degradation, light signaling, signal desensitizationHigher plants continually adapt to their light environments to promote photosynthesis for optimal growth and development. Natural light conditions are monitored by various plant photoreceptors, including red (R)/far-red (FR) photoreceptor phytochromes.^1,2 Phytochromes are dimeric chromoproteins covalently linked to tetrapyrrole chromophore phytochromobilin, and exist as two photo-interconvertible species, red-light absorbing Pr and far-red-light absorbing Pfr forms. Phytochromes are biosynthesized as the Pr form in the dark, and are transformed to the Pfr form upon exposure to red light. This photoactivation of phytochromes induces a highly regulated signaling network for photomorphogenesis in plants.^3,4 Recently, phosphorylation and dephosphorylation have been suggested to play important roles in phytochrome-mediated light signaling;^5,6 for instance, a few phytochrome-associated protein phosphatases have been shown to act as positive regulators of phytochrome signaling.^7–9 However, the functional roles of phytochrome phosphorylation remain to be explored.     

Phytochrome-mediated development in land plants: red light sensing evolves to meet the challenges of changing light environments

Phytochromes are photoreceptors that provide plants with circadian, seasonal, and positional information critical for the control of germination, seedling development, shade avoidance, reproduction, dormancy, and sleep movements. Phytochromes are unique among photoreceptors in their capacity to interconvert between a red-absorbing form (absorption maximum of approximately 660 nm) and a far-red absorbing form (absorption maximum of approximately 730 nm), which occur in a dynamic equilibrium within plant cells, corresponding to the proportions of red and far-red energy in ambient light. Because pigments in stems and leaves absorb wavelengths below about 700 nm, this provides plants with an elegant system for detecting their position relative to other plants, with which the plants compete for light. Certain aspects of phytochrome-mediated development outside of flowering plants are strikingly similar to those that have been characterized in Arabidopsis thaliana and other angiosperms. However, early diverging land plants have fewer distinct phytochrome gene lineages, suggesting that both diversification and subfunctionalization have been important in the evolution of the phytochrome gene family. There is evidence that subfunctionalization proceeded by the partitioning among paralogues of photosensory specificity, physiological response modes, and light-regulated gene expression and protein stability. Parallel events of duplication and functional divergence may have coincided with the evolution of canopy shade and the increasing complexity of the light environment. Within angiosperms, patterns of functional divergence are clade-specific and the roles of phytochromes in A. thaliana change across environments, attesting to the evolutionary flexibility and contemporaneous plasticity of phytochrome signalling in the control of development.     

Action and function of phytochrome family members revealed through the study of mutant and transgenic plants

The adaptation of plant growth and development to changes in the light environment is dependent upon photoperception by information transducing photoreceptors. The red/far-red light-absorbing phytochromes are perhaps the best characterized of these regulatory photoreceptors. Higher plants possess multiple, discrete phytochromes, the apoprotein components of which are the products of a small, divergent gene family. Different phytochromes have different biochemical and physiological properties, and are differentially expressed in the growing plant. This has led to the proposal that different phytochromes have different physiological roles. Mutations that disrupt the normal perception of light signals have proved to be a valuable resource in assigning physiological roles to different phytochromes as well as in identifying residues/domains critical for phytochrome function and in attempting to elucidate the signal transduction pathway(s) downstream of phytochromes. This article reviews some recent progress in these areas from the study of conventional and transgenic photomorphogenic mutants.     

The influence of de-etiolation on the sensitivity of Avena coleoptiles to the far-red absorbing form of phytochrome

In photoresponses regulated by phytochrome the effect of a red irradiation is not always reversed by far-red. This applies for instance to the influence of red light on the geotropic reactions of Avena coleoptiles. We could induce red/far-red reversibility by a short de-etiolating exposure to red light about 20 h prior to the experimental irradiations. This, was due to a decrease of the sensitivity to the low level of the far-red absorbing form of phytochrome that is established by far-red. Since etiolated plants react also to a wavelength of 520 nm (green light), it is advisable to expose the coleoptiles to a de-etiolating irradiation prior to manipulations in green safelight in order to prevent the plants from reacting to the green light.     

Photochemical characterization of phytochrome missense mutants

Phytochromes are photoreceptors that regulate many aspects of plant growth and development in response to red/far-red light signals from the environment. In this study, we analyzed chromophore ligation and photochromism of missense phytochrome mutants in the Per-Arnt-Sim (PAS)-related domain (PRD). Among the 14 mutants analyzed, the Gly768Asp mutant of Avena phytochrome A showed aberrant photochromism and dark reversion, suggesting that amino acid residues in the C-terminal domain affect the photochemical properties of the photosensory N-terminal domain.     

Influence of the Dietary Protein Deficiency on the Protein Synthetic Activity of Microsome and Soluble Fraction from Muscle and Liver of Rats

Phytochromes are photoreceptors that regulate many aspects of plant growth and development in response to red/far-red light signals from the environment. In this study, we analyzed chromophore ligation and photochromism of missense phytochrome mutants in the Per-Arnt-Sim (PAS)-related domain (PRD). Among the 14 mutants analyzed, the Gly768Asp mutant of Avena phytochrome A showed aberrant photochromism and dark reversion, suggesting that amino acid residues in the C-terminal domain affect the photochemical properties of the photosensory N-terminal domain.     

A phytochrome-associated protein phosphatase 2A modulates light signals in flowering time control in Arabidopsis

Reversible protein phosphorylation, which is catalyzed by functionally coupled protein kinases and protein phosphatases, is a major signaling mechanism in eukaryotic cellular functions. The red and far-red light-absorbing phytochrome photoreceptors are light-regulated Ser/Thr-specific protein kinases that regulate diverse photomorphogenic processes in plants. Here, we demonstrate that the phytochromes functionally interact with the catalytic subunit of a Ser/Thr-specific protein phosphatase 2A designated FyPP. The interactions were influenced by phosphorylation status and spectral conformation of the phytochromes. Recombinant FyPP efficiently dephosphorylated oat phytochrome A in the presence of Fe(2+) or Zn(2+) in a spectral form-dependent manner. FyPP was expressed predominantly in floral organs. Transgenic Arabidopsis plants with overexpressed or suppressed FyPP levels exhibited delayed or accelerated flowering, respectively, indicating that FyPP modulates phytochrome-mediated light signals in the timing of flowering. Accordingly, expression patterns of the clock genes in the long-day flowering pathway were altered greatly. These results indicate that a self-regulatory phytochrome kinase-phosphatase coupling is a key signaling component in the photoperiodic control of flowering.     

Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development.

Phytochromes are a family of plant photoreceptors that mediate physiological and developmental responses to changes in red and far-red light conditions. In Arabidopsis, there are genes for at least five phytochrome proteins. These photoreceptors control such responses as germination, stem elongation, flowering, gene expression, and chloroplast and leaf development. However, it is not known which red light responses are controlled by which phytochrome species, or whether the different phytochromes have overlapping functions. We report here that previously described hy3 mutants have mutations in the gene coding for phytochrome B (PhyB). These are the first mutations shown to lie in a plant photoreceptor gene. A number of tissues are abnormally elongated in the hy3(phyB) mutants, including hypocotyls, stems, petioles, and root hairs. In addition, the mutants flower earlier than the wild type, and they accumulate less chlorophyll. PhyB thus controls Arabidopsis development at numerous stages and in multiple tissues.     

Phytochrome-mediated effects of near-ultraviolet radiation in the induction of flowering in etiolated Lemna paucicostata T-101, a short-day plant

Induction of flowering of etiolated Lemna paucicostata Hegelm. T-101, a short-day plant, was inhibited by far-red (FR) or blue light (BL) applied at the beginning of a 72-h inductive dark period which was followed by two short days. In either case the inhibition was reversed by a subsequent exposure of the plants to near-ultraviolet radiation (NUV), with a peak of effectiveness near 380 nm. Inhibition by BL or FR and its reversion by NUV are repeatable, i.e., NUV is acting in these photoresponses like red light although with much lower effectiveness. Thus, it is considered that NUV acts through phytochrome and no specific BL and NUV photoreceptor is involved in photocontrol of floral induction on this plant.Abbreviations BL blue light - FR far-red light - NUV near ultraviolet radiation - P red-absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - R red light     

Missense mutation in the amino terminus of phytochrome A disrupts the nuclear import of the photoreceptor

Phytochromes are the red/far-red photoreceptors in higher plants. Among them, phytochrome A (PHYA) is responsible for the far-red high-irradiance response and for the perception of very low amounts of light, initiating the very-low-fluence response. Here, we report a detailed physiological and molecular characterization of the phyA-5 mutant of Arabidopsis (Arabidopsis thaliana), which displays hyposensitivity to continuous low-intensity far-red light and shows reduced very-low-fluence response and high-irradiance response. Red light-induced degradation of the mutant phyA-5 protein appears to be normal, yet higher residual amounts of phyA-5 are detected in seedlings grown under low-intensity far-red light. We show that (1) the phyA-5 mutant harbors a new missense mutation in the PHYA amino-terminal extension domain and that (2) the complex phenotype of the mutant is caused by reduced nuclear import of phyA-5 under low fluences of far-red light. We also demonstrate that impaired nuclear import of phyA-5 is brought about by weakened binding affinity of the mutant photoreceptor to nuclear import facilitators FHY1 (for FAR-RED ELONGATED HYPOCOTYL1) and FHL (for FHY1-LIKE). Finally, we provide evidence that the signaling and degradation kinetics of constitutively nuclear-localized phyA-5 and phyA are identical. Taken together, our data show that aberrant nucleo/cytoplasmic distribution impairs light-induced degradation of this photoreceptor and that the amino-terminal extension domain mediates the formation of the FHY1/FHL/PHYA far-red-absorbing form complex, whereby it plays a role in regulating the nuclear import of phyA.     

Crystallographic and Electron Microscopic Analyses of a Bacterial Phytochrome Reveal Local and Global Rearrangements during Photoconversion

Phytochromes are multidomain photoswitches that drive light perception in plants and microorganisms by coupling photoreversible isomerization of their bilin chromophore to various signaling cascades. How changes in bilin conformation affect output by these photoreceptors remains poorly resolved and might include several species-specific routes. Here, we present detailed three-dimensional models of the photosensing module and a picture of an entire dimeric photoreceptor through structural analysis of the Deinococcus radiodurans phytochrome BphP assembled with biliverdin (BV). A 1.16-Å resolution crystal structure of the bilin-binding pocket in the dark-adapted red light-absorbing state illuminated the intricate network of bilin/protein/water interactions and confirmed the protonation and ZZZssa conformation of BV. Structural and spectroscopic comparisons with the photochemically compromised D207A mutant revealed that substitutions of Asp-207 allow inclusion of cyclic porphyrins in addition to BV. A crystal structure of the entire photosensing module showed a head-to-head, twisted dimeric arrangement with bowed helical spines and a hairpin protrusion connecting the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) and phytochrome-specific (PHY) domains. A key conserved hairpin feature is its anti-parallel, two β-strand stem, which we show by mutagenesis to be critical for BphP photochemistry. Comparisons of single particle electron microscopic images of the full-length BphP dimer in the red light-absorbing state and the photoactivated far-red light-absorbing state revealed a large scale reorientation of the PHY domain relative to the GAF domain, which alters the position of the downstream histidine kinase output module. Together, our data support a toggle model whereby bilin photoisomerization alters GAF/PHY domain interactions through conformational modification of the hairpin, which regulates signaling by impacting the relationship between sister output modules.     

Phytochrome and photoperiodic induction

The photoreceptor phytochrome has been extensively characterized at the chromophore, protein and gene level. It consists of a family of red/far-red reversible molecules and the genes for three members have been sequenced. Phytochromes are chromoproteins, which probably exist as dimers in vivo. Photoperiodism in higher plants involves the interaction of phytochrome with an endogenous timekeeping system. The interaction is complex, and several distinct actions of light can be distinguished. The possible involvement of different phytochromes in different actions of light in both long-day plants and short-day plants is discussed. Potential roles for different members of the phytochrome family and homo-and hetero-dimers of phytochrome are proposed.     

Phytochromes are Pr-ipatetic kinases.

A series of new studies reveal how the red/far-red light photoreceptors called phytochromes act. Phytochrome A and phytochrome B each move to the nucleus when activated by light, and phytochrome A is a kinase. Phytochrome-interacting proteins provide candidate signal transduction components and a recent physiological study suggests how phyA may mediate responses to far-red light. Regulation of phytochrome nuclear localization and kinase activities creates multiple phytochrome species, which may each have different regulatory activities.     

FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor

The phytochrome family of red/far-red photoreceptors is involved in the regulation of a wide range of developmental responses in plants. The Arabidopsis genome contains five phytochromes (phyA-E), among which phyA and phyB play the most important roles. Phytochromes localize to the cytosol in the dark and accumulate in the nucleus under light conditions, inducing specific phytochrome-mediated responses. Light-regulated nuclear accumulation of the phytochrome photoreceptors is therefore considered a key regulatory step of these pathways. In fact, one of the most severe phyA signaling mutants, fhy1 (far red elongated hypocotyl 1), is strongly affected in nuclear accumulation of phyA. The fhy1 fhl (fhy1 like) double mutant, lacking both FHY1 and its only close homolog FHL, is virtually blind to far-red light like phyA null seedlings. Here we show that FHL accounts for residual amounts of phyA in the nucleus in a fhy1 background and that nuclear accumulation of phyA is completely inhibited in an fhy1 FHL RNAi knock-down line. Moreover, we demonstrate that FHL and phyA interact with each other in a light-dependent manner and that they co-localize in light-induced nuclear speckles. We also identify a phyA-binding site at the C-terminus of FHY1 and FHL, and show that the N-terminal 406 amino acids of phyA are sufficient for the interaction with FHY1/FHL.     

Arabidopsis thaliana TERMINAL FLOWER2 is involved in light-controlled signalling during seedling photomorphogenesis

Plants respond to changes in the environment by altering their growth pattern. Light is one of the most important environmental cues and affects plants throughout the life cycle. It is perceived by photoreceptors such as phytochromes that absorb light of red and far-red wavelengths and control, for example, seedling de-etiolation, chlorophyll biosynthesis and shade avoidance response. We report that the terminal flower2 (tfl2) mutant, carrying a mutation in the Arabidopsis thaliana HETEROCHROMATIN PROTEIN1 homolog, functions in negative regulation of phytochrome dependent light signalling. tfl2 shows defects in both hypocotyl elongation and shade avoidance response. Double mutant analysis indicates that mutants of the red/far-red light absorbing phytochrome family of plant photoreceptors, phyA and phyB, are epistatic to tfl2 in far-red and red light, respectively. An overlap between genes regulated by light and by auxin has earlier been reported and, in tfl2 plants light-dependent auxin-regulated genes are misexpressed. Further, we show that TFL2 binds to IAA5 and IAA19 suggesting that TFL2 might be involved in regulation of phytochrome-mediated light responses through auxin action.