Blue light and phytochrome-mediated stomatal opening in the npq1 and phot1 phot2 mutants of Arabidopsis

Recent studies have shown that blue light-specific stomatal opening is reversed by green light and that far-red light can be used to probe phytochrome-dependent stomatal movements. Here, blue-green reversibility and far-red light were used to probe the stomatal responses of the npq1 mutant and the phot1 phot2 double mutant of Arabidopsis. In plants grown at 50 micromol m-2 s-1, red light (photosynthetic)-mediated opening in isolated stomata from wild type (WT) and both mutants saturated at 100 micromol m-2 s-1. Higher fluence rates caused stomatal closing, most likely due to photo-inhibition. Blue light-specific opening, probed by adding blue light (10 micromol m-2 s-1) to a 100 micromol m-2 s-1 red background, was found in WT, but not in npq1 or phot1 phot2 double mutant stomata. Under 50 micromol m-2 s-1 red light, 10 micromol m-2 s-1 blue light opened stomata in both WT and npq1 mutant stomata but not in the phot1 phot2 double mutant. In npq1, blue light-stimulated opening was reversed by far-red but not green light, indicating that npq1 has a phytochrome-mediated response and lacks a blue light-specific response. Stomata of the phot1 phot2 double mutant opened in response to 20 to 50 micromol m-2 s-1 blue light. This opening was green light reversible and far-red light insensitive, indicating that stomata of the phot1 phot2 double mutant have a detectable blue light-specific response.     

Regulation of phytochrome B signaling by phytochrome A and FHY1 in Arabidopsis thaliana

Phytochrome A (phyA) and phytochrome B (phyB) share the control of many processes but little is known about mutual signaling regulation. Here, we report on the interactions between phyA and phyB in the control of the activity of an Lhcb1*2 gene fused to a reporter, hypocotyl growth and cotyledon unfolding in etiolated Arabidopsis thaliana. The very-low fluence responses (VLFR) induced by pulsed far-red light and the high-irradiance responses (HIR) observed under continuous far-red light were absent in the phyA and phyA phyB mutants, normal in the phyB mutant, and reduced in the fhy1 mutant that is defective in phyA signaling. VLFR were also impaired in Columbia compared to Landsberg erecta. The low-fluence responses (LFR) induced by red-light pulses and reversed by subsequent far-red light pulses were small in the wild type, absent in phyB and phyA phyB mutants but strong in the phyA and fhy1 mutants. This indicates a negative effect of phyA and FHY1 on phyB-mediated responses. However, a pre-treatment with continuous far-red light enhanced the LFR induced by a subsequent red-light pulse. This enhancement was absent in phyA, phyB, or phyA phyB and partial in fhy1. The levels of phyB were not affected by the phyA or fhy1 mutations or by far-red light pre-treatments. We conclude that phyA acting in the VLFR mode (i.e. under light pulses) is antagonistic to phyB signaling whereas phyA acting in the HIR mode (i.e. under continuous far-red light) operates synergistically with phyB signaling, and that both types of interaction require FHY1.     

Ultraviolet B radiation enhances a phytochrome-B-mediated photomorphogenic response in Arabidopsis

Ultraviolet B radiation (UV-B, 290-315 nm) can cause damage and induce photomorphogenic responses in plants. The mechanisms that mediate the photomorphogenic effects of UV-B are unclear. In etiolated Arabidopsis seedlings, a daily exposure to 2.5 h of UV-B enhanced the cotyledon opening response induced by a subsequent red light (R) pulse. An R pulse alone, 2.5 h of UV-B terminated with a far-red pulse, or 2.5 h of continuous R caused very little cotyledon opening. The enhancing effect of UV-B increased with fluence rate up to approximately 7.58 micromol m(-2) s(-1); at higher fluence rates the response to UV-B was greatly reduced. The phyA, phyA cry1, and cry1 cry2 mutants behaved like the wild type when exposed to UV-B followed by an R pulse. In contrast, phyB, phyB cry1, and phyB phyA mutants failed to open the cotyledons. Thus, phytochrome B was required for the cotyledon opening response to UV-B --> R treatments, whereas phytochrome A and cryptochromes 1 and 2 were not necessary under the conditions of our experiments. The enhancing effect of low doses of UV-B on cotyledon opening in uvr1 uvr2 and uvr1 uvr3 mutants, deficient in DNA repair, was similar to that found in the wild type, suggesting that this effect of UV-B was not elicited by signals derived from UV-B-induced DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts). We conclude that low doses of UV-B, perceived by a receptor system different from phytochromes, cryptochromes, or DNA, enhance a de-etiolation response that is induced by active phytochrome B.     

Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis

Chloroplasts move in a light-dependent manner that can modulate the photosynthetic potential of plant cells. Identification of genes required for light-induced chloroplast movement is beginning to define the molecular machinery that controls these movements. In this work, we describe plastid movement impaired 2 (pmi2), a mutant in Arabidopsis (Arabidopsis thaliana) that displays attenuated chloroplast movements under intermediate and high light intensities while maintaining a normal movement response under low light intensities. In wild-type plants, fluence rates below 20 micromol m(-2) s(-1) of blue light lead to chloroplast accumulation on the periclinal cell walls, whereas light intensities over 20 micromol m(-2) s(-1) caused chloroplasts to move toward the anticlinal cell walls (avoidance response). However, at light intensities below 75 micromol m(-2) s(-1), chloroplasts in pmi2 leaves move to the periclinal walls; 100 micromol m(-2) s(-1) of blue light is required for chloroplasts in pmi2 to move to the anticlinal cell walls, indicating a shift in the light threshold for the avoidance response in the mutant. The pmi2 mutation has been mapped to a gene that encodes a protein of unknown function with a large coiled-coil domain in the N terminus and a putative P loop. PMI2 shares sequence and structural similarity with PMI15, another unknown protein in Arabidopsis that, when mutated, causes a defect in chloroplast avoidance under high-light intensities.     

Fluence and wavelength requirements for Arabidopsis CAB gene induction by different phytochromes.

The roles of different phytochromes have been investigated in the photoinduction of several chlorophyll a/b-binding protein genes (CAB) of Arabidopsis thaliana. Etiolated seedlings of the wild type, a phytochrome A (PhyA) null mutant (phyA), a phytochrome B (PhyB) null mutant (phyB), and phyA/phyB double mutant were exposed to monochromatic light to address the questions of the fluence and wavelength requirements for CAB induction by different phytochromes. In the wild type and the phyB mutant, PhyA photoirreversibly induced CAB expression upon irradiation with very-low-fluence light of 350 to 750 nm. In contrast, using the phyA mutant, PhyB photoreversibly induced CAB expression with low-fluence red light. The threshold fluences of red light for PhyA- and PhyB-specific induction were about 10 nmol m-2 and 10 mumol m-2, respectively. In addition, CAB expression was photoreversibly induced with low-fluence red light in the phyA/phyB double mutant, revealing that another phytochrome(s) (PhyX) regulated CAB expression in a manner similar to PhyB. These data suggest that plants utilize different phytochromes to perceive light of varying wave-lengths and fluence, and begin to explain how plants respond so exquisitely to changing light in their environment.     

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.     

An Arabidopsis mutant hypersensitive to red and far-red light signals.

A new mutant called psi2 (for phytochrome signaling) was isolated by screening for elevated activity of a chlorophyll a/b binding protein-luciferase (CAB2-LUC) transgene in Arabidopsis. This mutant exhibited hypersensitive induction of CAB1, CAB2, and the small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) promoters in the very low fluence range of red light and a hypersensitive response in hypocotyl growth in continuous red light of higher fluences. In addition, at high- but not low-light fluence rates, the mutant showed light-dependent superinduction of the pathogen-related protein gene PR-1a and developed spontaneous necrotic lesions in the absence of any pathogen. Expression of genes responding to various hormone and environmental stress pathways in the mutant was not significantly different from that of the wild type. Analysis of double mutants demonstrated that the effects of the psi2 mutation are dependent on both phytochromes phyA and phyB. The mutation is recessive and maps to the bottom of chromosome 5. Together, our results suggest that PSI2 specifically and negatively regulates both phyA and phyB phototransduction pathways. The induction of cell death by deregulated signaling pathways observed in psi2 is reminiscent of retinal degenerative diseases in animals and humans.     

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.     

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.     

Seed germination of Arabidopsis thaliana phyA/phyB double mutants is under phytochrome control.

We examined the photocontrol of seed germination in the phyA/phyB double mutants of Arabidopsis thaliana seeds. Dormant phyA/phyB seeds showed a red/far-red light (R/FR)-reversible induction of seed germination. This suggests the involvement of at least one other phytochrome, phyC, D, and/or E, in controlling seed germination. We designated this spectrally active phytochrome in phyA/phyB as phyX. The full reversibility of the R-induced germination by subsequent FR pulses, and the observation that the response is reversible by FR, even after a 3-h R treatment, indicates that this phyX response belongs to the low-fluence-response type. Thus, this phyX response is functionally related to phyB-mediated responses. However, in contrast to phyB-controlled seed germination, this phyX-mediated response needs a prolonged imbibition period and exhibits reversibility kinetics different from that needed for phyB. Furthermore, this phyX response requires a prolonged irradiation time and shows a fluence rate response dependency, showing a similarity to the high irradiance response of photomorphogenesis. Thus, phyX, with regard to its control of seed germination, is a functionally new phytochrome that shares some characteristics of both phyA- and phyB-mediated responses.     

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.     

Immuno-electron microscopic quantification of the fucoxanthin chlorophyll a/c binding polypeptides Fcp2, Fcp4, and Fcp6 of Cyclotella cryptica grown under low- and high-light intensities.

The diatom Cyclotella cryptica was grown under low- and high-intensity white light of 50 and 500 micromol photons m-2 s-1, respectively. Western immunoblotting showed that the diatom adapted its light-harvesting apparatus, giving rise to different amounts of distinct fucoxanthin chlorophyll a/c binding polypeptides (Fcp). The amount of Fcp2 was approximately two-fold higher under low-light than under high-light conditions, whereas the amount of Fcp6 increased four- to five-fold under high-light conditions. For Fcp4, no significant differences were detected in response to either light regime. Cells of Cyclotella grown under high- and low-light intensity were subjected to immunoelectron microscopy. Quantification of the gold label, expressed as gold particles per microm2, confirmed the results obtained by Western immunoblotting. Exposure to low light resulted in the detection of approximately six times more Fcp2-bound gold particles per microm2 in thylakoid membranes, whereas in cells grown under high light the number of Fcp6-bound gold particles increased ten-fold. For Fcp4, similar amounts of gold particles per microm2 were counted under the two light regimes. These immunocytochemical results confirmed molecular data derived from phylogenetic analyses of the sequences of genes encoding fucoxanthin chlorophyll a/c binding polypeptides (fcp genes) and from measurements of steady-state fcp mRNA concentrations. The results show that Fcp2 and Fcp6 accumulate under low- and high-light intensity, respectively, whereas Fcp4 seems to be constitutively synthesized.     

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.     

Overexpression of Arabidopsis phytochrome B inhibits phytochrome A function in the presence of sucrose

Overexpression of phytochrome B (phyB) in Arabidopsis has previously been demonstrated to result in dominant negative interference of phytochrome A (phyA)-mediated hypocotyl growth inhibition in far-red (FR) light. This phenomenon has been examined further in this study and has been found to be dependent on the FR fluence rate and on the availability of metabolizable sugars in the growth medium. Poorly metabolized sugars capable of activating the putative hexokinase sensory function were not effective in eliciting the phytochrome interference response. Overexpressed phyB lacking the chromophore-binding site was also effective at inhibiting the phyA response, especially at higher fluence rates of FR. Overexpressed phyB produces the dominant negative phenotype without any apparent effect on phyA abundance or degradation. It is possible that phyA and phyB interact with a common reaction partner but that either the energy state of the cell or a separate sugar-signaling mechanism modulates the phytochrome-signaling interactions.     

Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice

We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering.     

Independent action of ELF3 and phyB to control hypocotyl elongation and flowering time

Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) mediates many responses to red light. In a screen for Arabidopsis mutants with phenotypes similar to those of phyB mutants, we isolated two new elf3 mutants. One has weaker morphological phenotypes than previously identified elf3 alleles, but still abolishes circadian rhythms under continuous light. Like phyB mutants, elf3 mutants have elongated hypocotyls and petioles, flower early, and have defects in the red light response. However, we found that elf3 mutations have an additive interaction with a phyB null mutation, with phyA or hy4 null mutations, or with a PHYB overexpression construct, and that an elf3 mutation does not prevent nuclear localization of phyB. These results suggest that either there is substantial redundancy in phyB and elf3 function, or the two genes regulate distinct signaling pathways.     

Phytochrome E influences internode elongation and flowering time in Arabidopsis.

From a screen of M2 seedlings derived from gamma-mutagenesis of seeds doubly null for phytochromes phyA and phyB, we isolated a mutant lacking phyE. The PHYE gene of the selected mutant, phyE-1, was found to contain a 1-bp deletion at a position equivalent to codon 726, which is predicted to result in a premature stop at codon 739. Immunoblot analysis showed that the phyE protein was undetectable in the phyE-1 mutant. In the phyA- and phyB-deficient background, phyE deficiency led to early flowering, elongation of internodes between adjacent rosette leaves, and reduced petiole elongation. This is a phenocopy of the response of phyA phyB seedlings to end-of-day far-red light treatments. Furthermore, a phyE deficiency attenuated the responses of phyA phyB seedlings to end-of-day far-red light treatments. Monogenic phyE mutants were indistinguishable from wild-type seedlings. However, phyB phyE double mutants flowered earlier and had longer petioles than did phyB mutants. The elongation and flowering responses conferred by phyE deficiency are typical of shade avoidance responses to the low red/far-red ratio. We conclude that in conjunction with phyB and to a lesser extent with phyD, phyE functions in the regulation of the shade avoidance syndrome.     

A plant-specific protein essential for blue-light-induced chloroplast movements

In Arabidopsis (Arabidopsis thaliana), light-dependent chloroplast movements are induced by blue light. When exposed to low fluence rates of light, chloroplasts accumulate in periclinal layers perpendicular to the direction of light, presumably to optimize light absorption by exposing more chloroplast area to the light. Under high light conditions, chloroplasts become positioned parallel to the incoming light in a response that can reduce exposure to light intensities that may damage the photosynthetic machinery. To identify components of the pathway downstream of the photoreceptors that mediate chloroplast movements (i.e. phototropins), we conducted a mutant screen that has led to the isolation of several Arabidopsis mutants displaying altered chloroplast movements. The plastid movement impaired1 (pmi1) mutant exhibits severely attenuated chloroplast movements under all tested fluence rates of light, suggesting that it is a necessary component for both the low- and high-light-dependant chloroplast movement responses. Analysis of pmi1 leaf cross sections revealed that regardless of the light condition, chloroplasts are more evenly distributed in leaf mesophyll cells than in the wild type. The pmi1-1 mutant was found to contain a single nonsense mutation within the open reading frame of At1g42550. This gene encodes a plant-specific protein of unknown function that appears to be conserved among angiosperms. Sequence analysis of the protein suggests that it may be involved in calcium-mediated signal transduction, possibly through protein-protein interactions.