The Signaling Mechanism of Arabidopsis CRY1 Involves Direct Interaction with COP1

Dark-grown transgenic Arabidopsis seedlings expressing the C-terminal domains (CCT) of the cryptochrome (CRY) blue light photoreceptors exhibit features that are normally associated only with light-grown seedlings, indicating that the signaling mechanism of Arabidopsis CRY is mediated through CCT. The phenotypic properties mediated by CCT are remarkably similar to those of the constitutive photomorphogenic1 (cop1) mutants. Here we show that Arabidopsis cryptochrome 1 (CRY1) and its C-terminal domain (CCT1) interacted strongly with the COP1 protein. Coimmunoprecipitation studies showed that CRY1 was bound to COP1 in extracts from both dark- and light-grown Arabidopsis. An interaction also was observed between the C-terminal domain of Arabidopsis phytochrome B and COP1, suggesting that phytochrome signaling also proceeds, at least in part, through direct interaction with COP1. These findings give new insight into the initial step in light signaling in Arabidopsis, providing a molecular link between the blue light receptor, CRY1, and COP1, a negative regulator of photomorphogenesis.     

Green light stimulates early stem elongation, antagonizing light-mediated growth inhibition

During the transition from darkness to light, the rate of hypocotyl elongation is determined from the integration of light signals sensed through the phototropin, cryptochrome, and phytochrome signaling pathways. In all light conditions studied, from UV to far-red, early hypocotyl growth is rapidly and robustly suppressed within minutes of illumination in a manner dependent upon light quality and quantity. In this study, it is shown that green light (GL) irradiation leads to a rapid increase in the growth rate of etiolated Arabidopsis seedlings. GL-mediated growth promotion was detected in response to constant irradiation or a short, single pulse of light with a similar time course. The response has a threshold between 10(-1) and 10(0) micromol m(-2), is saturated before 10(2) micromol m(-2) and obeys reciprocity. Genetic analyses indicate that the cryptochrome or phototropin photoreceptors do not participate in the response. The major phytochrome receptors influence the normal amplitude and timing of the GL response, yet the GL response is normal in seedlings grown for hours under constant dim-red light. Therefore, phytochrome activation enhances, but is not required for, the GL response. Seedlings grown under green, red, and blue light together are longer than those grown under red and blue alone. These data indicate that a novel GL-activated light sensor promotes early stem elongation that antagonizes growth inhibition.     

Chimeric proteins between cry1 and cry2 Arabidopsis blue light photoreceptors indicate overlapping functions and varying protein stability.

A blue light (cryptochrome) photoreceptor from Arabidopsis, cry1, has been identified recently and shown to mediate a number of blue light-dependent phenotypes. Similar to phytochrome, the cryptochrome photoreceptors are encoded by a gene family of homologous members with considerable amino acid sequence similarity within the N-terminal chromophore binding domain. The two members of the Arabidopsis cryptochrome gene family (CRY1 and CRY2) overlap in function, but their proteins differ in stability: cry2 is rapidly degraded under light fluences (green, blue, and UV) that activate the photoreceptor, but cry1 is not. Here, we demonstrate by overexpression in transgenic plants of cry1 and cry2 fusion constructs that their domains are functionally interchangeable. Hybrid receptor proteins mediate functions similar to cry1 and include inhibition of hypocotyl elongation and blue light-dependent anthocyanin accumulation; differences in activity appear to be correlated with differing protein stability. Because cry2 accumulates to high levels under low-light intensities, it may have greater significance in wild-type plants under conditions when light is limited.     

Light signals, phytochromes and cross-talk with other environmental cues

Plants have evolved highly complex sensory mechanisms to monitor their surroundings and adapt their growth and development to the prevailing environmental conditions. The integration of information from multiple environmental cues enables the co-ordination of development with favourable seasonal conditions and, ultimately, determines plant form. Light signals, perceived via the phytochrome, cryptochrome and phototropin photoreceptor families, are especially important environmental signals. Redundancy of function among phytochromes and their interaction with blue light photoreceptors enhance sensitivity to light signals, facilitating the accurate detection of, and response to, environmental fluctuations. In this review, current understanding of Arabidopsis phytochrome functions will be summarized, in particular, the interactions among the phytochromes and the integration of light signals with directional and temperature sensing mechanisms.     

Degradation of Arabidopsis CRY2 is regulated by SPA proteins and phytochrome A

The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.     

Action spectrum for cryptochrome-dependent hypocotyl growth inhibition in Arabidopsis

Cryptochrome blue-light photoreceptors are found in both plants and animals and have been implicated in numerous developmental and circadian signaling pathways. Nevertheless, no action spectrum for a physiological response shown to be entirely under the control of cryptochrome has been reported. In this work, an action spectrum was determined in vivo for a cryptochrome-mediated high-irradiance response, the blue-light-dependent inhibition of hypocotyl elongation in Arabidopsis. Comparison of growth of wild-type, cry1cry2 cryptochrome-deficient double mutants, and cryptochrome-overexpressing seedlings demonstrated that responsivity to monochromatic light sources within the range of 390 to 530 nm results from the activity of cryptochrome with no other photoreceptor having a significant primary role at the fluence range tested. In both green- and norflurazon-treated (chlorophyll-deficient) seedlings, cryptochrome activity is fairly uniform throughout its range of maximal response (390-480 nm), with no sharply defined peak at 450 nm; however, activity at longer wavelengths was disproportionately enhanced in CRY1-overexpressing seedlings as compared with wild type. The action spectrum does not correlate well with the absorption spectra either of purified recombinant cryptochrome photoreceptor or to that of a second class of blue-light photoreceptor, phototropin (PHOT1 and PHOT2). Photoreceptor concentration as determined by western-blot analysis showed a greater stability of CRY2 protein under the monochromatic light conditions used in this study as compared with broad band blue light, suggesting a complex mechanism of photoreceptor activation. The possible role of additional photoreceptors (in particular phytochrome A) in cryptochrome responses is discussed.     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.     

Conditional Synergism between Cryptochrome 1 and Phytochrome B Is Shown by the Analysis of phyA, phyB, and hy4 Simple, Double, and Triple Mutants in Arabidopsis

Wild-type or phyA, phyB, or hy4 mutant Arabidopsis seedlings lacking phytochrome A (phyA), phytochrome B (phyB), or cryptochrome 1 (cry1), respectively, and the double and triple mutants were used in combination with blue-light treatments given simultaneously with red or far-red light. We investigated the interaction between phytochromes and cry1 in the control of hypocotyl growth and cotyledon unfolding. Under conditions deficient for cry1 (short exposures to blue light) or phyB (far-red background), these photoreceptors acted synergistically: Under short exposures to blue light (3 h/d) added to a red-light background, cry1 activity required phyB (e.g. the hy4 mutant was taller than the wild type but the phyBhy4 mutant was not taller than the phyB mutant). Under prolonged exposures to blue light (24 h/d) added to a far-red light background, phyB activity required cry1 (e.g. the phyAphyB mutant was taller than the phyA mutant but the phyAphyBhy4 mutant was not taller than the phyAhy4 mutant). Under more favorable light inputs, i.e. prolonged exposures to blue light added to a red-light background, the effects of cry1 and phyB were independent. Thus, the synergism between phyB and cry1 is conditional. The effect of cry1 was not reduced by the phyA mutation under any tested light condition. Under continuous blue light the triple mutant phyAphyBhy4 showed reduced hypocotyl growth inhibition and cotyledon unfolding compared with the phyAphyB mutant. The action of cry1 in the phyAphyB double mutant was higher under the red-light than the far-red-light background, indicating a synergistic interaction between cry1 and phytochromes C, D, or E; however, a residual action of cry1 independent of any phytochrome is likely to occur.     

Hierarchical coupling of phytochromes and cryptochromes reconciles stability and light modulation of Arabidopsis development

In plants, development is a continuing process that takes place under strong fluctuations of the light environment. Here we show that in Arabidopsis thaliana plants grown under intense white light, coupling of the photoreceptor cryptochrome 2 to developmental processes is broader than previously appreciated. Compared to the wild type, the cry2 mutant showed reduced activity of a Lhcb1*2 promoter fused to a reporter, and delayed flowering. The cry2 mutation also reduced the inhibition of hypocotyl growth, the unfolding of the cotyledons, the rate of leaf production during the vegetative phase, and the pace of development after transition to the reproductive stage; but these effects were obvious only in the absence of cryptochrome 1 and in some cases phytochrome A and/or phytochrome B. Complementary, the cry2 mutation uncovered novel roles for cryptochrome 1 and phytochrome A. The activity of the Lhcb1*2 promoter was higher in the cry1 cry2 mutant than in the cry2 mutant, suggesting that cry1 could be involved in blue-light repression of photosynthetic genes. Surprisingly, the phyA cry1 cry2 triple mutant flowered earlier and showed better response to photoperiod than the cry1 cry2 double mutant, indicating that phyA is involved in light repression of flowering. Growth and development were severely impaired in the quadruple phyA phyB cry1 cry2 mutant. We propose that stability and light modulation of development are achieved by simultaneous coupling of phytochrome A, phytochrome B, cryptochrome 1 and cryptochrome 2 to developmental processes, in combination with context-dependent hierarchy of their relative activities.     

A gain-of-function mutation of Arabidopsis cryptochrome1 promotes flowering

Plants use different classes of photoreceptors to collect information about their light environment. Cryptochromes are blue light photoreceptors that control deetiolation, entrain the circadian clock, and are involved in flowering time control. Here, we describe the cry1-L407F allele of Arabidopsis (Arabidopsis thaliana), which encodes a hypersensitive cryptochrome1 (cry1) protein. Plants carrying the cry1-L407F point mutation have elevated expression of CONSTANS and FLOWERING LOCUS T under short-day conditions, leading to very early flowering. These results demonstrate that not only the well-studied cry2, with an unequivocal role in flowering promotion, but also cry1 can function as an activator of the floral transition. The cry1-L407F mutants are also hypersensitive toward blue, red, and far-red light in hypocotyl growth inhibition. In addition, cry1-L407F seeds are hypersensitive to germination-inducing red light pulses, but the far-red reversibility of this response is not compromised. This demonstrates that the cry1-L407F photoreceptor can increase the sensitivity of phytochrome signaling cascades. Molecular dynamics simulation of wild-type and mutant cry1 proteins indicated that the L407F mutation considerably reduces the structural flexibility of two solvent-exposed regions of the protein, suggesting that the hypersensitivity might result from a reduced entropic penalty of binding events during downstream signal transduction. Other nonmutually exclusive potential reasons for the cry1-L407F gain of function are the location of phenylalanine-407 close to three conserved tryptophans, which could change cry1's photochemical properties, and stabilization of ATP binding, which could extend the lifetime of the signaling state of cry1.     

Cryptochrome, phytochrome, and anthocyanin production

Anthocyanin production in cabbage (Brassica oleracea L.) and tomato (Lycopersicon esculentum Mill.) seedlings exposed to prolonged irradiations was studied under conditions that allowed discrimination, within certain limits, between the contribution of cryptochrome and phytochrome in the photoregulation of the response. The results of the study provide confirming evidence for the involvement of cryptochrome and direct evidence for a significant contribution of cryptochrome to the fluence rate dependence of the response to blue. The results provide some preliminary, direct indication for an interaction between cryptochrome and phytochrome in the photoregulation of anthocyanin production in seedlings exposed to the prolonged irradiations required for a high level of expression of the response. The type and degree of interaction between the two photoreceptors vary significantly, depending on the species and experimental conditions.     

shl, a New set of Arabidopsis mutants with exaggerated developmental responses to available red, far-red, and blue light

The interaction of light perception with development is the subject of intensive genetic analysis in the model plant Arabidopsis. We performed genetic screens in low white light-a threshold condition in which photomorphogenetic signaling pathways are only partially active-for ethyl methane sulfonate-generated mutants with altered developmental phenotypes. Recessive mutants with exaggerated developmental responses were obtained in eight complementation groups designated shl for seedlings hyperresponsive to light. shl1, shl2, shl5, and shl3 shl4 (double mutant) seedlings showed limited or no phenotypic effects in darkness, but showed significantly enhanced inhibition of hypocotyl elongation in low-white, red, far-red, blue, and green light across a range of fluences. These results reflect developmental hyper-responsiveness to signals generated by both phytochrome and cryptochrome photoreceptors. The shl11 mutant retained significant phenotypic effects on hypocotyl length in both the phyA mutant and phyB mutant backgrounds but may be dependent on CRY1 for phenotypic expression in blue light. The shl2 phenotype was partially dependent on PHYB, PHYA, and CRY1 in red, far-red, and blue light, respectively. shl2 and, in particular, shl1 were partially dependent on HY5 activity for their light-hyperresponsive phenotypes. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by CRY1, PHYA, and PHYB and other photoreceptors (CRY2, PHYC, PHYD, and PHYE).     

INTERACTION BETWEEN CRYPTOCHROME AND PHYTOCHROME IN HIGHER PLANT PHOTOMORPHOGENESIS

At least three photoreceptors are involved in the mediation of light action in higher plant photomorphogenesis: cryptochrome (UV-A/blue light photoreceptor), UV-B photoreceptor, and phytochrome. The degree of photoreceptor interaction in photomorphogenesis can apparently vary depending on the response, the species, and the stage of development of the biological system. In most cases of interaction studied so far, Pfr, the physiologically active form of phytochrome, is apparently required for the final expression of the response. In some systems, the cryptochrome and/or UV-B photoreceptor mediated action of UV/blue radiation is required to establish/enhance/maintain responsiveness toward Pfr. There is no evidence for photoreceptor interaction in some response-system combinations. It is not known for sure if this apparent lack of photoreceptor interaction represents a real situation or just a failure to detect it because of experimental limitations. Practically nothing is known about the mechanism of photoreceptor interaction at the molecular level.     

Interactive signalling by phytochromes and cryptochromes generates de-etiolation homeostasis in Arabidopsis thaliana

Single, double, triple and quadruple mutants of phyA, phyB, cry1 and cry2 were exposed to different sunlight irradiances and photoperiods to investigate the roll played by phytochrome A, phytochrome B, cryptochrome 1 and cryptochrome 2 during de-etiolation of Arabidopsis thaliana seedlings under natural radiation. Even the quadruple mutant retained some hypocotyl-growth inhibition by sunlight. Hypocotyl length was strongly affected by interactions among photoreceptors. Double phyA phyB, phyA cry1, and cry1 cry2 mutants were taller than expected from the additive action of single mutations. Some of these redundant interactions required the presence of phytochromes A and/or B. Interactions among photoreceptors resulted in a 44% reduction of the response to irradiance and a 70% reduction of the response to photoperiod. The complex network of interactions among photoreceptors is proposed to buffer de-etiolation against changes in irradiance and photoperiod, i.e light fluctuations not related to the positions of the shoot above or below soil level     

Mutant analyses define multiple roles for phytochrome C in Arabidopsis photomorphogenesis

The analysis of Arabidopsis mutants deficient in the A, B, D, and E phytochromes has revealed that each of these phytochrome isoforms has both distinct and overlapping roles throughout plant photomorphogenesis. Although overexpression studies of phytochrome C (phyC) have suggested photomorphogenic roles for this receptor, conclusive evidence of function has been lacking as a result of the absence of mutants in the PHYC locus. Here, we describe the isolation of a T-DNA insertion mutant of phyC (phyC-1), the subsequent creation of mutant lines deficient in multiple phytochrome combinations, and the physiological characterization of these lines. In addition to operating as a weak red light sensor, phyC may perform a significant role in the modulation of other photoreceptors. phyA and phyC appear to act redundantly to modulate the phyB-mediated inhibition of hypocotyl elongation in red light and to function together to regulate rosette leaf morphology. In addition, phyC performs a significant role in the modulation of blue light sensing. Several of these phenotypes are supported by the parallel analysis of a quadruple mutant deficient in phytochromes A, B, D, and E, which thus contains only active phyC. Together, these data suggest that phyC has multiple functions throughout plant development that may include working as a coactivator with other phytochromes and the cryptochrome blue light receptors.     

Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light-dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light-dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light-induced phosphorylation. These results demonstrate that blue light-dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation.