Resetting of the circadian clock by phytochromes and cryptochromes in Arabidopsis.

The authors sought to investigate the role of phytochromes A and B (phyA and phyB) and cryptochromes 1 and 2 (cryl and cry2) in the synchronization of the leaf position rhythm in Arabidopsis thaliana. The seedlings were transferred from white light-dark cycles to free-running conditions with or without exposure to a light treatment during the final hours of the last dark period. The phase advance caused by a far-red light treatment was absent in the phyA mutant, deficient in the fhy1 and fhy3 mutants involved in phyA signaling, and normal in the cryl and cryl cry2 mutants. The phase shift caused by blue light was normal in the cry2 mutant; reduced in the phyA, cryl, phyA cry1, and cry1 cry2 mutants; and abolished in the phyA cryl cry2 triple mutant. The phase shift caused by red light was partially retained by the phyA phyB double mutant. The authors conclude that cryl and cry2 participate as photoreceptors in the blue light input to the clock but are not required for the phyA-mediated effects on the phase of the circadian rhythm of leaf position. The signaling proteins FHY1 and FHY3 are shared by phyA-mediated photomorphogenesis and phyA input to the clock.     

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.     

Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.     

Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. In Arabidopsis thaliana, there are genes encoding at least five phytochromes, and it is of interest to learn if the different phytochromes have overlapping or distinct functions. To address this question for two of the phytochromes in Arabidopsis, we have compared light responses of the wild type with those of a phyA null mutant, a phyB null mutant, and a phyA phyB double mutant. We have found that both phyA and phyB mutants have a deficiency in germination, the phyA mutant in far-red light and the phyB mutant in the dark. Furthermore, the germination defect caused by the phyA mutation in far- red light could be suppressed by a phyB mutation, suggesting that phytochrome B (PHYB) can have an inhibitory as well as a stimulatory effect on germination. In red light, the phyA phyB double mutant, but neither single mutant, had poorly developed cotyledons, as well as reduced red-light induction of CAB gene expression and potentiation of chlorophyll induction. The phyA mutant was deficient in sensing a flowering response inductive photoperiod, suggesting that PHYA participates in sensing daylength. In contrast, the phyB mutant flowered earlier than the wild type (and the phyA mutant) under all photoperiods tested, but responded to an inductive photoperiod. Thus, PHYA and PHYB appear to have complementary functions in controlling germination, seedling development, and flowering. We discuss the implications of these results for possible mechanisms of PHYA and PHYB signal transduction.     

An emerging molecular map of the phytochromes

Molecular mapping studies and sequence comparisons are providing provocative new insights into regions of the phytochrome polypeptide important to the functional activities of the photoreceptor. The NH₂-terminal structural domain contains the determinants for photoperception, and for the differences in photosensory specificity and photolability between phyA and phyB. However, a contiguous COOH-terminal domain is also required for the transfer of perceived informational signals downstream to transduction pathway components and for PfrA-specific degradation to proceed. The COOH-terminal domains of phyA and phyB are functionally interchangeable in these processes and a core sequence at the proximal end of this domain contains determinants necessary for signal transfer from both phyA and phyB, suggesting a common biochemical mechanism of signal transfer for the two photoreceptors. Striking sequence similarity between the NH₂-terminal domain of a Synechocystis protein, ORF SLR0473, and the phytochromes indicates that the cyanobacteria contain phytochrome-related photoreceptors. The COOH-terminal domains of ORF SLR0473 and the phytochromes are also related to one another and both show sequence similarities to the sensor histidine kinases. These data raise the possibility that the cyanobacteria have a functional photoregulated histidine kinase signalling system and that the plant phytochromes represent remnants of that system.     

Arabidopsis phytochromes A and B synergistically repress SPA1 under blue light

In Arabidopsis, although studies have demonstrated that phytochrome A(phyA) and phyB are involved in blue light signaling, how blue light-activated phytochromes modulate the activity of the CONSTITUTIVELY PHOTOMORPHOGENIC1(COP1)-SUPPRESSOR OF PHYA-105(SPA1) E3 complex remains largely unknown. Here, we show that phyA responds to early and weak blue light, whereas phyB responds to sustainable and strong blue light. Activation of both phyA and phyB by blue light inhibits SPA1 activity.Specifically,...     

UV-B-induced photomorphogenesis in Arabidopsis thaliana

Relatively little is known about the types of photomorphogenic responses and signal transduction pathways that plants employ in response to ultraviolet-B (UV-B, 290–320 nm) radiation. In wild-type Arabidopsis seedlings, hypocotyl growth inhibition and cotyledon expansion were both reproducibly promoted by continuous UV-B. The fluence rate response of hypocotyl elongation was examined and showed a biphasic response. Whereas photomorphogenic responses were observed at low doses, higher fluences resulted in damage symptoms. In support of our theory that photomorphogenesis, but not damage, occurs at low doses of UV-B, photomorphogenic responses of UV-B sensitive mutants were indistinguishable from wild-type plants at the low dose. This allowed us to examine UV-B-induced photomorphogenesis in photoreceptor deficient plants and constitutive photomorphogenic mutants. The cry1 cryptochrome structural gene mutant, and phytochrome deficient hy1, phyA and phyB mutant seedlings resembled wild-type seedlings, while phyA/phyB double mutants were less sensitive to the photomorphogenic effects of UV-B. These results suggest that either phyA or phyB is required for UV-B-induced photomorphogenesis. The constitutive photomorphogenic mutants cop1 and det1 did not show significant inhibition of hypocotyl growth in response to UV-B, while det2 was strongly affected by UV-B irradiation. This suggests that COP1 and DET1 work downstream of the UV-B signaling pathway.     

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.     

The roles of phytochromes in elongation and gravitropism of roots

Gravitropic orientation and the elongation of etiolated hypocotyls are both regulated by red light through the phytochrome family of photoreceptors. The importance of phytochromes A and B (phyA and phyB) in these red light responses has been established through studies using phy mutants. To identify the roles that phytochromes play in gravitropism and elongation of roots, we studied the effects of red light on root elongation and then compared the gravitropic curvature from roots of phytochrome mutants of Arabidopsis (phyA, phyB, phyD and phyAB) with wild type. We found that red light inhibits root elongation approximately 35% in etiolated seedlings and that this response is controlled by phytochromes. Roots from dark- and light-grown double mutants (phyAB) and light-grown phyB seedlings have reduced elongation rates compared with wild type. In addition, roots from these seedlings (dark/light-grown phyAB and light-grown phyB) have reduced rates of gravitropic curvature compared with wild type. These results demonstrate roles for phytochromes in regulating both the elongation and gravitropic curvature of roots.     

Interactions of phytochromes A,B1 and B2 in light-induced competence for adventitious shoot formation in hypocotyl of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.)

The interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phytochrome B2 (phyB2) in light-dependent shoot regeneration from the hypocotyl of tomato was analysed using all eight possible homozygous allelic combinations of the null mutants. The donor plants were pre-grown either in the dark or under red or far-red light for 8 days after sowing; thereafter hypocotyl segments (apical, middle and basal portions) were transferred onto hormone-free medium for culture under different light qualities. Etiolated apical segments cultured in vitro under white light showed a very high frequency of regeneration for all of the genotypes tested besides phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants. Evidence is provided of a specific interference of phyB2 with phyA-mediated HIR to far-red and blue light in etiolated explants. Pre-treatment of donor plants by growth under red light enhanced the competence of phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants for shoot regeneration, whereas pre-irradiation with far-red light enhanced the frequency of regeneration only in the phyAphyB1 mutant. Multiple phytochromes are involved in red light- and far-red light-dependent acquisition of competence for shoot regeneration. The position of the segments along the hypocotyl influenced the role of the various phytochromes and the interactions between them. The culture of competent hypocotyl segments under red, far-red or blue light reduced the frequency of explants forming shoots compared to those cultured under white light, with different genotypes having different response patterns.Abbreviations HIR: High irradiance response - LFR: Low fluence response - Pfr: Far-red absorbing form of phytochrome - phyA: Phytochrome A - phyB1: Phytochrome B1 - phyB2: Phytochrome B2 - phyA(B1, B2): Phytochrome mutant deficient in phyA (B1, B2) - phyAphyB1(B1B2,AB2): Double phytochrome mutant deficient in phyA and phyB1(B1, B2) - phyAphyB1phyB2: Triple mutant deficient in phyA, phyB1 and phyB2 - VLFR: Very low fluence response - WT: Wild-type tomato Communicated by R. Reski     

Genetic dissection of blue-light sensing in tomato using mutants deficient in cryptochrome 1 and phytochromes A, B1 and B2

Several novel allelic groups of tomato (Solanum lycopersicum L.) mutants with impaired photomorphogenesis have been identified after gamma-ray mutagenesis of phyA phyB1 double-mutant seed. Recessive mutants in one allelic group are characterized by retarded hook opening, increased hypocotyl elongation and reduced hypocotyl chlorophyll content under white light (WL). These mutants showed a specific impairment in response to blue light (BL) resulting from lesions in the gene encoding the BL receptor cryptochrome 1 (cry1). Phytochrome A and cry1 are identified as the major photoreceptors mediating BL-induced de-etiolation in tomato, and act under low and high irradiances, respectively. Phytochromes B1 and B2 also contribute to BL sensing, and the relative contribution of each of these four photoreceptors differs according to the light conditions and the specific process examined. Development of the phyA phyB1 phyB2 cry1 quadruple mutant under WL is severely impaired, and seedlings die before flowering. The quadruple mutant is essentially blind to BL, but experiments employing simultaneous irradiation with BL and red light suggest that an additional non-phytochrome photoreceptor may be active under short daily BL exposures. In addition to effects on de-etiolation, cry1 is active in older, WL-grown plants, and influences stem elongation, apical dominance, and the chlorophyll content of leaves and fruit. These results provide the first mutant-based characterization of cry1 in a plant species other than Arabidopsis.     

Functional diversity of phytochrome family in the control of light and gibberellin‐mediated germination in Arabidopsis

In several species, seed germination is regulated by light in a way that restricts seedling emergence to the environmental conditions that are likely to be favourable for the success of the new individual, and therefore, this behaviour is recognized to have adaptive value. The phytochromes are one of the most relevant photoreceptors involved in light perception by plants. We explored the redundancy and diversity functions of the phytochrome family in the control of seed responsiveness to light and gibberellins (GA) by using a set of phytochrome mutants of Arabidopsis. Our data show that, in addition to the well‐known role of phyB in the promotion of germination in response to high red to far‐red ratios (R/FR), phyE and phyD stimulate germination at very low R/FR ratios, probably by promoting the action of phyA. Further, we show that phyC regulates negatively the seed responsiveness to light, unravelling unexpected functions for phyC in seed germination. Finally, we find that seed responsiveness to GA is mainly controlled by phyB, with phyC, phyD and phyE having relevant roles when acting in a phyB‐deficient background. Our results indicate that phytochromes have multiple and complex roles during germination depending on the active photoreceptor background.     

PKS1 plays a role in red-light-based positive phototropism in roots

Aerial parts of plants curve towards the light (i.e. positive phototropism), and roots typically grow away from the light (i.e. negative phototropism). In addition, Arabidopsis roots exhibit positive phototropism relative to red light (RL), and this response is mediated by phytochromes A and B (phyA and phyB). Upon light stimulation, phyA and phyB interact with the phytochrome kinase substrate (PKS1) in the cytoplasm. In this study, we investigated the role of PKS1, along with phyA and phyB, in the positive phototropic responses to RL in roots. Using a high-resolution feedback system, we studied the phenotypic responses of roots of phyA, phyB, pks1, phyA pks1 and phyB pks1 null mutants as well as the PKS1-overexpressing line in response to RL. PKS1 emerged as an intermediary in the signalling pathways and appears to promote a negative curvature to RL in roots. In addition, phyA and phyB were both essential for a positive response to RL and act in a complementary fashion. However, either photoreceptor acting without the other results in negative curvature in response to red illumination so that the mode of action differs depending on whether phyA and phyB act independently or together. Our results suggest that PKS1 is part of a signalling pathway independent of phyA and phyB and that PKS1 modulates RL-based root phototropism.     

The Light-Response BTB1 and BTB2 Proteins Assemble Nuclear Ubiquitin Ligases That Modify Phytochrome B and D Signaling in Arabidopsis

Members of the Bric-a-Brac/Tramtrack/Broad Complex (BTB) family direct the selective ubiquitylation of proteins following their assembly into Cullin3-based ubiquitin ligases. Here, we describe a subfamily of nucleus-localized BTB proteins encoded by the LIGHT-RESPONSE BTB1 (LRB1) and LRB2 loci in Arabidopsis (Arabidopsis thaliana) that strongly influences photomorphogenesis. Whereas single lrb1 and lrb2 mutants are relatively normal phenotypically, double mutants are markedly hypersensitive to red light, but not to far-red or blue light, and are compromised in multiple photomorphogenic processes, including seed germination, cotyledon opening and expansion, chlorophyll accumulation, shade avoidance, and flowering time. This red light hypersensitivity can be overcome by eliminating phytochrome B (phyB) and phyD, indicating that LRB1/2 act downstream of these two photoreceptor isoforms. Levels of phyB/D proteins but not their messenger RNAs are abnormally high in light-grown lrb1 lrb2 plants, implying that their light-dependent turnover is substantially dampened. Whereas other red light-hypersensitive mutants accumulate phyA protein similar to or higher than the wild type in light, the lrb1 lrb2 mutants accumulate less, suggesting that LRB1/2 also positively regulate phyA levels in a phyB/D-dependent manner. Together, these data show that the BTB ubiquitin ligases assembled with LRB1/2 function redundantly as negative regulators of photomorphogenesis, possibly by influencing the turnover of phyB/D.     

Negative interference of endogenous phytochrome B with phytochrome A function in Arabidopsis

To study negative interactions between phytochromes, phytochrome B (phyB) overexpressor lines, the mutants phyA-201, phyB-4, phyB-5, phyD-1, phyA-201 phyB-5, phyA-201 phyD-1, and phyB-5 phyD-1 of Arabidopsis were used. Endogenous phyB, but not phytochrome D (phyD), partly suppressed phytochrome A (phyA)-dependent inhibition of hypocotyl elongation in far-red light (FR). Dichromatic irradiation demonstrated that the negative effect of phyB was largely independent of the photoequilibrium, i.e. far-red light absorbing form of phytochrome formation. Moreover, phyB-4, a mutant impaired in signal transduction, did not show a loss of inhibition of phyA by phyB. Overexpression of phyB, conversely, resulted in an enhanced inhibition of phyA function, even in the absence of supplementary carbohydrates. However, overexpression of a mutated phyB, which cannot incorporate the chromophore, had no detectable effect on phyA action. In addition to seedling growth, accumulation of anthocyanins in FR, another manifestation of the high irradiance response, was strongly influenced by phyB holoprotein. Induction of seed germination by FR, a very low fluence response, was suppressed by both endogenous phyB and phyD. In conclusion, we show that both classical response modes of phyA, high irradiance response, and very low fluence response are subject to an inhibitory action of phyB-like phytochromes. Possible mechanisms of the negative interference are discussed.     

Nuclear accumulation of the phytochrome A photoreceptor requires FHY1

The phytochrome family of red/far-red (R/FR)-responsive photoreceptors plays a key role throughout the life cycle of plants . Arabidopsis has five phytochromes, phyA-phyE, among which phyA and phyB play the most predominant functions . Light-regulated nuclear accumulation of the phytochromes is an important regulatory step of this pathway, but to this date no factor specifically required for this event has been identified . Among all phyA signaling mutants, fhy1 and fhy3 (far-red elongated hypocotyl 1 and 3) have the most severe hyposensitive phenotype, indicating that they play particularly important roles . FHY1 is a small plant-specific protein of unknown function localized both in the nucleus and the cytoplasm . Here we show that FHY1 is specifically required for the light-regulated nuclear accumulation of phyA but not phyB. Moreover, phyA accumulation is only slightly affected in fhy3, indicating that the diminished nuclear accumulation of phyA observed in fhy1 seedlings is not simply a general consequence of reduced phyA signaling. By in vitro pull-down and yeast two-hybrid analyses, we demonstrate that FHY1 physically interacts with phyA, preferentially in its active Pfr form. Furthermore, FHY1 and phyA colocalize in planta. We therefore identify the first component required for light-regulated phytochrome nuclear accumulation.     

Cryptochromes,Phytochromes, and COP1 Regulate Light-Controlled Stomatal Development in Arabidopsis

In Arabidopsis thaliana, the cryptochrome (CRY) blue light photoreceptors and the phytochrome (phy) red/far-red light photoreceptors mediate a variety of light responses. COP1, a RING motif–containing E3 ubiquitin ligase, acts as a key repressor of photomorphogenesis. Production of stomata, which mediate gas and water vapor exchange between plants and their environment, is regulated by light and involves phyB and COP1. Here, we show that, in the loss-of-function mutants of CRY and phyB, stomatal development is inhibited under blue and red light, respectively. In the loss-of-function mutant of phyA, stomata are barely developed under far-red light. Strikingly, in the loss-of-function mutant of either COP1 or YDA, a mitogen-activated protein kinase kinase kinase, mature stomata are developed constitutively and produced in clusters in both light and darkness. CRY, phyA, and phyB act additively to promote stomatal development. COP1 acts genetically downstream of CRY, phyA, and phyB and in parallel with the leucine-rich repeat receptor-like protein TOO MANY MOUTHS but upstream of YDA and the three basic helix-loop-helix proteins SPEECHLESS, MUTE, and FAMA, respectively. These findings suggest that light-controlled stomatal development is likely mediated through a crosstalk between the cryptochrome-phytochrome-COP1 signaling system and the mitogen-activated protein kinase signaling pathway.     

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.