Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways

Cryptochromes are blue light receptors with multiple signaling roles in plants and animals. Plant cryptochrome (cry1 and cry2) biological activity has been linked to flavin photoreduction via an electron transport chain comprising three evolutionarily conserved tryptophan residues known as the Trp triad. Recently, it has been reported that cry2 Trp triad mutants, which fail to undergo photoreduction in vitro, nonetheless show biological activity in vivo, raising the possibility of alternate signaling pathways. Here, we show that Arabidopsis thaliana cry2 proteins containing Trp triad mutations indeed undergo robust photoreduction in living cultured insect cells. UV/Vis and electron paramagnetic resonance spectroscopy resolves the discrepancy between in vivo and in vitro photochemical activity, as small metabolites, including NADPH, NADH, and ATP, were found to promote cry photoreduction even in mutants lacking the classic Trp triad electron transfer chain. These metabolites facilitate alternate electron transfer pathways and increase light-induced radical pair formation. We conclude that cryptochrome activation is consistent with a mechanism of light-induced electron transfer followed by flavin photoreduction in vivo. We further conclude that in vivo modulation by cellular compounds represents a feature of the cryptochrome signaling mechanism that has important consequences for light responsivity and activation.     

Cryptochromes and phytochromes synergistically regulate Arabidopsis root greening under blue light

To increase their fitness, plants sense ambient light conditions and modulate their developmental processes by utilizing multiple photoreceptors such as phytochrome, cryptochrome and phototropin. Even roots, which are normally not exposed to light, express photoreceptors and can respond to light by developing chloroplasts. In the present study, root greening was observed in Arabidopsis thaliana. Seedlings were grown under monochromatic light and chlorophyll levels in the roots were determined. It was found that blue light was far more effective at inducing chloroplast development in Arabidopsis roots than was red light, and this response was under the control of a strong synergistic interaction between phytochromes and cryptochromes. As expected, the cry1 mutant was deficient in this response. Interestingly, the phyAphyB double mutant failed to respond to blue light under these conditions. This strongly suggests that either phytochrome A or phytochrome B, in addition to cryptochrome, was required for this blue light response. It was further demonstrated that the expression of photosynthetic genes was regulated in the same way. Dichromatic irradiation experiments indicated that this interaction depends on the level of phyB P(FR). Analysis of the cop1, det1 and hy5 mutants indicated that the corresponding factors were involved in the response.     

Cryptochromes are required for phytochrome signaling to the circadian clock but not for rhythmicity

The circadian clock is entrained to the daily cycle of day and night by light signals at dawn and dusk. Plants make use of both the phytochrome (phy) and cryptochrome (cry) families of photoreceptors in gathering information about the light environment for setting the clock. We demonstrate that the phytochromes phyA, phyB, phyD, and phyE act as photoreceptors in red light input to the clock and that phyA and the cryptochromes cry1 and cry2 act as photoreceptors in blue light input. phyA and phyB act additively in red light input to the clock, whereas cry1 and cry2 act redundantly in blue light input. In addition to the action of cry1 as a photoreceptor that mediates blue light input into the clock, we demonstrate a requirement of cry1 for phyA signaling to the clock in both red and blue light. Importantly, Arabidopsis cry1 cry2 double mutants still show robust rhythmicity, indicating that cryptochromes do not form a part of the central circadian oscillator in plants as they do in mammals.     

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.     

Opposing roles of phytochrome A and phytochrome B in early cryptochrome-mediated growth inhibition

The cryptochrome 1 (cry1) photoreceptor is responsible for the majority of the inhibitory effect of blue light on hypocotyl elongation, but phytochrome photoreceptors also contribute to the response through a phenomenon known as coaction. In Arabidopsis thaliana the participation of phytochromes A and B (phyA and phyB) in the early phase of cry1 action was investigated by determining the effects of phyA, phyB and hy1 mutations on a cry1-dependent membrane depolarization, which is caused by the activation of plasma-membrane anion channels within seconds of blue light treatment. High-resolution growth measurements were also performed to determine the timing of the requirement for phytochrome in cry1-mediated growth inhibition, which is causally linked to the preceding anion-channel activation. A null mutation in PHYA impaired the membrane depolarization and prevented the early cry1-dependent phase of growth inhibition as effectively and with the same time course as mutations in CRY1. Thus, phyA is necessary for cry1/cry2 to activate anion channels within the first few seconds of blue light and to suppress hypocotyl elongation for at least 120 min. This finding furthers the notion of an intimate mechanistic association between the cry and phy receptors in mediating light responses. The absence of phyB did not affect the depolarization or growth inhibition during this time frame. Instead, double mutant analyses showed that the phyB mutation suppressed the early growth phenotypes of both phyA and cry1 seedlings. This result is consistent with the emerging view that the prevailing growth rate of a stem is a compromise between light-dependent inhibitory and promotive influences. It appears that phyB opposes the cry1/phyA-mediated inhibition by promoting growth during at least the first 120 min of blue light treatment.     

Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.     

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.     

Cryptochrome 1 contributes to blue-light sensing in pea

Cryptochromes are widespread in higher plants but their physiological roles as blue-light photoreceptors have been examined in relatively few species. Screening in a phyA null mutant background has identified several blue-light response mutants in pea (Pisum sativum), including one that carries a substitution of a highly conserved glycine residue in the N-terminal photolyase-homologous domain of the pea CRY1 gene. Analyses of cry1, phyA, and phyB mutants show that all three photoreceptors contribute to seedling photomorphogenesis under high-irradiance blue light, whereas phyA is the main photoreceptor active under low irradiances. Triple phyA phyB cry1 mutants grown under high-irradiance blue light are indistinguishable from dark-grown wild-type plants in length and leaf expansion but show a small residual response to higher-irradiance white light. Monogenic cry1 mutants have little discernable phenotype at the seedling stage, but later in development are more elongated than wild-type plants. In addition, the loss of cry1 moderates the short-internode phenotype of older phyA mutants, suggesting an antagonism between phyA and cry1 under some conditions. Pea cry1 has a small inhibitory effect on flowering under long and short days. However, the phyA cry1 double mutant retains a clear promotion of flowering in response to blue-light photoperiod extensions, indicating a role for one or more additional blue-light photoreceptors in the control of flowering in pea.     

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.     

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.     

Interactions within a network of phytochrome, cryptochrome and UV-B phototransduction pathways regulate chalcone synthase gene expression in Arabidopsis leaf tissue

The Arabidopsis gene encoding the key flavonoid biosynthesis enzyme chalcone synthase (CHS) is regulated by several environmental and endogenous stimuli. Here we dissect the network of light signalling pathways that control CHS expression in mature leaves using cryptochrome (cry) and phytochrome (phy) deficient mutants. The UV-A/blue light induction of CHS is mediated principally by cry1, but neither cry1 nor cry2 is involved in UV-B induction or in the UV-A and blue light signalling pathways that interact synergistically with the UV-B pathway to enhance CHS expression. Moreover, these synergistic responses do not require phyA or phyB. Phytochrome is a positive regulator of the cry1 inductive pathway, mediating distinct potentiation and coaction effects. A red light pretreatment enhances subsequent cry1-mediated CHS induction. This potentiation is unaltered in phyA and phyB mutants but much reduced in a phyA phyB double mutant, indicating that it requires principally phyA or phyB. In contrast, the cry1-mediated induction of CHS, without pretreatment, is much reduced in phyB but not phyA mutants, indicating coaction between cry1 and phyB. Further experiments with phy-deficient mutants demonstrate that phyB is a negative regulator of the UV-B inductive pathway. We further show that phyB acts upstream of the points of interaction of the UV-A and blue synergism pathways with the UV-B pathway. We propose that phyB functions to balance flux through the cry1 and UV-B signalling pathways.