Stress responsive gene CIPK14 is involved in phytochrome A-mediated far-red light inhibition of greening in Arabidopsis

In this study, we show that CIPK14, a stress responsive CBL-interacting protein kinase gene, is involved in phytochrome A-mediated far-red light inhibition of greening in Arabidopsis seedlings. The CIPK14-impairment mutant cipk14 grown in continuous far-red (FR) light did not show greening when exposed to white light illumination for 15 h. By contrast, the FR-grown phytochrome A null mutant phyA greened within 0.5 h of exposure to white light. Although greening of Col-4 (wild-type) was not completely abolished by FR, it exhibited a significantly decreased greening capacity compared with that of phyA. Further analyses demonstrated that the expression of protochlorophyllide reductase (POR) genes was correlated with the greening ability of the genotypes. In addition, CIPK14 appeared to be regulated by both the circadian clock and PhyA. Taken together, these results suggest that CIPK14 plays a role in PhyA-mediated FR inhibition of seedling greening, and that a Ca-related kinase may be involved in a previously undefined branch point in the phytochrome A signaling pathway.     

Photoconversion and nuclear trafficking cycles determine phytochrome A's response profile to far-red light

Phytochrome A (phyA) is the only photoreceptor in plants, initiating responses in far-red light and, as such, essential for survival in canopy shade. Although the absorption and the ratio of active versus total phyA are maximal in red light, far-red light is the most efficient trigger of phyA-dependent responses. Using a joint experimental-theoretical approach, we unravel the mechanism underlying this shift of the phyA action peak from red to far-red light and show that it relies on specific molecular interactions rather than on intrinsic changes to phyA's spectral properties. According to our model, the dissociation rate of the phyA-FHY1/FHL nuclear import complex is a principle determinant of the phyA action peak. The findings suggest how higher plants acquired the ability to sense far-red light from an ancestral photoreceptor tuned to respond to red light.     

Glutathione S-transferase interacting with far-red insensitive 219 is involved in phytochrome A-mediated signaling in Arabidopsis

Far-red (FR) insensitive 219 (FIN219) was previously shown to be involved in phytochrome A-mediated FR light signaling. To further understand its function and regulatory relation with other light-signaling components, a yeast two-hybrid approach was used to isolate FIN219-interacting partners. Here, we demonstrate that FIN219-interacting protein 1 (FIP1) interacts with FIN219 in vitro and in vivo and is composed of 217 amino acids that belong to the tau class of the large glutathione S-transferase gene family. FIP1 was further shown to have glutathione S-transferase activity. The gain of function and partial loss of function of FIP1 resulted in a hyposensitive hypocotyl phenotype under continuous FR (cFR) light and a delayed flowering phenotype under long-day conditions, which suggests that FIP1 may exist in a complex to function in the regulation of Arabidopsis (Arabidopsis thaliana) development. In addition, FIP1 mRNA was down-regulated in the suppressor of phytochrome A-105 1 mutant and differentially expressed in constitutive photomorphogenic 1-4 (cop1-4) and cop1-5 mutants under cFR. Intriguingly, FIP1 expression was up-regulated in the fin219 mutant under all light conditions, except cFR. Furthermore, promoter activity assays revealed that FIP1 expression was light dependent, mainly associated with vascular tissues, and developmentally regulated. Subcellular localization studies revealed that the beta-glucuronidase-FIP1 fusion protein was localized in the nucleus and cytoplasm. Taken together, these data indicate that FIP1 may interact with FIN219 to regulate cell elongation and flowering in response to light.     

Control of mitochondrial activities by phytochrome during greening

Mitochondria isolated from 7-day old darkgrown Avena sativa L. (var. Arnold) laminae given 5 min illumination of red light, followed by varying lengths of darkness up to 3 h, showed at least a twofold increase in the rates of both NADH-dependent oxygen consumption and respiratory chain phosphorylation over those of mitochondria isolated from unilluminated tissue. Similar organelles, isolated from tissue given either far-red or red followed by far-red pretreatment, exhibited rates of both functions of between 25% and 75% below those of the mitochondria from unilluminated tissue. The induction-reversion criteria for phytochrome control of respiration and oxidative phosphorylation were satisfied under all experimental conditions during the greening process.Treatment with continuous far-red light, acting presumably through the high irradiance reaction of phytochrome, served to disengage phytochrome activity from photosynthesis. The stimulation of oxidative phosphorylation still occurred under these conditions, slightly slower but much more prolonged in the absence of ATP from photophosphorylation.Abbreviations BSA bovine serum albumen - DAD diaminodurene - EDTA ethylene-diaminetetra-acetic acid - HEPES N-2-hydroxy-ethyl-piperazine-N-2-ethane-sulphonic acid - P_fr phytochrome in the active form     

Far-red light blocks greening of Arabidopsis seedlings via a phytochrome A-mediated change in plastid development.

We have characterized a far-red-light response that induces a novel pathway for plastid development in Arabidopsis seedlings. This response results in the inability of cotyledons to green upon subsequent white light illumination, and the response is suppressed by exogenous sucrose. Studies with mutants showed that this far-red block of greening is phytochrome A dependent and requires an intact downstream signaling pathway in which FHY1 and FHY3 may be components but in which HY5 is not. This highlights a previously undefined branchpoint in the phytochrome signaling pathway. Ultrastructural analysis showed that the far-red block correlates with both the failure of plastids to accumulate prolamellar bodies and the formation of vesicles in the stroma. We present evidence that the far-red block of greening is the result of severe repression of protochlorophyllide reductase (POR) genes by far-red light coupled with irreversible plastid damage. This results in the temporal separation of phytochrome-mediated POR; repression from light-dependent protochlorophyllide reduction, two processes that normally occur in coordination in white light.     

Correlation between far-red absorbing phytochrome and response in phytochrome-mediated anthocyanin synthesis

Abstract Mustard seedlings were light treated at 24 h after sowing (25°C) to induce phytochrome-mediated anthocyanin synthesis in cotyledons and hypocotylar hook. All light treatments were performed within the range of the reciprocity law. The in situ photoconversion kinetics of phytochrome (P_r→ P_fr) were measured under the same light treatment. It was found that between 0.4 and 1.0 relative P_fr level the amount of anthocyanin extracted from the organs at 52 h after sowing was linearily correlated with the amount of P_fr produced at 24 h in cotyledons and hypocotylar hook. It is concluded that an explanation of the fluence response function for red light mediated anthocyanin synthesis in the mustard seedling does not require the concept of active vs. bulk phytochrome.     

Analysis of far-red light-regulated genome expression profiles of phytochrome A pathway mutants in Arabidopsis

Phytochrome A (phyA) is the primary photoreceptor responsible for various far-red (FR) light-mediated responses. Previous studies have identified multiple phyA signaling mutants, including both positive and negative regulators of the phyA-mediated responses. How these defined intermediates act to mediate FR light responses is largely unknown. Here a cDNA microarray was used to examine effects of those mutations on the far-red light control of genome expression. Clustering analysis of the genome expression profiles supports the notion that phyA signaling may entail a network with multiple paths, controlling overlapping yet distinct sets of gene expression. FHY1, FAR1 and FHY3 most likely act upstream in the phyA signaling network, close to the phyA photoreceptor itself. FIN219, SPA1 and REP1 most likely act somewhere more downstream in the network and control the expression of smaller sets of genes. Further, this study also provides genomics evidence for the partial functional redundancy between FAR1 and FHY3. These two homologous proteins control the expression of a largely overlapping set of genes, and likely act closely together in the phyA-mediated FR light responses.     

Stability of phytochrome concentration in dicotyledonous tissues under continuous far-red light

Summary The phytochrome concentration in dark-grown seedlings of Pisum sativum, Phaseolus aureus and Sinapis alba remained constant under continuous far-red illumination for periods of up to 6 hours. Similar treatment of Zea mays seedlings reduced the phytochrome concentration by more than 60 percent. The results in the dicotyledonous seedlings may be due to the reversion of P_fr to P_r at a rate sufficient to prevent P_fr destruction; no evidence for reversion has been detected in Zea. Typical photomorphogenic responses were observed in the dicotyledonous seedlings in the absence of P_fr destruction.Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission.     

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [³5S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [³⁵S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.     

GIGANTEA regulates phytochrome A-mediated photomorphogenesis independently of its role in the circadian clock

GIGANTEA (GI) is a nuclear protein involved in the promotion of flowering by long days, in light input to the circadian clock, and in seedling photomorphogenesis under continuous red light but not far-red light (FR). Here, we report that in Arabidopsis (Arabidopsis thaliana) different alleles of gi have defects in the hypocotyl-growth and cotyledon-unfolding responses to hourly pulses of FR, a treatment perceived by phytochrome A (phyA). This phenotype is rescued by overexpression of GI. The very-low-fluence response of seed germination was also reduced in gi. Since the circadian clock modulates many light responses, we investigated whether these gi phenotypes were due to alterations in the circadian system or light signaling per se. In experiments where FR pulses were given to dark-incubated seeds or seedlings at different times of the day, gi showed reduced seed germination, cotyledon unfolding, and activity of a luciferase reporter fused to the promoter of a chlorophyll a/b-binding protein gene; however, rhythmic sensitivity was normal in these plants. We conclude that while GI does not affect the high-irradiance responses of phyA, it does affect phyA-mediated very-low-fluence responses via mechanisms that do not obviously involve its circadian functions.     

Both subunits of the dimeric plant photoreceptor phytochrome require chromophore for stability of the far-red light-absorbing form

The dimeric plant photoreceptor phytochrome is converted from its inactive red light-absorbing form (Pr) into the active far-red light-absorbing form (Pfr) upon light absorption. Dynamics of Pfr generation and of thermal Pfr-to-Pr conversion are of fundamental importance for inducing adequate responses to light signals. Here, we analyzed the role of subunit interactions on spectroscopic properties of dimeric phytochrome A. Using a coexpression system and affinity chromatography, we prepared mixed phytochrome dimers that can incorporate the essential chromophore only in one subunit. We demonstrate that such mixed dimers have unaltered difference spectra. In contrast, dark reversion differed greatly between Pfr-Pfr homodimers and Pfr-Pr heterodimers, the former being about 100-fold more stable. Temperature dependence of reaction rates revealed an additional stabilization of about 4 kcal/mol in homodimers. Consequences of these findings are discussed in relation to the biological function of, and functional diversification between, phytochrome family members.     

The influence of de-etiolation on the sensitivity of Avena coleoptiles to the far-red absorbing form of phytochrome

In photoresponses regulated by phytochrome the effect of a red irradiation is not always reversed by far-red. This applies for instance to the influence of red light on the geotropic reactions of Avena coleoptiles. We could induce red/far-red reversibility by a short de-etiolating exposure to red light about 20 h prior to the experimental irradiations. This, was due to a decrease of the sensitivity to the low level of the far-red absorbing form of phytochrome that is established by far-red. Since etiolated plants react also to a wavelength of 520 nm (green light), it is advisable to expose the coleoptiles to a de-etiolating irradiation prior to manipulations in green safelight in order to prevent the plants from reacting to the green light.     

Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development.

Phytochromes are a family of plant photoreceptors that mediate physiological and developmental responses to changes in red and far-red light conditions. In Arabidopsis, there are genes for at least five phytochrome proteins. These photoreceptors control such responses as germination, stem elongation, flowering, gene expression, and chloroplast and leaf development. However, it is not known which red light responses are controlled by which phytochrome species, or whether the different phytochromes have overlapping functions. We report here that previously described hy3 mutants have mutations in the gene coding for phytochrome B (PhyB). These are the first mutations shown to lie in a plant photoreceptor gene. A number of tissues are abnormally elongated in the hy3(phyB) mutants, including hypocotyls, stems, petioles, and root hairs. In addition, the mutants flower earlier than the wild type, and they accumulate less chlorophyll. PhyB thus controls Arabidopsis development at numerous stages and in multiple tissues.     

A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing.

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-1 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. Immunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-1 lines and of phyB- PHYD+ and phyB- phyD- lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is approximately 80% identical in amino acid sequence to phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-1 mutation indicates that phyD is not essential in some natural environments.