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UPDATE ON BLUE LIGHT SIGNALING

Blue Light Signaling through the Cryptochromes and Phototropins. So That's What the Blues Is All About¹

Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211

Sunlight is the ultimate energy source for nearly all life on Earth. Yet, its importance to life extends far beyond a source of energy because it is also a critical information carrier. Plants and animals sample the light environment to gain information about local surroundings, time of day, and season of the year. Although animals can use opsin-based visual systems to capture information from their light environments, plants don't have eyes in a metazoan sense. How, then, do plants sample their light environment? In short, plants have evolved a number of diverse, nonopsin photoreceptors capable of perceiving a broad range of light qualities and intensities. Just like children who sort M&Ms into different color groups before eating them, plants utilize various photoreceptors to sort the colors of incident light. However, unlike children who ultimately eat all the M&Ms independent of color and prior sorting, plants have preference for particular colors of light, making them more selective than the child in what they consume. For example, if one considers just the visible and near-infrared regions of the light spectrum (400–850 nm), plants have evolved three major classes of photoreceptors—the cryptochromes (cry), the phototropins (phot), and the phytochromes (phy)—capable of absorbing the blue (400–500 nm) and red/far-red (600–800 nm) portions in particular (Quail, 2002). Thus, like parents who have an apparent proclivity for green M&Ms, these plant photoreceptors have a consumption preference with blue and red being the colors of choice.

On the surface, it appears that plants have restricted their information gathering capacity relative to the light environment. However, it makes adaptive sense to tune ones morphogenic and developmental program to the same light qualities, blue and red wavelengths, utilized by the photosynthetic apparatus. In fact, a majority of the photomorphogenesis (light-dependent changes in morphology or development) occurring in plants is induced by blue or red/far-red signals through the aforementioned three classes of photoreceptors. Gene duplication and selection events have led to the evolution of multiple cry, phot, and phy receptors in higher plants that allow efficient sampling of the blue and red/far-red portions of the spectrum over a wide range of environmental and developmental conditions. Recent developments in our understanding of red/far-red light-dependent photomorphogenic events induced by the phy class of receptors are the subject of another Update. Here, we wish to highlight some of the exciting advances that have occurred over the past couple of years in the area of blue light sensing and response associated with the cry and phot classes of receptors. Although this Update is focused on cry and phot signaling, it is important for the reader to keep in mind that other BL-absorbing pigments, such as xanthophylls (see Talbott et al., 2003 in this issue), although not predominant in their action, can influence cry- and phot-dependent and -independent processes.

	CRYS. A NEW CLASS, NEW MECHANISTIC INSIGHTS, AND NEW DOWNSTREAM SIGNALING ELEMENTS

TOP CRYS. A NEW CLASS,... PHOTS. NEW BIOLOGY, NEW... FUTURE PROSPECTS LITERATURE CITED

 
The cry family of blue light receptors regulates various aspects of plant development, most notably seedling de-etiolation—the transition from a pale nonautotrophic seedling to a green photosynthetically competent one, entrainment of the circadian clock, and day length-sensitive timing of flowering (Lin and Shalitin, 2003). Crys are members of a larger blue light-absorbing chromoprotein superfamily that also includes the DNA photolyases. All members of the cry/photolyase family share an amino-terminal photolyase-related (PHR) domain that is responsible for chromophore binding (both a primary/catalytic flavin and a second light-harvesting deazaflavin or pterin) and light-absorbing capacity (Fig. 1). What, then, separates a cry from a photolyase? In short, crys from both plants and animals lack DNA repair activity that is the hallmark of the photolyases (Sancar, 2003). A recent crystallographic study has provided structural information that may explain this catalytic difference between crys and photolyases (Brudler et al., 2003).

View larger version (8K): [in this window] [in a new window]   Figure 1. Domain features of the plant crys and phots. Most crys can be divided into two major domains: the PHR and cry carboxylterminal (CCT) domains. The PHR domain is the sensor domain and binds two chromophores: (a) a light-harvesting deazaflavin (D) or pterin (P), and (b) a primary/catalytic FAD. The CCT is thought to represent the output domain (see Fig. 2). Within the CCT, there is a sub-domain of conserved sequence designated DAS for the presence of three amino clusters: (a) DQXVP near the amino end of the CCT, (b) a region containing varying numbers of acid residues (E or D), and (c) STAES and GGXVP at the carboxyl end separated by a short non-conserved spacer. The recently discovered cry DASH is the exception to this general domain organization because it lacks the CCT and is essentially just a PHR domain (not shown). The phots can also be divided into two major regions: the LOV domain region that functions as the sensor domain and a Ser/Thr protein kinase domain that functions as the signal output domain. All phots described to date have two LOV domains, each of which bind one FMN as a chromophore.

View larger version (21K): [in this window] [in a new window]   Figure 2. Model for cry-mediated signaling through modulation of gene expression. In dark-and light-grown wild-type plants crys (black/blue protein labeled with PHR and CCT) are proposed to interact with COP1 via their CCT. In darkness, COP1 associates with photomorphogenesis-promoting transcription factors, such as HY5, and the COP10/CSN complex. The latter promotes the ubiquitination of the transcription factors, targeting them for degradation by the proteasome. Thus, in darkness, photomorphogenesis-promoting genes are not transcribed because the proper positive regulators are not present. In blue light, the PHR domain of crys is activated resulting in a presumed structural change that allows phosphorylation of the CCT. This structural change and/or phosphorylation event leads to inactivation of COP1, preventing its interaction with transcriptional activator proteins and the COP10/CSN complex, which in turn allows the transcriptional activators to accumulate, bind to light-responsive promoter elements), and promote transcription of genes necessary for photomorphogenesis. Plants expressing a GUSCCT fusion protein exhibit a constitutive photomorphogenic phenotype that results from a failure of the plants to properly regulate turnover of the COP1 targets. Because of these phenotypes and the fact that the GUS-CCT is constitutively phosphorylated, it is assumed that the CCT portion of GUS-CCT fusion protein assumes a structure similar to that of the CCT from wild-type cry exposed to blue light.

Although it is generally accepted, based on comparisons of sequences and domain architectures, that the crys arose from a photolyase progenitor, it has remained an open question whether all crys have a common progenitor and whether the split between animal and plant CRY evolution occurred before or after the appearance of eukaryotes (Kanai et al., 1997; Cashmore et al., 1999). However, as will be discussed in the following subsection, recent identification of a new class of crys provides new clarity on this issue.

cry DASH, a New Class of DNA-Binding crys That Lack a CCT

Genome sequencing projects provided the impetus for the identification of a new class of crys, dubbed cry DASH because orthologs are present in fruitfly (Drosophila melanogaster), Arabidopsis, Synechocystis sp., and humans (Homo sapiens; Brudler et al., 2003). Synechocystis sp. PCC6803 cry DASH has been shown to be structurally most similar to class I CPD photolyases. Interestingly, both Synechocystis sp. cry DASH and Arabidopsis cry DASH (also called cry3) bind DNA in a sequence-independent fashion, although neither exhibit DNA repair activity (Brudler et al., 2003; Kleine et al., 2003). Although Arabidopsis cry2 has been shown to associate with chromatin (Cutler et al., 2000), cry3 is the only plant cry with clear DNA-binding activity. Arabidopsis cry3 is also distinct from other plant crys examined to date in that it lacks the CCT and is targeted to chloroplasts and mitochondria (Kleine et al., 2003). Relative to intracellular targeting, Arabidopsis cry2 appears to be constitutively nuclear in residence, whereas cry1 can either be nuclear (in darkness) or cytoplasmic (in light; Lin and Shalitin, 2003).

But what do cry DASH holoproteins do? Comparison of microarray expression profiles between Synechocystis sp. wild-type and cry DASH knockout mutant strains suggests that cry DASH functions as a transcriptional repressor (Brudler et al., 2003). Nothing is known at present about the function of Arabidopsis cry3; however, it is intriguing to speculate that, given its localization and ability to bind DNA, it functions as a transcriptional regulator of chloroplast and mitochondrial-encoded genes. The regulation of transcription by crys appears not to be unique to the cry DASH subfamily. For example, mouse CRY1 and CRY2 appear to repress CLOCK:BMAL1-activated transcription in the circadian clock (Kume et al., 1999). More relevant to plants, Arabidopsis cry1 and cry2 appear to derepress gene transcription by interacting with and repressing COP1 activity (Wang et al., 2001; Yang et al., 2001; see below).

Do the cry DASH proteins provide any clarity with respect to the evolutionary history of the cry family? Some. The discovery of cry DASH in the Synechocystis sp., a cyanobacterium, supports the idea that crys evolved from photolyases before (Kanai et al., 1997), rather than after (Cashmore et al., 1999), the divergence of prokaryotes and eukaryotes. However, the possibility of convergent evolution within the cry family still remains. For example, Kleine et al. (2003) have speculated that plant crys evolved from two independent horizontal transfer events, with CRY1 and CRY2 having an endosymbiotic -proteobacteria-like ancestry and CRY3 having an endosymbiotic cyanobacteria-like ancestor.

cry Activity Modulation by Intramolecular Electron Transfer and Phosphorylation

How does light alter cry activity? This is one of the fundamental questions one faces when studying any photoreceptor system. Fortunately for cry investigators, a vast photolyase literature exists that can be used as a springboard to ask very direct questions about cry photochemistry. For example, comparisons of the catalytic FAD-binding pocket of Synechocystis sp. cry DASH and class I CPD photolyase from Escherichia coli demonstrate the structural conservation of three Trp residues required for electron transfer from an external reducing agent to the photoexcited flavin in photolyases (Aubert et al., 2000; Brudler et al., 2003). Results from a recent time-resolved difference spectroscopy study suggest that the reduction of photoexcited FAD in Arabidopsis cry1 also requires intramolecular electron transfer from a Trp residue (Giovani et al., 2003). So, what consequences do these photochemical events have on the function of crys? Two obvious possibilities exist. First, the photoreduction of the excited flavin in photolyase provides the electron that is used to isomerize the pyrimidine dimer in damaged DNA (Sancar, 2003). Although crys do not catalyze repair of damaged DNA, and, with the exception of cry DASH, plant crys do not bind DNA, a similar mechanism of electron transfer to a non-DNA substrate molecule could be essential for the induction of cry-dependent photomorphogenesis. Alternatively, an intramolecular redox reaction—a transfer of electrons to or from the flavin— could result in structural changes within cry itself that allow for signal transmission.

Although no direct evidence exists in support of either of these possibilities, results from studies utilizing Arabidopsis cry1 and cry2 CCTs have been interpreted as supporting an intramolecular redox model (Yang et al., 2001). It has been shown that transgenic Arabidopsis plants expressing cry1 and cry2 CCTs fused to -glucuronidase (GUS) exhibit a constitutive photomorphogenic response in darkness (Yang et al., 2000). This phenotype appears to result from the suppression of COP1 activity through the direct interaction with the CCTs (Wang et al., 2001; Yang et al., 2001). COP1 is an E3 ubiquitin ligase that tags positive regulators of photomorphogenesis, such as the transcription factors HY5 and LAF1, for proteolysis in darkness (Wang and Deng, 2002; Seo et al., 2003; Fig. 2, top). In the light, COP1 activity is repressed, resulting in the accumulation of the positive regulators and transcriptional activation of genes encoding proteins necessary for seedling de-etiolation (Wang and Deng, 2002; Fig. 2, middle). Both fulllength cry and CCT constructs bind to COP1 with similar efficacy independent of light condition, indicating that cry-COP1 interaction itself is not sufficient for inactivation of COP1 (Wang et al., 2001; Yang et al., 2001). It appears, then, that in wild-type plants, the COP1 inactivation requires a light-dependent change in the CCT already bound to COP1 (Fig. 2, middle). Yang et al. (2001) have proposed that an intramolecular redox reaction between the photoactivated PHR and the CCT results in a conformational change that allows cry to inactivate COP1. What, then, is happening in the GUS-CCT transgenic plants? Given the constitutive photomorphogenic phenotypes of the GUS-CCT plants, one must assume that the structure assumed by the GUSCCT fusion proteins mimics that of the CCT in a normal full-length cry that has been activated by light (Fig. 2, bottom). One simple explanation that is consistent with all the data is that the PHR domain functions as a repressor of the CCT and that a light-dependent change in conformation, whether induced by an intramolecular redoc reaction or not, releases this repression. Although these interpretations are speculative, results discussed below suggest that the GUS-CCT fusion protein is biochemically more similar to the "light-activated" CCT then it is to the "ground state" CCT in a wild-type cry.

What other mechanisms might modulate cry function in plants? Shalitin et al. (2002) have demonstrated recently that the CCT of Arabidopsis cry2 becomes phosphorylated in response to blue light irradiation, whereas in darkness, it exists mostly in an unphosphorylated form. The temporal and fluence rate dependencies of phosphorylation suggest that this response is important to the generation of "active" cry2. In addition to being a signal for activation, phosphorylation may also play a role in desensitization as phosphorylated cry2 is targeted for proteolysis (Shalitin et al., 2002), an aspect of cry2 regulation that already had been demonstrated to be light dependent (Guo et al., 1999). It is interesting to note that in contrast to native cry2, transgenically expressed GUS-CCT2 (cry2 CCT) fusion protein is phosphorylated in both dark- and light-grown plants (Shalitin et al., 2002). These results suggest that the constitutive photomorphogenic phenotypes of the GUS-CCT lines discussed above may result from the constitutive phosphorylation of the CCT fusion protein. What, if any, relationship is there between these phosphorylation events and the proposed redox changes that occur in crys? One can speculate that in wild-type plants, a blue light-induced intramolecular electron transfer between the PHR and the CCT results in a conformational change that, although not great enough to dramatically affect cry-COP1 interaction, exposes sites on the CCT to a constitutively active protein kinase and that phosphorylation of the cry promotes COP1 inactivation (Fig. 2, middle). In the GUS-CCT fusion protein, the phosphorylation sites are already exposed; thus, light is not required for COP1 inactivation (Fig. 2, bottom).

Three New Regulators of cry Signaling, One Specific and Two Shared with phyA

From the studies discussed above, it should be obvious that a relatively short transduction chain can exist between crys and their ultimate target responses—changes in gene expression. Minimally, the cry1 and cry2 signaling pathways mediating seedling deetiolation require the receptors themselves, COP1, the COP10/CSN (COP9 signalosome) complex, and targets of COP1 such as HY5 (Fig. 2). Given this information, it is probably not surprising that very few cry-signaling intermediates have been found. Yet, recent studies have identified three new crysignaling loci: HFR1, SUB1, and PP7. HFR1 (also called RSF1 and REP1) encodes a basic helix-loophelix transcription factor that appears to function as a positive regulator of photomorphogenesis downstream of both cry1 and phyA, although how HFR1 functions in both pathways is currently unknown (Duek and Fankhauser, 2003). SUB1 encodes a Ca²⁺ binding protein that appears to function as a repressor of light-dependent HY5 accumulation (Guo et al., 2001). Like HFR1, SUB1 functions both in cry and phyA signaling. Although the precise mechanism by which SUB1 functions is unknown, the knowledge that crys can directly interact with and repress COP1, which stimulates the turnover of HY5, suggests that SUB1 might act as a "rheostat" to fine-tune crydependent accumulation of HY5 and other COP1-targeted factors. PP7 encodes a Ser/Thr protein phosphatase with high sequence homology to the fruitfly retinal degeneration C phosphatase that appears to function as a positive and specific regulator of cry signaling (Møller et al., 2003). The biochemical function of PP7 raises an obvious question: Is PP7 action related to cry2 phosphorylation status and function? This appears unlikely because PP7 knockdown lines exhibit reduced rather than increased blue light responsiveness as might be predicted because phosphorylated cry2 is apparently the "active" form. Møller et al. (2003) have proposed that PP7 may regulate the function of a nuclear signaling intermediate to modulate the flux of signaling downstream of crys.

	PHOTS. NEW BIOLOGY, NEW MECHANISTIC INSIGHTS, AND NEW DOWNSTREAM SIGNALING ELEMENTS

TOP CRYS. A NEW CLASS,... PHOTS. NEW BIOLOGY, NEW... FUTURE PROSPECTS LITERATURE CITED

 
The phot blue light receptors regulate a number of blue light-induced responses in plants including chloroplast relocalization, stomatal opening, and phototropism (Briggs and Christie, 2002). What do all these responses have in common? Each requires the movement of some part of the plant: Chloroplast relocalization involves the movement of an organelle to either the periclinal or anticlinal walls depending upon the intensity of blue light (Wada et al., 2003), stomatal opening involves the tugor-driven "movement" of two opposing guard cells in response to relatively high-intensity blue light (Schroeder et al., 2001), and phototropism involves the movement of whole-plant organs toward or away from directional blue light (Liscum, 2002). However, as will be discussed shortly, these are not the only processes dependent upon phot action.

The phots are the founding members of a larger superfamily known as the LOV domain family that includes such proteins as WHITE COLLAR-1 of Neurospora crassa and YtvA of Bacillus subtillis (Crosson et al., 2003). The hallmark feature of the phot/LOV-domain superfamily is the LOV (light, oxygen, and voltage) domain itself—an approximately 110-amino acid motif that is responsible for chromophore (flavin)-binding and light-sensing capacity in photoactive LOV domain-containing proteins (Fig. 1). What, then, separates the phots from other members of the phot/LOV domain superfamily? First, the phots are the only members of the family to contain two LOV domains (LOV1 and LOV2); all other members contain a single LOV domain (Crosson et al., 2003). The significance of having two LOV domains in the phots is not fully understood. However, as will be discussed below, some important insights have been made relative to the functions of LOV1 and LOV2. Second, although a Ser/Thr protein kinase domain is coupled to the LOV domain region in the phots (Fig. 1), other members of the superfamily couple diverse and unrelated output domains to the LOV sensor domain (Crosson et al., 2003). Thus, although all members of the phot/LOV domain superfamily are capable of light sensing through the LOV domain, output signals to initiate downstream events vary from one member to another. Although no downstream phosphorylation targets have been identified to date, it is clear that activity of the protein kinase domain of phot1 is necessary for proper transduction of phototropic signals (Christie et al., 2002). How exactly is the activated state of a phot transduced? Some recent developments in the identification of downstream signaling events will be discussed below.

phots. Not Just for Movement Anymore

The last couple of years have seen a number of "orphan" blue light responses, relative to what BL-receptor controls them, move to the "photdependent" list of responses. First, it has been demonstrated that the rapid, transient inhibition of hypocotyl growth by blue light that precedes the cry1-, cry2-, and phyA-dependent inhibition response requires phot1 activity (Folta and Spalding, 2001; Parks et al., 2001). Although phot1 deficiency has no detectable effect on the resultant "end point" phenotype of seedlings grown in several days of continuous blue light (Liscum and Briggs, 1995), phot1 mutants do exhibit a slight delay in cry1-dependent growth inhibition only observable through high-resolution kinetic analyses (Folta and Spalding, 2001). Second, Folta and Kaufman (2003) have shown that phot1 is essential for the blue light-dependent destabilization of the Lhcb and rbcL transcripts in Arabidopsis. Consistent with this finding, low-light-acclimated phot1 mutants continue to accumulate LHCB and RCBL proteins when transferred to high-light conditions, whereas wild-type and cry1 mutants slow the accumulation of these proteins (Weston et al., 2000). Third, phot has been shown to be required for multiple aspects of sexual development in Chlamydomonas reinhardtii that are dependent upon blue light, including pregamete to gamete conversion, up-regulation of gene expression during late gametogenesis, maintenance of mating competence, and zygote germination (Huang and Beck, 2003). What is particularly striking about these findings in C. reinhardtii is that these are the types of developmental responses that have been traditionally associated with cry function in higher plants.

phot Activity Modulation by a Self-Contained Photocycle, LOV Domain Conformational Change, and Autophosphorylation

How does light absorption by the LOV domains alter phot activity? Given the knowledge that both phots and crys utilize a flavin as the primary chromophore, we might expect these two classes of blue light receptors to exhibit conserved photochemical properties. Add a few more pieces of information, however, and we'll come to a different conclusion. For example, although crys utilize FAD as their primary chromophore (Lin and Shalitin, 2003), phots apparently bind FMN exclusively (Crosson et al., 2003; Fig. 1). Not only is the species of flavin chromophore different between the crys and phots, but the amino acid sequences of their flavin-binding domains are also distinct. These differences contribute dramatically to the production of two very different holoprotein structures that impart different photochemical properties to the two classes of chromoproteins (Crosson et al., 2003; Sancar, 2003).

What properties define the light reaction of a phot LOV domain? In brief, phot LOV domains undergo a self-contained photocycle in which a stable yet darkreversible covalent adduct is formed between the Cys within the conserved NCRFL motif in the LOV domain and the C(4a) atom of the FMN chromophore (Fig. 3). This photocycle was first described by Salomon et al. (2000) using solution spectrographic methods and was later confirmed by crystallographic studies (Crosson and Moffat, 2001, 2002; Federov et al., 2003). Importantly, the formation of this cysteinyl-flavin adduct in LOV2 is absolutely essential for the phototropic function of phot1 in Arabidopsis seedlings (Christie et al., 2002). Curiously, phot1 function does not appear to require adduct formation in LOV1.

View larger version (22K): [in this window] [in a new window]   Figure 3. Proposed photocycle of LOV domains from phots. In darkness, each phot LOV domain binds, non-covalently, one molecule of ground-state FMN (white structure on black background). Absorption of blue light results in the generation of the singlet excited state FMN (blue asterisk). Intersystem crossing results in the formation of the triplet-state (blue T), and this excited state is stabilized by protonation of the N5 atom of the isoalloxazine ring through the abstraction of a proton from the conserved Cys in the LOV domain polypeptide. This protonation of the triplet state FMN increases the electrophilicity of the C(4a) atom and promotes nucleophilic attack of the thiol anion at this position, resulting in the formation of the cysteinyl-FMN adduct. This adduct is completely reversible in darkness. The catalytically reactive N5 and C(4a) atoms are highlighted on the ground state molecule, and their excited orbitals are highlighted in blue on the singlet and triplet state molecules. This model is adapted from that published by Kennis et al. (2003).

The LOV domain photocycle reflects a fundamentally different photoreaction for the phot/LOV domain family compared with other light-responsive flavin-binding proteins. For example, activation of most photoreactive flavoproteins requires electron transfer to generate an excited flavin radical, such as occurs in the cry/photolyase family (Sancar, 2003). In contrast, electron transfer processes appear unnecessary for the photoactivation of phots (Kennis et al., 2003), although an excited neutral flavin radical (FMNH) can be formed if the critical Cys is mutated to prevent adduct formation (Kay et al., 2003). The normal primary photoproduct in the phots appears to be a FMN triplet state that is stabilized by proton transfer from the conserved Cys to the N5 atom of the excited FMN (Corchnoy et al., 2003; Kennis et al., 2003). This protonation event triggers thiolate attack at C(4a), resulting in the cysteinyl-flavin adduct (Fig. 3).

How does this cysteinyl-flavin adduct promote signal transduction? Crystallographic comparisons of dark- and photo-activated states of a LOV2 domain from Adiantum capillus-veneris suggests that only minor conformational changes accompany the cysteinyl-flavin adduct formation (Crosson and Moffat, 2002). These results have been interpreted as indicating that phot signal transmission occurs via dynamical changes in the protein rather than gross conformational changes (Crosson et al., 2003). However, vibrational and NMR spectroscopy studies of a recombinant LOV2 domain from oat (Avena sativa) suggest that fairly substantial structural changes can occur (Salomon et al., 2001; Swartz et al., 2002). These latter studies differ from the crystallographic studies in that spectroscopy was done using a recombinant LOV domain fused to the calmodulin-binding domain from myosin light chain kinase. It seems likely, then, that the protein context in which the LOV domain resides determines, in part, the structural changes that occur in response to light absorption. Whatever the structural consequence, cysteinylflavin adduct formation ultimately leads to the activation of the protein kinase domain of the phots and subsequent transduction of signal (Crosson et al., 2003).

At present, there is only one known substrate for the phot kinase—the phots themselves. As would be expected from the discussion of the photoactivation mechanism, phot autophosphorylation is light dependent (Briggs and Christie, 2002). A recent study has demonstrated that the autophosphorylation of phot1a from oat is hierarchical (Salomon et al., 2003). In particular, low-fluence (1 µmol m^-2) blue light stimulates phosphorylation of four Ser residues, two amino-terminal and two slightly carboxyl-terminal to LOV1, whereas higher fluences (10 µmol m^-2) stimulate phosphorylation of four Ser residues, all carboxyl-terminal to the previous four but still amino-terminal to LOV2. Why should there be low- and high-fluence-responsive phosphorylation sites in phots? Phosphorylation of the low-fluence sites might initiate phototropic signaling by electrostatically altering protein-protein interactions with downstream signaling components such as NPH3 (Motchoulski and Liscum, 1999). In contrast, phoshorylation at higher fluence sites may be prerequisite for receptor desensitization (Liscum, 2002; Salomon et al., 2003). It remains to be seen whether these hypotheses have validity and whether or not phots have other phosphorylation substrates.

phot-Activated Ion Channels. Key Players in phot Signaling?

The regulation of ion channel activity by light has long been proposed to play a central role in photomorphogenesis, including several blue light responses now known to be under phot control (Spalding, 2000). Given this information and the plasma membrane localization of both the phots (Christie et al., 2002; Sakamoto and Briggs, 2002) and a majority of the channels linked to blue light responses, it is not unreasonable to suspect that phots might regulate channel activity. However, does any evidence for such an association exist? Four studies, using three independent approaches, have recently demonstrated that there are phot-activated Ca²⁺ channels in Arabidopsis.

First, Baum et al. (1999) have used transgenic Arabidopsis expressing the Ca²⁺-sensitive fluorescent protein, apoaequorin, to show that a blue light-dependent transient increase in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) occurs in an otherwise wildtype seedling but is dramatically attenuated in a phot1 mutant. In a second study utilizing apoaequorin transgenic plants, Harada et al. (2003) found that both phot1 and phot2 mediate a blue light-dependent increase in [Ca²⁺]_cyt in Arabidopsis leaves. Moreover, the authors were able to use pharmacological agents to show that both phot1 and phot2 mediate Ca²⁺ influx from the apoplast, whereas only phot2 is capable of mediating an increase in [Ca²⁺]_cyt through mobilization from internal stores, likely via a phospholipase C-dependent pathway. Microelectrode ion flux measurements in cotyledons and hypocotyls of wild-type and phot mutants were used in a third study to show that a rapid phot1-dependent increase in [Ca²⁺]_cyt occurs in response to blue light exposure (Babourina et al., 2002). This study, like that of Harada et al. (2003), also suggested that phot2 mediates movement of Ca²⁺ from internal stores, whereas the phot1 effects are on apoplastic stores exclusively. Last, Stoelzle et al. (2003) have used patch-clamping techniques to show that blue light activation of a plasma membranelocalized Ca²⁺ channel is dramatically reduced in leaf mesophyll cells from phot1 single mutants and is essentially eliminated in phot1 phot2 double mutants. Although each of these studies clearly shows a requirement for phots in the blue light regulation of Ca²⁺ channel activity, none provides direct causal evidence for a role of Ca²⁺ fluxes in any of the known phot-dependent responses. However, the tissue/organ locations of the observed fluxes are certainly consistent with the locations of each of the known phot responses.

A more causal connection between phots, ion channel activity, and physiological response has been provided by a study of phot1/phot2-dependent stomatal opening (Kinoshita et al., 2001). In this study, it was shown that epidermal strips from a phot1 phot2 double mutant, which essentially fails to exhibit blue light-induced stomatal opening, fail to extrude protons in response to blue light. This proton extrusion is known to be mediated by an H⁺-ATPase and to be essential for stomatal opening (Schroeder et al., 2001). It is intriguing to note that blue light activation of this H⁺-ATPase occurs via phosphorylation (Kinoshita and Shimazaki, 1999). Is the H⁺-ATPase a substrate for phot kinase activity? This remains one of the open questions facing phot researchers.

	FUTURE PROSPECTS

TOP CRYS. A NEW CLASS,... PHOTS. NEW BIOLOGY, NEW... FUTURE PROSPECTS LITERATURE CITED

 
The future of photobiology is limited only by the imaginations of its researchers. With the new tools of genomics and proteomics, targets of photobiological pathways will not remain elusive indefinitely. Structural biology is also playing an important role in the advancement of cry and phot research. Clearly, having crystal structures for proteins of interest allows one to design experiments aimed at addressing very precise questions. As discussed in this Update, information gained from structural and photochemical studies is providing important insight into cry and phot function. By coupling these technologies with genetics, we will continue to make important inroads into understanding cry and phot signaling. Lest we forget Darwin, comparisons of cry/photolyase and phot/LOV domain family members across different taxa are also playing significant roles in the current research activities. Phylogenetic studies across not just sequence but also structure and function (dare we say Evo-Devo?) will provide new insights otherwise not accessible. This truly is an exciting time to study photobiology—maybe it's time for your rendezvous with the blues!

	ACKNOWLEDGMENTS

 
We would like to thank Vera Quecini, Alex Esmon, and Bethany Stone for helpful discussions and critical comments. We regret that because of space constraints and the desire to engage young readers from broad training background in plant biology, many of the recent studies on blue light signaling, especially with respect to structure-function of the phots, were not discussed here. We apologize to all of our colleagues whose works were not discussed here, but we encourage all readers who have found this Update interesting to delve deeper into the rich BL signaling literature.

Received July 22, 2003; returned for revision August 21, 2003; accepted September 19, 2003.

	FOOTNOTES

 
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.030601.

¹ This work was supported by the National Science Foundation (grant nos. MCB–0077312 and IBN–0114992 to E.L.) and by the University of Missouri Life Sciences Program (predoctoral fellowships to T.J.C. and D.W.H.).

^* Corresponding author; e-mail liscume{at}missouri.edu; fax 573–882–0123.

	LITERATURE CITED

TOP CRYS. A NEW CLASS,... PHOTS. NEW BIOLOGY, NEW... FUTURE PROSPECTS LITERATURE CITED

 
Aubert C, Vos MH, Mathis P, Eker APM, Brettel K (2000) Intraprotein radical transfer during photoactivation of DNA photolyase. Nature 405: 586-590[CrossRef][Medline]

Babourina O, Newman I, Shabala S (2002) Blue light-induced kinetics of H⁺ and Ca²⁺ fluxes in etiolated wild-type and phototropin-mutant Arabidopsis seedlings. Proc Natl Acad Sci USA 99: 2433-2438[Abstract/Free Full Text]

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