Review. 1 Cryptochrome Signaling in Plants ABSTRACT INTRODUCTION

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1 P H P B Dispatch: Journal: PHP CE: Saranraj Journal Name Manuscript No. Author Received: No. of pages: 8 PE: Saravanan Photochemistry and Photobiology, 2007, 83: 1 8 Review 1 Cryptochrome Signaling in Plants Qing-Hua Li 1 and Hong-Quan Yang* 1,2 1 National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China 2 School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai, China Received 28 February 2006; accepted 25 September 2006; DOI: j x ABSTRACT Cryptochromes are blue light receptors that mediate various light-induced responses in plants and animals. They share sequence similarity to photolyases, flavoproteins that catalyze the repair of UV light-damaged DNA, but do not have photolyase activity. Arabidopsis cryptochromes work together with the red far-red light receptor phytochromes to regulate various light responses, including the regulation of cell elongation and photoperiodic flowering, and are also found to act together with the blue light receptor phototropins to mediate blue light regulation of stomatal opening. The signaling mechanism of Arabidopsis cryptochromes is mediated through negative regulation of COP1 by direct CRY COP1 interaction through CRY C-terminal domain. Arabidopsis CRY dimerized through its N-terminal domain and dimerization of CRY is required for light activation of the photoreceptor activity. Recently, significant progresses have been made in our understanding of cryptochrome functions in other dicots such as pea and tomato and lower plants including moss and fern. This review will focus on recent advances in functional and mechanism characterization of cryptochromes in plants. It is not intended to cover every aspect of the field; readers are referred to other review articles for historical perspectives and a more comprehensive understanding of this photoreceptor (1 8). INTRODUCTION Cryptochromes were first characterized through the isolation of a T-DNA-tagged allele of hy4 (9), an Arabidopsis mutant deficient in its response to blue UV-A light (10). When Arabidopsis seedlings are grown under light, they have a shortened hypocotyl (the stem) relative to dark-grown seedlings. The hy4 cry1 mutant has a long hypocotyl when grown under blue or UV-A light (9,10). By contrast, the mutant appears like wild-type when grown under red or far-red light under these conditions, the light-induced inhibition of hypocotyl growth is mediated by the phytochrome family of photoreceptors (11). CRY1, the protein encoded by the HY4 CRY1 gene, showed sequence similarity to photolyases, a family of proteins that mediate repair of UV-damaged DNA. Photolyases are a rare class of flavoproteins that mediate a redox reaction in response to the absorption of light (12). The Arabidopsis CRY1 protein also affects anthocyanin production, as well as chalcone synthase gene expression, and mutant cry1 plants are deficient in these responses (13). Overexpression of the CRY1 photoreceptor results in hypersensitivity to blue UV-A light, with transgenic seedlings exhibiting unusually short hypocotyls and high levels of anthocyanin (14). CRY2, the second member of the Arabidopsis cryptochrome family, also affects hypocotyl elongation (15). The CRY2 protein is light-labile and, in accordance with this, the long hypocotyl phenotype associated with the cry2 mutant is most discernible under low-intensity blue light. Both cry1 and cry2 mutations affect flowering time (16,17). Indeed cry2 is allelic to fha, first characterized as a late-flowering Arabidopsis mutant (18). There is now evidence for a third CRY (CRY3) in Arabidopsis, the function of which is presently unknown. The sequence of CRY3 is similar to synechocystis CRY DASH (for Drosophila Arabidopsis Synechocystis Human), functioning as a transcriptional repressor (19). Unlike classical plant cryptochrome, CRY3 lacks a C-terminal extension and it has signal sequence that directs CRY3 into the mitochondrial and chloroplast (20). Genetic studies have demonstrated that cryptochromes interact with phytochromes in the regulation of photomorphogenic development, floral initiation, and in the entrainment of the circadian clock in Arabidopsis (21 25), and biochemical studies have confirmed that cryptochromes physically interact with phytochromes (26,27). Most plant cryptochromes have an N-terminal PHR (for photolyase-related) domain that shares sequence similarity to photolyase and are commonly characterized by a distinguishing C-terminal domain, not present in photolyases (1). Insight into the signaling mechanism of Arabidopsis CRY was obtained through the demonstration that transgenic plants expressing the C-terminal domain of either CRY1 (CCT1) or CRY2 (CCT2) fused to b-glucuronidase (GUS) display a constitutive photomorphogenic (COP) phenotype (28), which is similar to that of mutants of both COP1 and COP9 signalosome, the negative regulators of photomorphogenesis This invited paper is part of the Symposium-in-Print: Photobiology in Asia. *Corresponding author hqyang@sibs.ac.cn (Hong-Quan Yang) Ó 2007 American Society for Photobiology /07 1

2 2 Qing-Hua Li and Hong-Quan Yang (29,30). Both CCT1 and CCT2 were shown to bind to COP1 (31,32), indicating that the signaling mechanism of Arabidopsis CRY1 and CRY2 is mediated through negative regulation of COP1 by direct CRY COP1 interaction. It is now demonstrated that Arabidopsis CRY1 N-terminal domain mediates homodimerization, which is required for light activation of CCT1 (33). A role for cryptochromes in the regulation of flowering time in Arabidopsis Insight into the involvement of cryptochromes in the regulation of floral induction was obtained through the demonstration that a mutation in the Arabidopsis cry2 mutant is allelic to the late-flowering mutant fha (18). It is shown that the flowering time of the cry1 cry2 double mutant is similar to that of the cry2 single mutant in both long-day (LD) and short-day (SD) photoperiods, indicating no interaction of CRY1 and CRY2 in photoperiod flowering (24). However, the cry1 cry2 double mutant showed delayed flowering under monochromatic blue light, whereas neither cry1 nor cry2 single mutant displayed late flowering in blue light, suggesting that CRY2 acts redundantly with CRY1 in promoting flowering induction. The phyb mutant was found to flower early, and studies of the cry2 phyb double mutant demonstrated that it flowered as early as the phyb mutant, indicating antagonistic actions of CRY2 and phyb in the regulation of floral initiation (24). In Arabidopsis, CONSTANS (CO) plays a central role in the photoperiod regulation of flowering. It encodes a nuclear protein containing zinc fingers (34,35), and its mrna abundance is regulated by the circadian clock and accumulates late in the day in plants growing under LDs (36). Under these conditions, CO activates transcription of the FT gene, which encodes a RAF-kinase-inhibitor-like protein that accelerates flowering (36 39). Activation of FT transcription is proposed to depend on posttranscriptional regulation of CO that is triggered by light, and therefore flowering under LDs occurs because of the coincidence of circadian clock-controlled transcription of CO and light-mediated posttranscriptional regulation (36,37,40). It is shown that light stabilizes nuclear CO protein in the evening, whereas in the morning or in darkness the protein is degraded by the proteasome (41). Consistent with the antagonistic actions of cryptochrome and phytochrome in the regulation of flowering time, cryptochromes act to enhance CO stability, whereas phyb acts to promote CO degradation (41). This antagonistic action of cryptochrome and phyb results in the generation of daily rhythms in CO abundance. A role for cryptochromes in the entrainment of the circadian clock in Arabidopsis It is postulated that the circadian system includes three primary components: (1) the input pathways that synchronize the clock mechanism to daily cycles of light and dark, (2) the central oscillator that generates the 24-h time-keeping mechanism, and the output pathways that regulate particular processes (42,43) and (3) cryptochrome and phytochrome photoreceptors act in the input pathway (22). It has been shown that cry1 mutant had longer period lengths relative to wild-type in both high and low intensities blue light, and only in relatively low intensities of blue light cry2 mutant showed a slight change in period length (22). Although cry2 mutant is day length-insensitive and flowers late when grown in white light, the effects of CRY2 on flowering time is independent of the effects on period length (17,22). Further studies have demonstrated that CRY1 and CRY2 act redundantly in blue light input to the clock and CRY1 is required for phya signaling to the clock in both the red and the blue lights (44). A role for cryptochromes in the regulation of stomatal opening in Arabidopsis The stomatal pores of higher plants act as ports that tightly regulate the uptake of CO 2 for photosynthesis and the evaporation of water for transpiration. Situated in the epidermis, they are surrounded by a pair of guard cells, which regulate their opening in response to environmental and internal signals, including light, humidity, CO 2, phytohormones, calcium and reactive oxygen species (45 49). Stomata are closed in darkness but open in response to blue light. The blue light receptor phototropins (PHOT1 and PHOT2) are demonstrated to mediate phototropism, blue light-induced chloroplast migration, blue light-dependent regulation of stomatal opening and photomorphogenic response including initial rapid inhibition of hypocotyl elongation and cotyledon expansion (50 56). A more recent study has shown that CRY-mediated signaling is also involved in the regulation of stomatal opening in Arabidopsis (57). The cry1 cry2 double mutant plants were found to be highly drought-tolerant. In accordance with this finding, the stomata of the cry1 cry2 double mutant showed reduced blue light response, whereas those of the CRY1-overexpressing plants showed hypersensitive response to blue light. Based on a study of the cry1 cry2 phot1 phot2 quadruple mutant, it was demonstrated that stomatal opening of the phot1phot2 mutant remained responsive to blue light, but those of the cry1 cry2 phot1 phot2 quadruple mutant failed to respond to blue light, indicating that CRY functions additively with PHOT in mediating blue lightinduced stomatal opening. Strikingly, in contrast with stomata of the wild-type plants, which are closed in darkness, those of the cop1 mutant were constitutively open in darkness, indicating that COP1 acts as a repressor of stomatal opening. Furthermore, stomata of the cry1 cry2 cop1 triple mutants were open as wide as those of the cop1 single mutant under blue light (57). These studies suggest that CRY COP1 signaling system is used to regulate both photomorphogenesis and blue light regulation of stomatal opening. Since stomatal opening of the cop1 mutant is independent of light, COP1 might also lie downstream of phototropins in mediating this process. Indeed, genetic interaction studies demonstrated that the stomata of the phot1 phot2 cop1 triple mutants are open as wide as those of the cop1 single mutant under blue light, implying both cryptochrome- and phototropin-mediated signaling might converge to COP1 in the mediation of blue light regulation of stomatal opening. In addition to regulating of stomatal opening, cryptochromes have been shown to enhance phototropism under low fluence rate blue light and mediate blue light-dependent, random hypocotyl-bending (56,58).

3 Photochemistry and Photobiology, 2007, 83 3 A role for cryptochromes in the regulation of root development in Arabidopsis Though normally growing underground and being not exposed to light, roots express cryptochromes in Arabidopsis (14,59). Light affects many aspects of root development including root extension, geosensitivity and lateral root formation, and blue light is much more effective than red light in inducing chloroplast development in Arabidopsis roots (60). Indeed, CRY1 was found to be the major photoreceptor for root greening under blue light, although it needs the synergistic action of phytochromes (61). It has been shown recently that cryptochrome plays an essential role in the regulation of primary root elongation growth under blue light. Through analysis of both cry mutants and CRY-overexpressing lines in blue light, it was demonstrated that the root length of the cry1 mutant is significantly less than that of wild-type, whereas the root length of CRY1-overexpressing lines is dramatically longer than that of wild-type. By contrast, the cry2 mutant showed greater stimulation of root growth while CRY2- overexpressing lines exhibited a decrease in root elongation (59), indicating that CRY1 and CRY2 act antagonistically in primary root elongation. Functions of cryptochromes in other dicots Tomato cryptochromes. Tomato is the second higher plant species in which cryptochromes have been cloned and characterized. Four CRY genes have been found in tomato Lycopersicon esculentum: LeCRY1a, LeCRY1b, LeCRY2, and LeCRY3. The amino acid sequences of LeCRY1a or Le- CRY1b and LeCRY2 are more similar to their Arabidopsis counterparts than to each other (62) (Fig. 1) and the functions of LeCRY1a and LeCRY2 have been characterized. Based on an anti-sense transgenic study, it was demonstrated that transgenic tomato plants expressing reduced level of LeCRY1a or cry1a had reduced response of blue light inhibition of long hypocotyls and blue light stimulation of anthocyanin accumulation (63). A genetic study in the phya phyb1 double mutant background facilitated isolation of the phyb2 and cry1 mutants (64). Analysis of the single, double, and triple mutant of cry1, phya, and phyb indicated that LeCRY1 act together with phya and phyb to promote photomorphogenesis and enhance anthocyanin accumulation, which is consistent with the findings made in Arabidopsis. Most recently, the function of LeCRY2 has been characterized through a combination of transgenic overexpression and virus-induced gene silencing (VIGS) (65). Compared with the phenotypes of Arabidopsis CRY2 overexpressors, which showed reduced hypocotyl elongation under low fluence blue light, LeCRY2 overexpressors exhibited shortened hypocotyl and internode under both low- and high-fluence blue light. Furthermore, overexpression of LeCRY2 in tomato resulted in overproduction of anthocyanins and chlorophyll in leaves and of flavonoids and lycopene in fruits. Interestingly, the LeCRY2 overexpressors displayed an unexpected late flowering phenotype under both SD and LD conditions, and an increased outgrowth of axillary branches. Virus-induced gene silencing of LeCRY2 reversed the phenotypes observed for the LeCRY2 overexpressors. Figure 1. Phylogenic tree of plant cryptochromes. The phylogenetic analysis of plant CRY protein families was conducted by MEGA 3.1 and the clustalx1.83 program was used for alignment of multiple sequences. Amino acid sequences of AtCRY1 (AAB28724), AtCRY2 (AAD09837) and AtCRY3 (AAL57669) from Arabidopsis; LeCRY1a (AAD44161), LeCRY1b (AAL02092), LeCRY2 (AAF72556) and LeCRY3 (ABB01166) from tomato; PsCRY1 (AAS79662), PsCRY2a (AAS79666) and PsCRY2b (AAS79667) from pea; BnCRY1 (CAG28805) from rape; OsCRY1a (BAB70686), OsCRY1b (BAB70688) and OsCRY2 (BAC56984) from rice; PpCRY1a (BAA83338) and PpCRY1b (BAB70665) from Moss; AcCRY1 (BAA32810), AcCRY2 (BAA32811), AcCRY3 (BAA32812), AcCRY4 (BAA88423) and AcCRY5 (BAA88424) from Fern were obtained from the National Center for Biotechnology information (NCBI) database. Pea cryptochromes. Three expressed cryptochrome genes have been characterized in the model legume garden pea Pisum sativum: one CRY1 orthologue and two distinct CRY2 genes (PsCRY2a and PsCRY2b) (Fig. 1). These genes encode a fulllength CRY protein that contains the photolyase-related domain (PHR) and the C-terminal domain, characteristic of other higher plant cryptochromes. However, the C-terminal domain of pea and Medicago CRY2b is shorter than that of CRY2 from other species. Dramatic differences in light regulation of the three genes are demonstrated. In particular, PsCRY2b expression shows a high-amplitude diurnal cycling and rapid repression in seedlings transferred from darkness to blue light (66). The role for PsCRY1 in pea development was obtained through characterization of the pea cry1 mutant, which was initially made in the phya mutant background (67). At a moderate fluence rate of blue light, the pea cry1 mutant plants show a substantial reduction in leaflet expansion, but do not show a significant difference in internode elongation. However, the cry1 phya double mutant exhibits an increase in internode elongation under blue light, indicating coactions of

4 4 Qing-Hua Li and Hong-Quan Yang these photoreceptors. Genetic analysis using various double and triple mutants of cry1, phya, and phyb further confirmed the interaction between these photoreceptors. De-etiolation in response to red and far-red light in pea is regulated exclusively by phya and phyb, as the phya phyb double mutant is fully etiolated under both conditions. Consistently, the phya phyb cry1 triple mutant is essentially insensitive to blue, red, and farred light, respectively. The striking difference in internode elongation under blue light between the phya phyb cry1 triple mutant and the phya phyb, phya cry1, and phyb cry1 double mutants indicates a large degree of overlapping role among all these three photoreceptors in the regulation of this response. Only at high fluence rate of white light is the triple mutant found to show a small enhancement in leaflet expansion and retain a small induction of CAB gene expression, indicating that other phytochromes or cryptochromes are involved in this minor response. PsCRY1 is shown to play a minor role in the promotion of flowering in pea, since the cry1 mutant plants grown in SD flowered at the same time as the wild-type plants, and under the extended day treatment flowered slightly earlier than wild-type plants. Because of the unavailability of the cry2 mutant, it remains unknown whether PsCRY2 plays a major role in the regulation of floral induction in pea. Rape cryptochrome. Brassica napus is a close relative of Arabidopsis (Fig. 1). Though it is a natural allotetraploid crop, it has a single copy of CRY1 gene (BnCRY1) on the genome (68). Through an anti-sense expression approach, it has demonstrated that BnCRY1 functions similarly to Arabidopsis CRY1. Namely, the anti sense-bncry1 Brassica transgenic seedlings displayed a blue light-dependent elongated hypocotyl phenotype and the anthocyanin accumulation of these seedlings was significantly reduced, and the adult antisense-bncry1 plants were taller than the wild-type, indicating that BnCRY1 functions as a blue light receptor in the regulation of photomorphogenic development in Brassica species. Monocot cryptochromes. Compared with dicots, monocots do not have hypocotyls and cotyledons. Instead, they have mesocotyls and coleoptiles, whose elongation has been shown to be inhibited by blue light in oat (69), maize (70), and rice (71). Three cryptochromes have been found in barley (72) and rice (4,73): duplicated copies of CRY1 (CRY1a and CRY1b) and CRY2. Rice CRY1 (OsCRY1) shows a high degree of similarity to AtCRY1 and OsCRY2 demonstrates greater homology with AtCRY2 (74,75) (Fig. 1). OsCRY1 fused to green fluorescent protein (GFP) confers short hypocotyl and enhanced anthocyanin phenotype in Arabidopsis (73). It has been demonstrated recently that transgenic rice plants overexpressing either OsCRY1 or OsCRY2 showed a blue light-dependent short coleoptile, leaf sheath and leaf blade phenotype, and that OsCRY2 anti-sense transgenic rice plants flower later than wild-type under both LD and SD (74,75). On fusion with GUS, either the C-terminal domain of OsCRY1 (OsCCT1a and OsCCT1b) or the full-length OsCRY1b displays a constitutive photomorphogenic (COP) phenotype in rice, whereas OsCCT1b mutants corresponding to mis-sense mutations in previously described Arabidopsis cry1 alleles failed to confer a COP phenotype. OsCCT1b interacted physically with rice COP1 (OsCOP1) in yeast cells, and OsCCT1b and OsCOP1 are shown to be co-localized in the living plant cells (74), indicating that the signaling mechanism of OsCRY1 involves direct interaction with OsCOP1. Taken together, these studies suggest that both OsCRY1 and OsCRY2 are implicated in blue-light inhibition of coleoptile and leaf elongation during early seedling development in rice, and that OsCRY2 is involved in the promotion of flowering time in rice. Function of cryptochromes in lower plants Moss cryptochromes. The moss Physcomitrella patens has recently become known as the only plant species in which gene replacement occurs at a high frequency by homologous recombination. This approach was used to generate the Physcomitrella patens cryptochrome mutants: Ppcry1a and Ppcry1b. Using the single and double mutants of Ppcry1a and Ppcry1b, it has been demonstrated that the cryptochromemediated signals regulate multiple developmental processes in moss, including induction of side branch formation on protonema and gametophore induction and development, and repress auxin response through suppression of auxininduced gene expression (76). Detailed photobiological analyses indicated that fluence rate and duration of blue light positively regulate branch formation (77). Moreover, under the blue light background, illumination of red light clearly increases branch number, indicating that the blue light receptors, including cryptochromes and phototropins, might act together with the red light receptor phytochromes to regulate branch development in moss. Fern cryptochromes. Blue light mediates a variety of physiological responses in gametophytes of the fern Adiantum capillus-veneris, including inhibition of spore germination (78), phototropism (79), inhibition of tip growth, apical swelling and subsequent cell division (80,81), and the orientational movements of chloroplasts organelle movements (50,82). Five cryptochrome genes, AcCRY1 to AcCRY5, have been isolated from fern (83). Thus, fern seems to have the largest cryptochrome gene family in plants studied so far (Fig. 1). At present, only AcCRY4 and AcCRY5 have been characterized in details (84). It is demonstrated that expression of both the AcCRY4 and the AcCRY5 genes is regulated by light and phytochromes. Cellular localization studies using fern gametophytes transiently expressing AcCRY fused to GUS indicated that the AcCRY proteins have distinct distribution patterns. Clear nuclear localization has been observed for AcCRY3 and AcCRY4, but not for either AcCRY1 or AcCRY2 or AcCRY5. Furthermore, the nuclear accumulation of AcCRY3 seems blue light-dependent, whereas that of AcCRY4 appears light-independent. These cellular distribution properties observed for AcCRY3 and AcCRY4 imply that these photoreceptors might be responsible for mediating inhibition of spore germination by blue light in fern. Signaling mechanism of Arabidopsis cryptochromes A role for CRY C-terminal domain in CRY signaling. It was demonstrated that transgenic plants expressing the C-terminal domain of either CRY1 (CCT1) or CRY2 (CCT2) fused to b-glucuronidase (GUS) display a constitutive photomorphogenic (COP) phenotype, similar to mutants of both the COP1 and the COP9 signalosome complex (28 30). This COP phenotype was not observed for transgenic plants expressing

5 Photochemistry and Photobiology, 2007, 83 5 mutant GUS-CCT1 proteins corresponding to loss-of-function cry1 alleles, indicating that the COP phenotype observed for GUS-CCT1 is physiologically meaningful. These data suggest that CRY1 and CRY2 signaling in response to light activation is mediated through their C-terminal domains. The striking similarities between the transgenic lines expressing either GUS- CCT1 or GUS-CCT2 and the cop1 mutant, including the COP phenotype, chloroplast development in darkness, genetic epistasis to mutants of multiple photoreceptors, dwarfism of adult plants, and early flowering, led to the molecular demonstration of the mechanism of cryptochrome signaling. Through yeast two-hybrid and co-immunoprecipitation studies, it is shown that both CCT1 and CRY1 interact strongly with COP1 in a light-independent manner (31). Similarly, both CCT2 and CRY2 are shown to interact with COP1 (32). Thus, these findings have given new insight into the initial step in Arabidopsis cryptochrome signaling, providing a molecular link between the blue light receptor CRY1 and CRY2, and COP1. A role for CRY1 N-terminal domain in CRY1 signaling. CRY1 N-terminal domain (CNT1) lacks photolyase activity although it shares sequence homology with photolyase (9,87). Like photolyases, cryptochromes contain two noncovalently bound chromophores, flavin adenine dinucleotide (FAD) as a key cofactor to carry out initial biological function upon photoexcitation and methenyltetrahydrofolate (MTHF) as a lightharvesting antenna to enhance biological efficiency. It has been shown that primary light reactions in CRY1 involve intraprotein electron transfer from tryptophan and tyrosine residues to its flavin cofactor FAD (88). Recently, flavin photoreduction and autophosphorylation analyses of the mutant CRY1 proteins with substitution of two conserved tryptophans (CRY1-W324F and CRY1-W400F) have demonstrated that light-induced flavin reduction via the tryptophan chain may be the primary step in the cryptochrome signaling pathway (89). Analysis of the crystal structure of Arabidopsis CNT1 suggests that it is very similar to photolyase (90). CNT1 is shown to bind ATP in the presence of Mg 2+, and the crystal structure data indicate that the surface cavity near the bound FAD cofactor of CNT1 can bind a single molecule of an ATP analog (90,91). Substitution of GUS for CNT1 in CRY1 is shown to be able to mediate a COP phenotype in darkness, implying that CNT1 performs an inhibitory effect on CCT1. Ethyl methanesulfonate mutagenesis screens have given rise to at least thirteen cry1 mutant alleles that contain mutations that are distributed throughout CNT1, indicating an essential role of CNT1 in CRY1 action (13,86). It is demonstrated that overexpression of CNT1 in wild-type Arabidopsis conferred a cry1 mutant-like phenotype through a dominant-negative mechanism (33). Yeast two-hybrid, in vitro binding, in vivo chemical cross-linking, gel filtration, and co-immunoprecipitation studies indicate that CRY1 homodimerizes in a light-independent manner. Mutagenesis and transgenic studies demonstrate that CNT1-mediated dimerization is required for light activation of the C- terminal domain of CRY1 (CCT1). Transgenic data and native gel electrophoresis studies suggest that multimerization of GUS is both responsible and required for mediating a COP phenotype on fusion to CCT1. These results indicate that the properties of the GUS multimer are analogous to those of the light-modified CNT1 dimer. Cryptochrome phosphorylation Light-dependent protein phosphorylation is important for the biological activity of cryptochromes. It has been reported that recombinant CRY1 protein is phosphorylated by the recombinant oat phya protein in vitro and the phosphorylation occurs in the C-terminal domain (26). Recently, both Arabidopsis CRY1 and human CRY1 have been shown to be lightdependent phosphorylated in vitro (91). In addition, CRY1 is phosphorylated when plants exposed to blue light from darkness and phosphorylation increases in response to increased fluence of blue light. CRY1 phosphorylation appears only in blue light but not in red light or far-red light, and none of phytochrome mutants tested had an effect on the phosphorylation of CRY1. The Arabidopsis CRY1 protein purified from insect cells can be phosphorylated in vitro without the addition of a protein kinase in blue light (86). All these results suggest that cryptochromes may possess blue light-dependent autophosphorylation activity. Mis-sense cry1 mutants that express full-length CRY1 apoprotein and show little blue light inhibition of hypocotyl elongation fail to be phosphorylated in blue light (86). Because many of these mutations (S66 N, G347R, and A462 V) eliminated CNT1 ability to dimerize, the integral structure of the CNT1 dimer may be critical for CCT1 phosphorylation (33). These results suggest that CRY1 phosphorylation is essential to the function or regulation of the photoreceptor, and the integral structure of CRY1 is critical to its phosphorylation. Like CRY1, CRY2 phosphorylaion is not detected in etiolated seedling, but it is detected shortly after seedlings are exposed to blue light. The CRY2 phosphorylation is dependent on both the fluence rate of blue light and the exposure time (85). It has been shown that CRY2 is degraded in blue light, especially under high-fluence blue light (15,26,92). When etiolated seedlings are exposed to blue light, the level of phosphorylated CRY2 begins to increase, then decreases when it increases to a certain level. These results suggest that the phosphorylated CRY2 may be degraded. In contrast to the endogenous CRY2 that is phosphorylated in a blue-lightdependent manner, the GUS CCT2 fusion protein is constitutively phosphorylated. Residues of the GUS CCT2 fusion protein that are phosphorylated must be in CCT2 because no phosphorylation was detected in the GUS protein. Based on these studies, it was proposed that the constitutive light response found in transgenic plants expressing the GUS CCT2 fusion protein is because of a constitutive phosphorylation in CCT2 and that CRY2 activity is dependent on the phosphorylation of its C-terminal domain (85). PROSPECTS Genetic, transgenic and biochemical studies have greatly advanced our understanding of cryptochrome functions and signaling mechanism in plants. However, these progresses have primarily made in Arabidopsis. Because of the lack of various cry mutants in other higher plants, the precise roles for cryptochromes need to be further examined in future studies. Although we have obtained some understanding of cryptochrome function in lower plants, the signaling mechanism remains poorly understood. In Arabidopsis, even though it has been demonstrated that COP1 is the downstream interacting

6 6 Qing-Hua Li and Hong-Quan Yang partner of CRY and that CRY1 is required for blue light regulation of the cytoplasm localization property of COP1 (93), the exact mechanism for CRY1-regulated cellular localization of COP1 is not clear. To date, there have been only two characterized downstream interacting partners for Arabidopsis CRY1: COP1 and ZTL ADO1 (94,95). Thus, it will be worth exploring whether there are other CRY1 signaling components that constitute additional layers of regulation of CRY1- mediated signaling. Although we have known that the Arabidopsis CRY1 C-terminal domain transduces light signal downstream and that the N-terminal domain mediates dimerization of the CRY1 protein, we do not understand how light modulates the properties of the CNT1 dimer and ultimately activates CCT1. Future studies of crystallized full-length CRY1 and GUS-CCT1 fusion proteins should be able to gain insights into this problem. Since the plant development program is controlled by multiple signaling pathways, for example, both auxin and light are implicated in the regulation of plant morphogenesis, it raises the question of whether cryptochrome- (and phytochrome-) mediated signaling interacts with auxin signaling pathway to regulate photomorphogenic development. If so, what mechanism could account for the cross-talking? With regard to the regulation of stomatal movement, it is well understood that the phytohormone ABA acts to induce stomatal closing, and now we know that COP1 also functions to promote stomatal closing and that blue light regulation of stomatal opening by cryptochromes is mediated through negative regulation of COP1 by CRY (57). The FT and SOC1 are shown to be common components of two flowering-time pathways that lie downstream of both cryptochrome and FCA (96), an ABA receptor characterized most recently (97). Thus, it will be interesting to investigate whether COP1 lies downstream of both cryptochrome- and ABAmediated signaling pathways in the regulation of stomatal opening. Recent progress has established the CRY-COP-HY5 signaling system for regulation of photomorphogenic development, in which HY5 is a bzip transcription factor that positively regulates photomorphogenesis (98,99) and is targeted for proteasome-mediated degradation in the nucleus through COP1-HY5 interaction (100), it will be very interesting in future studies to determine whether the same signaling system involves the regulation of stomatal opening. Acknowledgments We thank lab members for helpful discussions and the anonymous reviewer for critical yet constructive comments. This work was supported by grants from the National Natural Science Foundation of China to H.-Q.Y. ( , , and ), Chinese Academy of Sciences, and Shanghai Government. REFERENCES 1. Cashmore, A. R., J. A. Jarillo, Y. J. Wu and D. Liu (1999) Cryptochromes: blue light receptors for plants and animals. Science 284, Cashmore, A. R. (2003) Cryptochromes: enabling plants and animals to determine circadian time. Cell 114, Lin, C. (2002) Blue light receptors and signal transduction. Plant Cell 14, S Lin, C. T. and D. Shalitin (2003) Cryptochrome structure and signal transduction. Annu. Rev. Plant Biol. 54, Sancar, A. (2000) Cryptochrome: the second photoactive pigment in the eye and its role in circadian photoreception. Annu. Rev. Biochem. 69, Partch, C. L. and A. Sancar (2005) Photochemistry and photobiology of cryptochrome blue-light photopigments: the search for a photocycle. Photochem. Photobiol. 81, Ahmad, M. (1999) Seeing the world in red and blue: insight into plant vision and photoreceptors. Curr. Opin. Plant Biol. 2, Banerjee, R. and A. Batschauer (2005) Plant blue-light receptors. Planta 220, Ahmad, M. and A. R. Cashmore (1993) HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366, Koornneef, M., E. Rolff and C. J. P. Spruit (1980) Genetic control of light-inhibited hypocotyl elongation in Arabidopsis thaliana (L.) Heynh. Z. Pflanzenphysiol. Bd. 100, Deng, X. W. and P. H. Quail (1999) Signalling in light-controlled development. Semin. Cell Dev. Biol. 10, Sancar, A. (1994) Structure and function of DNA photolyase. Biochemistry 33, Ahmad, M., C. Lin and A. R. Cashmore (1995) Mutations throughout an Arabidopsis blue-light photoreceptor impair bluelight-responsive anthocyanin accumulation and inhibition of hypocotyl elongation. Plant J. 8, Lin, C., M. Ahmad and A. R. Cashmore (1996) Arabidopsis cryptochrome 1 is a soluble protein mediating blue light-dependent regulation of plant growth and development. Plant J. 10, Lin, C., H. Yang, H. Guo, T. Mockler, J. Chen and A. R. Cashmore (1998) Enhancement of blue-light sensitivity of Arabidopsis seedlings by a blue light receptor cryptochrome 2. Proc. Natl Acad. Sci. USA 95, Bagnall, D. J., R. W. King and R. P. Hangarter (1996) Blue-light promotion of flowering is absent in hy4 mutants of Arabidopsis. Planta 200, Guo, H., H. Yang, T. C. Mockler and C. Lin (1998) Regulation of flowering time by Arabidopsis photoreceptors. Science 279, Koornneef, M., C. J. Hanhart and J. H. van der Veen (1991) A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana. Mol. Gen. Genet. 229, Brudler, R., K. Hitomi, H. Daiyasu, H. Toh, K. Kucho, M. Ishiura, M. Kanehisa, V. A. Roberts, T. Todo, J. A. Tainer and E. D. Getzoff (2003) Identification of a new cryptochrome class. Structure, function, and evolution. Mol. Cell 11, Kleine, T., P. Lockhart and A. Batschauer (2003) An Arabidopsis protein closely related to Synechocystis cryptochrome is targeted to organelles. Plant J. 35, Casal, J. J. and M. A. Mazzella (1998) 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. Plant Physiol. 118, Somers, D. E., P.F. Devlin and S. A. Kay (1998) Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock. Science 282, Neff M. M. and J. Chory (1998) Genetic interactions between phytochrome A, phytochrome B, and cryptochrome 1 during Arabidopsis development. Plant Physiol. 118, Mockler, T. C., H. Guo, H. Yang, H. Duong and C. Lin (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in the regulation of floral induction. Development 126, Hennig, L., M. Funk, G. C. Whitelam and E. Schafer (1999) Functional interaction of cryptochrome 1 and phytochrome D. Plant J. 20, Ahmad, M., J. A. Jarillo, O. Smirnova and A. R. Cashmore (1998) The CRY1 blue light photoreceptor of Arabidopsis interacts with phytochrome A in vitro. Mol. Cell 1, Mas, P., P. F. Devlin, S. Panda and S. A. Kay (2000) Functional interaction of phytochrome B and cryptochrome 2. Nature 408, Yang, H. Q., Y. J. Wu, R. H. Tang, D. Liu, Y. Liu and A. R. Cashmore (2000) The C-termini of Arabidopsis cryptochromes mediate a constitutive light response. Cell 103, Deng, X. W., M. Matsui, N. Wei, D. Wagner, A. M. Chu, K. A. Feldmann and P. H. Quail (1992) COP1, an Arabidopsis regula-

7 Photochemistry and Photobiology, 2007, 83 7 tory gene, encodes a protein with both a zinc-binding motif and a G beta homologous domain. Cell 71, Wei, N., D. A. Chamovitz and X. W. Deng (1994) Arabidopsis COP9 is a component of a novel signaling complex mediating light control of development. Cell 78, Yang, H. Q., R. H. Tang and A. R. Cashmore (2001) The signaling mechanism of Arabidopsis CRY1 involves direct interaction with COP1. Plant Cell 13, Wang, H., L. G. Ma, J. M. Li, H. Y. Zhao and X. W. Deng (2001) Direct interaction of Arabidopsis cryptochromes with COP1 in light control development. Science 294, Sang, Y., Q. H. Li, V. Rubio, Y. C. Zhang, J. Mao, X. W. Deng and H. Q. Yang (2005) N-terminal domain-mediated homodimerization is required for photoreceptor activity of Arabidopsis CRYPTOCHROME 1. Plant Cell 17, Putterill, J., F. Robson, K. Lee, R. Simon and G. Coupland (1995) The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors. Cell 80, Coupland, G., M. I. Igeno, R. Simon, R. Schaffer, G. Murtas, P. Reeves, F. Robson, M. Pineiro, M. Costa, K. Lee and P. Suarez-Lopez (1998) The regulation of flowering time by daylength in Arabidopsis. Symp. Soc. Exp. Biol. 51, Suarez-Lopez, P. and G. Coupland (1998) Plants see the blue light. Science 279, Yanovsky, M. J. and S. A. Kay (2002) Molecular basis of seasonal time measurement in Arabidopsis. Nature 419, Kardailsky, I., V. K. Shukla, J. H. Ahn, N. Dagenais, S. K. Christensen, J. T. Nguyen, J. Chory, M. J. Harrison and D. Weigel (1999) Activation tagging of the floral inducer FT. Science 286, Kobayashi, Y., H. Kaya, K. Goto, M. Iwabuchi and T. Araki (1999) A pair of related genes with antagonistic roles in mediating flowering signals. Science 286, Roden, L. C., H. R. Song, S. Jackson, K. Morris and I. A. Carre (2002) Floral responses to photoperiod are correlated with the timing of rhythmic expression relative to dawn and dusk in Arabidopsis. Proc. Natl Acad. Sci. USA 99, Valverde, F., A. Mouradov, W. Soppe, D. Ravenscroft, A. Samach and G. Coupland (2004) Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303, Roenneberg, T. and M. Merrow (2000) Circadian clocks: Omnes viae Romam ducunt. Curr. Biol. 10, R McClung, C. R. (2001) Circadian Rhythms in Plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, Devlin, P. F. and S. A. Kay (2000) Cryptochromes are required for phytochrome signaling to the circadian clock but not for rhythmicity. Plant Cell 12, Blatt, M. R. (2000) Cellular signaling and volume control in stomatal movements in plants. Annu. Rev. Cell Dev. Biol. 16, Assmann, S. M. and X. Q. Wang (2001) From milliseconds to millions of years: guard cells and environmental responses. Curr. Opin. Plant Biol. 4, Dietrich, P., D. Sanders and R. Hedrich (2001) The role of ion channels in light-dependent stomatal opening. J. Exp. Bot. 52, Schroeder, J. I., G. J. Allen, V. Hugouvieux, J. M. Kwak and D. Waner (2001) Guard Cell Signal Transduction. Annu Rev Plant Physiol Plant Mol Biol, 52, Roelfsema, M. R., S. Hanstein, H. H. Felle and R. Hedrich (2002) CO 2 provides an intermediate link in the red light response of guard cells. Plant J. 32, Kagawa, T. and M. Wada (1994) Brief irradiation with red or blue light induces orientational movement of chloroplasts in darkadapted prothallial cells of the fern Adiantum. J. Plant Res. 107, Sakai, T., T. Kagawa, M. Kasahara, T. E. Swartz, J. M. Christie, W. R. Briggs, M. Wada and K. Okada (2001) Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation. Proc. Natl Acad. Sci. USA 98, Jarillo, J. A., H. Gabrys, J. Capel, J. M. Alonso, J. R. Ecker and A. R. Cashmore (2001) Phototropin-related NPL1 controls chloroplast relocation induced by blue light. Nature 410, Briggs, W. R. and E. Huala (1999) Blue-light photoreceptors in higher plants. Annu. Rev. Cell Dev. Biol. 15, Kinoshita, T., M. Doi, N. Suetsugu, T. Kagawa, M. Wada and K. Shimazaki (2001) Phot1 and phot2 mediate blue light regulation of stomatal opening. Nature 414, Folta, K. M. and E. P. Spalding (2001) Unexpected roles for cryptochrome 2 and phototropin revealed by high-resolution analysis of blue light-mediated hypocotyl growth inhibition. Plant J. 26, Ohgishi, M., K. Saji, K. Okada and T. Sakai (2004) Functional analysis of each blue light receptor, cry1, cry2, phot1, and phot2, by using combinatorial multiple mutants in Arabidopsis. Proc. Natl Acad. Sci. USA 101, Mao, J., Y. C. Zhang, Y. Sang, Q. H. Li and H. Q. Yang (2005) A role for Arabidopsis cryptochromes and COP1 in the regulation of stomatal opening. Proc. Natl Acad. Sci. USA 102, Whippo, C. W. and R. P., Hangarter (2003) Second positive phototropism results from coordinated co-action of the phototropins and cryptochromes. Plant Physiol. 132, Canamero, R. C., N. Bakrim, J. P. Bouly, A. Garay, E. E. Dudkin, Y. Habricot and M. Ahmad (2006) Cryptochrome photoreceptors cry1 and cry2 antagonistically regulate primary root elongation in Arabidopsis thaliana. Planta DOI /s Feldman, L. J. (1984) Regulation of root development. Annu. Rev. Plant Physiol. 35, Usami, T., N. Mochizuki, M. Kondo, M. Nishimura and A. Nagatani (2004) Cryptochromes and phytochromes synergistically regulate Arabidopsis root greening under blue light. Plant Cell Physiol. 45, Perrotta, G., L. Ninu, F. Flamma, J. L. Weller, R.E. Kendrick, E. Nebuloso and G. Giuliano (2000) Tomato contains homologues of Arabidopsis cryptochromes 1 and 2. Plant Mol. Biol. 42, Ninu, L., M. Ahmad, C. Miarelli, A. R. Cashmore and G. Giuliano (1999) Cryptochrome 1 controls tomato development in response to blue light. Plant J. 18, Weller, J. L., G. Perrotta, M. E. Schreuder, A. van Tuinen, M. Koornneef, G. Giuliano and R. E. Kendrick (2001) Genetic dissection of blue-light sensing in tomato using mutants deficient in cryptochrome 1 and phytochromes A, B1 and B2. Plant J. 25, Giliberto, L., G. Perrotta, P. Pallara, J. L. Weller, P. D. Fraser, P. M. Bramley, A. Fiore, M. Tavazza and G. Giuliano (2005) Manipulation of the blue light photoreceptor cryptochrome 2 in tomato affects vegetative development, flowering time, and fruit antioxidant content. Plant Physiol. 137, Platten, J. D., E. Foo, F. Foucher, V. Hecht, J. B. Reid and J. L. Weller (2005) The cryptochrome gene family in pea includes two differentially expressed CRY2 genes. Plant Mol. Biol. 59, Platten, J. D., E. Foo, R. C. Elliott, V. Hecht, J. B. Reid and J. L. Weller (2005) Cryptochrome 1 contributes to blue-light sensing in pea. Plant Physiol. 139, Chatterjee, M., P. Sharma and J. P. Khurana (2006) Cryptochrome 1 from Brassica napus is up-regulated by blue light and controls hypocotyl stem growth and anthocyanin accumulation. Plant Physiol. 141, Thornton, R. M. and K. V. Thimann (1967) Transient effects of light on auxin transport in the Avena coleoptile. Plant Physiol. 111, Wang, X. and M., Iino (1997) Blue-light-induced shrinking of protoplasts from maize coleoptiles and its relationship to coleoptile growth. Plant Physiol. 114, Biswas, K. K., R., Neumann, K., Haga, O., Yatoh and M., Iino (2003) Photomorphogenesis of rice seedlings: a mutant impaired in phytochrome-mediated inhibition of coleoptile growth. Plant Cell Physiol. 44, Perrotta, G. Y. G., E. Nebuloso, L. Renzi and G. Giuliano (2001) Tomato and barley contain duplicated copies of cryptochrome 1. Plant Cell Environ. 24, Matsumoto, N., T. Hirano, T. Iwasaki and N. Yamamoto (2003) Functional analysis and intracellular localization of rice cryptochromes. Plant Physiol. 133,

8 8 Qing-Hua Li and Hong-Quan Yang 74. Zhang, Y. C., S. F. Gong, Q. H. Li, Y. Sang and H. Q. Yang (2006) Functional and signaling mechanism analysis of rice CRYPTOCHROME 1. Plant J. 46, Hirose, F., T. Shinomura, T. Tanabata, H. Shimada and M. Takano (2006) Involvement of rice cryptochromes in de-etiolation responses and flowering. Plant Cell Physiol. 47, Imaizumi, T., A. Kadota, M. Hasebe and M. Wada (2002) Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens. Plant Cell 14, Uenaka, H., M. Wada and A. Kadota (2005) Four distinct photoreceptors contribute to light-induced side branch formation in the moss Physcomitrella patens. Planta 222, Sugai, M. and M. Furuya (1985) Action spectrum in ultraviolet and blue light region for the inhibition of red-light-induced spore germination in Adiantum capillus-veneris L. Plant Cell Physiol. 26, Hayami, J., A. Kadota and M. Wada (1986) Blue light-induced phototropic response and the intracellular photoreceptive site in Adiantum protonemata. Plant Cell Physiol. 27, Wada, M. and M., Furuya (1972) Phytochrome action on the timing of cell division in Adiantum gametophytes. Plant Physiol. 49, Miyata, M., M. Wada and M. Furuya (1979) Effects of phytochrome and blue-near ultraviolet light-absorbing pigment on duration of component phases of the cell cycle in Adiantum gametophytes. Dev. Growth Differ. 21, Yatsuhashi, H., A. Kadota and M. Wada (1985) Blue- and redlight action in photoorientation of chloroplasts in Adiantum protonemata. Planta 165, Kanegae, T. and M. Wada (1998) Isolation and characterization of homologues of plant blue-light photoreceptor (cryptochrome) genes from the fern Adiantum capillus-veneris. Mol. Gen. Genet. 259, Imaizumi, T., T. Kanegae and M. Wada (2000) Cryptochrome nucleocytoplasmic distribution and gene expression are regulated by light quality in the fern Adiantum capillus-veneris. Plant Cell 12, Shalitin, D., H. Yang, T. C. Mockler, M. Maymon, H. Guo, G. C. Whitelam and C. Lin (2002) Regulation of Arabidopsis cryptochrome 2 by blue-light-dependent phosphorylation. Nature 417, Shalitin, D., X. Yu, M. Maymon, T. Mockler and C. Lin (2003) Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1. Plant Cell 15, Lin, C., D. E. Robertson, M. Ahmad, A. A. Raibekas, M. S. Jorns, P. L. Dutton and A. R. Cashmore (1995) Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1. Science 269, Giovani, B., M. Byrdin, M. Ahmad and K. Brettel (2003) Lightinduced electron transfer in a cryptochrome blue-light photoreceptor. Nat. Struct. Biol. 10, Zeugner, A., M. Byrdin, J. P. Bouly, N. Bakrim, B. Giovani, K. Brettel and M. Ahmad (2005) Light-induced electron transfer in Arabidopsis cryptochrome 1 correlates with in vivo function. J. Biol. Chem. 280, Brautigam, C. A., B. S. Smith, Z. Ma, M. Palnitkar, D. R. Tomchick, M. Machius and J. Deisenhofer (2004) Structure of the photolyase-like domain of cryptochrome 1 from Arabidopsis thaliana. Proc. Natl Acad. Sci. U S A 101, Bouly, J. P., B. Giovani, A. Djamei, M. Mueller, A. Zeugner, E. A. Dudkin, A. Batschauer and M. Ahmad (2003) Novel ATP-binding and autophosphorylation activity associated with Arabidopsis and human cryptochrome-1. Eur. J. Biochem. 270, Guo, H., H. Duong, N. Ma and C. Lin (1999) The Arabidopsis blue light receptor cryptochrome 2 is a nuclear protein regulated by a blue light-dependent post-transcriptional mechanism. Plant J. 19, Osterlund, M. T. and X. W. Deng (1998) Multiple photoreceptors mediate the light-induced reduction of GUS-COP1 from Arabidopsis hypocotyl nuclei. Plant J. 16, Somers, D. E., T. F. Schultz, M. Milnamow and S. A. Kay (2000) ZEITLUPE encodes a novel clock-associated PAS protein from Arabidopsis. Cell 101, Jarillo, J. A., J. Capel, R. H. Tang, H. Q. Yang, J. M. Alonso, J. R. Ecker and A.R. Cashmore (2001) An Arabidopsis circadian clock component interacts with both CRY1 and phyb. Nature 410, Samach, A., H. Onouchi, S. E. Gold, G. S. Ditta, Z. Schwarz- Sommer, M. F. Yanofsky and G. Coupland (2000) Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288, Razem, F. A., A. El-Kereamy, S. R. Abrams and R. D. Hill (2006) The RNA-binding protein FCA is an abscisic acid receptor. Nature 439, Oyama, T., Y. Shimura and K. Okada (1997) The Arabidopsis HY5 gene encodes a bzip protein that regulates stimulus-induced development of root and hypocotyl. Genes Dev. 11, Ang, L. H., S. Chattopadhyay, N. Wei, T. Oyama, K. Okada, A. Batschauer and X. W. Deng (1998) Molecular interaction between COP1 and HY5 defines a regulatory switch for light control of Arabidopsis development. Mol. Cell 1, Osterlund, M. T., C. S. Hardtke, N. Wei and X. W. Deng (2000) Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature 405,