Blue Light Responses Research Articles

Intramolecular co-action of two independent photosensory modules in the fern phytochrome 3

Fern phytochrome3/neochrome1 (phy3/neo1) is a chimeric photoreceptor composed of a phytochrome-chromophore binding domain and an almost full-length phototropin. phy3 thus contains two different light-sensing modules; a red/far-red light receptor phytochrome and a blue light receptor phototropin. phy3 induces both red light- and blue light-dependent phototropism in phototropin-deficient Arabidopsis thaliana (phot1 phot2) seedlings. The red-light response is dependent on the phytochrome module of phy3, and the blue-light response is dependent on the phototropin module. We recently showed that both the phototropin-sensing module and the phytochrome-sensing module mediate the blue light-dependent phototropic response. Particularly under low-light conditions, these two light-sensing modules cooperate to induce the blue light-dependent phototropic response. This intramolecular co-action of two independent light-sensing modules in phy3 enhances light sensitivity, and perhaps allowed ferns to adapt to the low-light canopy conditions present in angiosperm forests.

Photoreceptor Specificity in the Light-Induced and COP1-Mediated Rapid Degradation of the Repressor of Photomorphogenesis SPA2 in Arabidopsis.

The Arabidopsis COP1/SPA E3 ubiquitin ligase is a key negative regulator that represses light signaling in darkness by targeting transcription factors involved in the light response for degradation. The COP1/SPA complex consists of COP1 and members of the four-member SPA protein family (SPA1-SPA4). Genetic analysis indicated that COP1/SPA2 function is particularly strongly repressed by light when compared to complexes carrying the other three SPAs, thereby promoting a light response after exposure of plants to extremely low light. Here, we show that the SPA2 protein is degraded within 5–15 min after exposure of dark-grown seedlings to a pulse of light. Phytochrome photoreceptors are required for the rapid degradation of SPA2 in red, far-red and also in blue light, whereas cryptochromes are not involved in the rapid, blue light-induced reduction in SPA2 protein levels. These results uncover a photoreceptor-specific mechanism of light-induced inhibition of COP1/SPA2 function. Phytochrome A (phyA) is required for the severe blue light responsiveness of spa triple mutants expressing only SPA2, thus confirming the important role of phyA in downregulating SPA2 function in blue light. In blue light, SPA2 forms a complex with cryptochrome 1 (cry1), but not with cryptochrome 2 (cry2) in vivo, indicating that the lack of a rapid blue light response of the SPA2 protein is only in part caused by a failure to interact with cryptochromes. Since SPA1 interacts with both cry1 and cry2, these results provide first molecular evidence that the light-regulation of different SPA proteins diverged during evolution. SPA2 degradation in the light requires COP1 and the COP1-interacting coiled-coil domain of SPA2, supporting that SPA2 is ubiquitinated by COP1. We propose that light perceived by phytochromes causes a switch in the ubiquitination activity of COP1/SPA2 from ubiquitinating downstream substrates to ubiquitinating SPA2, which subsequently causes a repression of COP1/SPA2 function.

Stomatal Blue Light Response Is Present in Early Vascular Plants.

Light is a major environmental factor required for stomatal opening. Blue light (BL) induces stomatal opening in higher plants as a signal under the photosynthetic active radiation. The stomatal BL response is not present in the fern species of Polypodiopsida. The acquisition of a stomatal BL response might provide competitive advantages in both the uptake of CO2 and prevention of water loss with the ability to rapidly open and close stomata. We surveyed the stomatal opening in response to strong red light (RL) and weak BL under the RL with gas exchange technique in a diverse selection of plant species from euphyllophytes, including spermatophytes and monilophytes, to lycophytes. We showed the presence of RL-induced stomatal opening in most of these species and found that the BL responses operated in all euphyllophytes except Polypodiopsida. We also confirmed that the stomatal opening in lycophytes, the early vascular plants, is driven by plasma membrane proton-translocating adenosine triphosphatase and K(+) accumulation in guard cells, which is the same mechanism operating in stomata of angiosperms. These results suggest that the early vascular plants respond to both RL and BL and actively regulate stomatal aperture. We also found three plant species that absolutely require BL for both stomatal opening and photosynthetic CO2 fixation, including a gymnosperm, C. revoluta, and the ferns Equisetum hyemale and Psilotum nudum.

Activation of the General Stress Response of Bacillus subtilis by Visible Light.

A key challenge for microbiology is to understand how evolution has shaped the wiring of regulatory networks. This is amplified by the paucity of information of power-spectra of physicochemical stimuli to which microorganisms are exposed. Future studies of genome evolution, driven by altered stimulus regimes, will therefore require a versatile signal transduction system that allows accurate signal dosing. Here, we review the general stress response of Bacillus subtilis, and its upstream signal transduction network, as a candidate system. It can be activated by red and blue light, and by many additional stimuli. Signal integration therefore is an intricate function of this system. The blue-light response is elicited via the photoreceptor YtvA, which forms an integral part of stressosomes, to activate expression of the stress regulon of B.subtilis. Signal transfer through this network can be assayed with reporter enzymes, while intermediate steps can be studied with live-cell imaging of fluorescently tagged proteins. Different parts of this system have been studied invitro, such that its computational modeling has made significant progress. One can directly relate the microscopic characteristics of YtvA with activation of the general stress regulon, making this system a very well-suited system for network evolution studies.

Blue enhanced vertically stacked quad junction photodetector with opto window

A vertically stacked quad junction photodetector (PD) with an enhanced spectral responsivity for blue light is presented. This PD is implemented in a 0.35 µm high voltage complementary metal-oxide semiconductor (CMOS) technology. In addition, an optical window, an antireflection coating and a modified p + doping are used to attain the enhancement for blue light. The proposed PD is compared against other state of the art implementations and characterised for its spectral response.

Comparison and Correction of the Light Sensor Output from 48 Wearable Light Exposure Devices by Using a Side-by-Side Field Calibration Method

ABSTRACTMeasurement of personal light exposures and activity has gained popularity in studies of the circadian rhythm and its effects on human health. Calibration of a batch of measuring devices may be needed, especially before initiating interventional studies, but manufactory calibration of devices before every initiated study is costly for the researcher and therefore often left out. Still, knowledge of inter-equipment variability is essential and seldom provided by the manufactory. The aim of the present study was to develop and test a method for field calibration of Actiwatch Spectrum devices. We tested 48 Actiwatch devices side by side under various light sources and present the red, green, blue, and white light response variability among the Actiwatches. The influence of different spatial and spectral light environments on the white light response when compared with the output from a calibrated photometer is discussed. In agreement with previous studies by Price and others [2012] and Figueiro and others [2013], we confirm the devices’ white light responses to be highly dependent on both the spatial and the spectral composition of the light. The white light response represents photopic illuminance only to a minor degree and light source-specific calibration may therefore be needed in some cases. Moreover, light responses were found to vary between devices by up to 60%. Implications are that the results of light effects on health issues in studies using Actiwatches are blurred by the equipment variability. To compensate for inter-equipment variability we stress the need for a field calibration procedure. When light exposure devices of lower grade quality are used in spectrally and spatially changing light environments, daylight from a diffused overcast sky is suggested to be used for side-by-side calibration of Actiwatches and similar personal light exposure devices. We suggest that the calibration methods presented can be used for calibration of other practical field devices, with respect to the various sensors already on the market and devices that will be introduced in the future.

Hormonal diterpenoids derived from ent-kaurenoic acid are involved in the blue-light avoidance response of Physcomitrella patens

Gibberellins (GAs) are diterpenoid hormones that regulate growth and development in flowering plants. The moss Physcomitrella patens has part of the GA biosynthetic pathway from geranylgeranyl diphosphate to ent-kaurenoic acid via ent-kaurene, but it does not produce GA. Disruption of the ent-kaurene synthase gene in P. patens suppressed caulonemal differentiation. Application of ent-kaurene or ent-kaurenoic acid restored differentiation, suggesting that derivative(s) of ent-kaurenoic acid, but not GAs, are endogenous regulator(s) of caulonemal cell differentiation. The protonemal growth of P. patens shows an avoidance response under unilateral blue light. Physiological studies using gene mutants involved in ent-kaurene biosynthesis confirmed that diterpenoid(s) regulate the blue-light response. Here, we discuss the implications of these findings, and provide data for the ent-kaurene oxidase gene-disrupted mutant.

Effect of vegetational shade and its components on stomatal responses to red, blue and green light in two deciduous tree species with different shade tolerance

Stomatal responses to red, blue and green light were measured in shade-midtolerant Nothofagus alpina and shade-avoidant Betula pendula seedlings grown in full sunlight (control, C-set), simulated vegetational shade (IUW-set), or with a single component of vegetational shade: decreased light intensity (I-set) or attenuated UV irradiance (U-set). In N. alpina, stomatal opening response to red light switch-on was stronger in IUW-set and U-set, than in C-set. The addition of medium blue light into the red (stomatal opening) induction light increased the stomatal response, and the stomata also responded sensitively to weak blue light in the IUW- and U-sets. The addition of green light into the red induction light did not change the stomatal response in any set. Yet in the U-set, the addition of green light into the red+(medium) blue or weak blue induction light decreased the stomatal response. Thus, vegetational shade had increased the photosynthesis-mediated stomatal light response, activated an additional stomatal response through blue light receptors, and let stomata insensitive to green light. The attenuated UV irradiance had the same effects as vegetational shade on the stomatal photosynthesis-mediated response and the additional blue light receptors-mediated response, but it also activated the green light reversal of blue light response. The stomata of leaves grown under decreased irradiance exhibited the same stomatal responses as the full-light-grown leaves. In B.pendula, stomatal responses to all the different types of light were remarkably weaker than those in N.alpina, and did not differ between themselves or between the different shade treatments. Thus, the modulation of stomatal light sensitivity participates in shade acclimation in shade-tolerating, but not in shade-avoiding deciduous tree(s).

Genome-wide expression analysis of soybean NF-Y genes reveals potential function in development and drought response

Nuclear factor-Y (NF-Y), a heterotrimeric transcription factor, is composed of NF-YA, NF-YB and NF-YC proteins. In plants, there are usually more than 10 genes for each family and their members have been identified to be key regulators in many developmental and physiological processes controlling gametogenesis, embryogenesis, nodule development, seed development, abscisic acid (ABA) signaling, flowering time, primary root elongation, blue light responses, endoplasmic reticulum (ER) stress response and drought tolerance. Taking the advantages of the recent soybean genome draft and information on functional characterizations of nuclear factor Y (NF-Y) transcription factor family in plants, we identified 21 GmNF-YA, 32 GmNF-YB, and 15 GmNF-YC genes in the soybean (Glycine max) genome. Phylogenetic analyses show that soybean’s proteins share strong homology to Arabidopsis and many of them are closely related to functionally characterized NF-Y in plants. Expression analysis in various tissues of flower, leaf, root, seeds of different developmental stages, root hairs under rhizobium inoculation, and drought-treated roots and leaves revealed that certain groups of soybean NF-Y are likely involved in specific developmental and stress responses. This study provides extensive evaluation of the soybean NF-Y family and is particularly useful for further functional characterization of GmNF-Y proteins in seed development, nodulation and drought adaptation of soybean.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-014-0978-2) contains supplementary material, which is available to authorized users.

Structural Biochemistry of a Fungal LOV Domain Photoreceptor Reveals an Evolutionarily Conserved Pathway Integrating Light and Oxidative Stress

Fungal LOV proteins facilitate photoadaptation via blue light regulation of dimer formation. Despite considerable homology of these proteins in closely related fungi, deviations in signaling exist. Here wereport the crystal structure of ENVOY (ENV1), a homolog of N.crassa VVD in the fungus T.reesei, amodel organism for plant cell wall degradation. Structural studies contradict a model of reversible competitive dimerization. Rather, evolutionary pressures have facilitated a two-residue shift in the position of a key Cys residue (Cys96) that enables theintegration of environmental stress and light responses. A Cys96Thr variant abolishes adaptive responses to light and oxidative stress in a carbon source-dependent manner invivo. Phylogenetic analysis verifies an evolutionary relevance of the Cys residue shift in different orders within Sordariomycetes. In this manner, we identified a widespread oxidative stress signaling mechanism that couples metabolic sensing and blue light responses not previously identified in LOV proteins.

A Dominant Mutation in the Light-Oxygen and Voltage2 Domain Vicinity Impairs Phototropin1 Signaling in Tomato

In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A'α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A'α helix region in phototropic signaling of tomato.

Phototropism: Some History, Some Puzzles, and a Look Ahead

Though few and far between, phototropism studies through 1937 established a number of important principles. (1) Blue light is the active spectral region. (2) The phototropic stimulus is perceived by the coleoptile tip, and the consequences of the stimulation progress down into the growing region. (3) Lateral transport of auxin mediates the curvature response. (4) The reciprocity law holds for first positive curvature, whereas second positive curvature is time dependent. (5) Red light treatment had a major effect on phototropic sensitivity. Later studies established the following. (6) Seven blue light receptors (cryptochromes, phototropins, and three F-box proteins) were identified and characterized. (7) A flavin was established as the photoreceptor chromophore for all seven. (8) The chromophore domain, designated the LOV domain (for light, oxygen, or voltage), carries out a unique photochemistry. (9) LOV domains must be truly ancient chromophore domains. There remain some puzzles. The fluence-response threshold level for first positive curvature is far below that for phototropin photochemistry. Likewise, the fluence-response threshold level for the red light effect on coleoptile phototropism is far below those for phytochrome phototransformation. Cytological effects of red light are also very insensitive compared with the physiological effects of red light. What is the mechanism allowing for this extraordinary photosensitivity? How is phototropin specificity controlled? What are the functions of the phytochrome kinase substrate proteins in both phytochrome and phototropin responses? What mechanism leads to lateral auxin transport? Finally, are LOV domain proteins true photoreceptors in all of the bacteria in which they occur? If so, what is their biological function? Even in the ancient world, astute observers noted that plants could turn to face the sunlight. What was originally designated heliotropism for plants that followed the sun eventually became divided into two distinct response categories: solar tracking (the real heliotropism), a repetitive and completely reversible turgor-driven process; and phototropism, an irreversible directional growth response determined by light direction. Over the past 200 years, a large number of brilliant biologists, including Julius Sachs (1864), Charles Darwin (1881), Frits Went (1928), and Kenneth Thimann (Went and Thimann, 1937) have applied their talents to examining and elucidating the mechanisms accounting for both of these responses. The entire history of phototropism and solar tracking parallels and is intertwined with that for a number of other blue light responses, found not just in higher plants but in bryophytes, ferns, algae, fungi, and, most recently bacteria. For a detailed account of this history, see Briggs (2006). Progress in research on blue light-activated processes over the last half century was severely hampered by a lack of knowledge of the relevant blue light receptors.

The Arabidopsis NF-YA3 and NF-YA8 Genes Are Functionally Redundant and Are Required in Early Embryogenesis

Nuclear factor Y (NF-Y) is a trimeric transcription factor composed of three distinct subunits called NF-YA, NF-YB and NF-YC. In Arabidopsis thaliana, NF-Y subunits are known to play roles in many processes, such as gametogenesis, embryogenesis, seed development, drought resistance, ABA signaling, flowering time, primary root elongation, Endoplasmic Reticulum (ER) stress response and blue light responses. Here, we report that the closely related NF-YA3 and NF-YA8 genes control early embryogenesis. Detailed GUS and in situ analyses showed that NF-YA3 and NF-YA8 are expressed in vegetative and reproductive tissues with the highest expression being during embryo development from the globular to the torpedo embryo stage. Plants from the nf-ya3 and nf-ya8 single mutants do not display any obvious phenotypic alteration, whereas nf-ya3 nf-ya8 double mutants are embryo lethal. Morphological analyses showed that the nf-ya3 nf-ya8 embryos fail to undergo to the heart stage and develop into abnormal globular embryos with both proembryo and suspensor characterized by a disordered cell cluster with an irregular shape, suggesting defects in embryo development. The suppression of both NF-YA3 and NF-YA8 gene expression by RNAi experiments resulted in defective embryos that phenocopied the nf-ya3 nf-ya8 double mutants, whereas complementation experiments partially rescued the abnormal globular nf-ya3 nf-ya8 embryos, confirming that NF-YA3 and NF-YA8 are required in early embryogenesis. Finally, the lack of GFP expression of the auxin responsive DR5rev::GFP marker line in double mutant embryos suggested that mutations in both NF-YA3 and NF-YA8 affect auxin response in early developing embryos. Our findings indicate that NF-YA3 and NF-YA8 are functionally redundant genes required in early embryogenesis of Arabidopsis thaliana.

Effect of alkali treatment on the spectral response of silicon-nanowire solar cells

The effect of alkali treatment of Si nanowires (SiNWs) on the spectral response of solar cells was investigated using monochromatic incident photon-to-electron conversion efficiency spectroscopy. SiNWs were prepared on a substrate by metal-assisted etching and were then treated with NaOH/isopropanol. The results show that alkali treatment of SiNWs for 30s obviously improved the cell conversion efficiency. This was attributed to enhancement of the red light response and a decrease in surface reflectivity from 6% to ~2%. However, SiNW alkali treatment led to poor blue light response, which is a major limiting factor for efficient SiNW solar cells. To improve the photovoltaic properties of SiNW cells, a near-complete response over the whole solar spectrum is essential.

Arabidopsis Casein Kinase1 Proteins CK1.3 and CK1.4 Phosphorylate Cryptochrome2 to Regulate Blue Light Signaling

Casein kinase1 (CK1) plays crucial roles in regulating growth and development via phosphorylating various substrates throughout the eukaryote kingdom. Blue light is crucial for normal growth of both plants and animals, and blue light receptor cryptochrome2 (CRY2) undergoes blue light-dependent phosphorylation and degradation in planta. To study the function of plant CK1s, systematic genetic analysis showed that deficiency of two paralogous Arabidopsis thaliana CK1s, CK1.3 and CK1.4, caused shortened hypocotyls, especially under blue light, while overexpression of either CK1.3 or CK1.4 resulted in the insensitive response to blue light and delayed flowering under long-day conditions. CK1.3 or CK1.4 act dependently on CRY2, and overexpression of CK1.3 or CK1.4 significantly suppresses the hypersensitive response to blue light by CRY2 overexpression. Biochemical studies showed that CK1.3 and CK1.4 directly phosphorylate CRY2 at Ser-587 and Thr-603 in vitro and negatively regulate CRY2 stability in planta, which are stimulated by blue light, further confirming the crucial roles of CK1.3 and CK1.4 in blue light responses through phosphorylating CRY2. Interestingly, expression of CK1.3 and CK1.4 is stimulated by blue light and feedback regulated by CRY2-mediated signaling. These results provide direct evidence for CRY2 phosphorylation and informative clues on the mechanisms of CRY2-mediated light responses.

Modeling the functioning of YtvA in the general stress response in Bacillus subtilis

The blue-light photoreceptor YtvA activates the general stress response (GSR) of Bacillus subtilis by activating a large protein complex (the stressosome). We have constructed a model for the YtvA's photocycle, and derived an equation for the fraction of YtvA in the light-induced signaling state at a given light intensity. The model was verified experimentally in vitro on wild type YtvA and on an R63K mutant with faster recovery kinetics. Application of the model to the activation of the GSR at various light intensities in vivo revealed that the GSR is more sensitive to light than would be expected based on YtvA's in vitro kinetics. These results were confirmed with the R63K mutant and a slower-recovering V28I mutant. Additionally, we have demonstrated the presence of a near-UV-light-induced branching reaction that converts the signaling state of YtvA to the dark state. Extension of the model with this reaction shows that it does not contribute significantly to the in vivo blue-light response. The model represents an important step towards a complete systems biology model of the GSR.

Characterization of Pupil Responses to Blue and Red Light Stimuli in Autosomal Dominant Retinitis Pigmentosa due toNR2E3Mutation

We characterized the pupil responses that reflect rod, cone, and melanopsin function in a genetically homogeneous cohort of patients with autosomal dominant retinitis pigmentosa (adRP). Nine patients with Gly56Arg mutation of the NR2E3 gene and 12 control subjects were studied. Pupil and subjective visual responses to red and blue light flashes over a 7 log-unit range of intensities were recorded under dark and light adaptation. The pupil responses were plotted against stimulus intensity to obtain red-light and blue-light response curves. In the dark-adapted blue-light stimulus condition, patients showed significantly higher threshold intensities for visual perception and for a pupil response compared to controls (P = 0.02 and P = 0.006, respectively). The rod-dependent, blue-light pupil responses decreased with disease progression. In contrast, the cone-dependent pupil responses (light-adapted red-light stimulus condition) did not differ between patients and controls. The difference in the retinal sensitivity to blue and red stimuli was the most sensitive parameter to detect photoreceptor dysfunction. Unexpectedly, the melanopsin-mediated pupil response was decreased in patients (P = 0.02). Pupil responses of patients with NR2E3-associated adRP demonstrated reduced retinal sensitivity to dim blue light under dark adaptation, presumably reflecting decreased rod function. Rod-dependent pupil responses were quantifiable in all patients, including those with non-recordable scotopic electroretinogram, and correlated with the extent of clinical disease. Thus, the chromatic pupil light reflex can be used to monitor photoreceptor degeneration over a larger range of disease progression compared to standard electrophysiology.

A Protein Phosphatase 2A Catalytic Subunit Modulates Blue Light-Induced Chloroplast Avoidance Movements through Regulating Actin Cytoskeleton in Arabidopsis

Chloroplast avoidance movements mediated by phototropin 2 (phot2) are one of most important physiological events in the response to high-fluence blue light (BL), which reduces damage to the photosynthetic machinery under excess light. Protein phosphatase 2A-2 (PP2A-2) is an isoform of the catalytic subunit of PP2A, which regulates a number of developmental processes. To investigate whether PP2A-2 was involved in high-fluence BL-induced chloroplast avoidance movements, we first analyzed chloroplast migration in the leaves of the pp2a-2 mutant in response to BL. The data showed that PP2A-2 might act as a positive regulator in phot2-mediated chloroplast avoidance movements, but not in phot1-mediated chloroplast accumulation movements. Then, the effect of okadaic acid (OA) and cantharidin (selective PP2A inhibitors) on high-fluence BL response was further investigated in Arabidopsis thaliana mesophyll cells. Within a certain concentration range, exogenously applied OA or cantharidin inhibited the high-fluence BL-induced chloroplast movements in a concentration-dependent manner. Actin depolymerizing factor (ADF)/cofilin phosphorylation assays demonstrated that PP2A-2 can activate/dephosphorylate ADF/cofilin, an actin-binding protein, in Arabidopsis mesophyll cells. Consistent with this observation, the experiments showed that OA could inhibit ADF1 binding to the actin and suppress the reorganization of the actin cytoskeleton after high-fluence BL irradiation. The adf1 and adf3 mutants also exhibited reduced high-fluence BL-induced chloroplast avoidance movements. In conclusion, we identified that PP2A-2 regulated the activation of ADF/cofilin, which, in turn, regulated actin cytoskeleton remodeling and was involved in phot2-mediated chloroplast avoidance movements.

Identification of a Regulatory Subunit of Protein Phosphatase 1 Which Mediates Blue Light Signaling for Stomatal Opening

Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase comprised of a catalytic subunit (PP1c) and a regulatory subunit that modulates catalytic activity, subcellular localization and substrate specificity. PP1c positively regulates stomatal opening through blue light signaling between phototropins and the plasma membrane H(+)-ATPase in guard cells. However, the regulatory subunit functioning in this process is unknown. We identified Arabidopsis PRSL1 (PP1 regulatory subunit2-like protein1) as a regulatory subunit of PP1c. Tautomycin, a selective inhibitor of PP1c, inhibited blue light responses of stomata in the single mutants phot1 and phot2, supporting the idea that signals from phot1 and phot2 converge on PP1c. We obtained PRSL1 based on the sequence similarity to Vicia faba PRS2, a PP1c-binding protein isolated by a yeast two-hybrid screen. PRSL1 bound to Arabidopsis PP1c through its RVxF motif, a consensus PP1c-binding sequence. Arabidopsis prsl1 mutants were impaired in blue light-dependent stomatal opening, H(+) pumping and phosphorylation of the H(+)-ATPase, but showed normal phototropin activities. PRSL1 complemented the prsl1 phenotype, but not if the protein carried a mutation in the RVxF motif, suggesting that PRSL1 functions through binding PP1c via the RVxF motif. PRSL1 did not affect the catalytic activity of Arabidopsis PP1c but it stimulated the localization of PP1c in the cytoplasm. We conclude that PRSL1 functions as a regulatory subunit of PP1 and regulates blue light signaling in stomata.

Arabidopsis cryptochrome 2 (CRY2) functions by the photoactivation mechanism distinct from the tryptophan (trp) triad-dependent photoreduction.

Cryptochromes are blue-light receptors mediating various light responses in plants and animals. The photochemical mechanism of cryptochromes is not well understood. It has been proposed that photoactivation of cryptochromes involves the blue-light-dependent photoreduction of flavin adenine dinucleotide via the electron transport chain composed of three evolutionarily conserved tryptophan residues known as the "trp triad." We investigated this hypothesis by analyzing the photochemical and physiological activities of Arabidopsis cryptochrome 2 (CRY2) mutations altered in each of the three trp-triad residues. We found that all trp-triad mutations of CRY2 tested lost photoreduction activity in vitro but retained the physiological and biochemical activities in vivo. Some of the trp-triad mutations of CRY2 remained responsive to blue light; others, such as CRY2(W374A), became constitutively active. In contrast to wild-type CRY2, which undergoes blue-light-dependent interaction with the CRY2-signaling proteins SUPPRESSOR OF PHYA 1 (SPA1) and cryptochrome-interaction basic helix-loop-helix 1 (CIB1), the constitutively active CRY2(W374A) interacts with SPA1 and CIB1 constitutively. These results support the hypothesis that cryptochromes mediate blue-light responses via a photochemistry distinct from trp-triad-dependent photoreduction and that the trp-triad residues are evolutionarily conserved in the photolyase/cryptochrome superfamily for reasons of structural integrity rather than for photochemistry per se.