The genetics of phytochrome signalling in Arabidopsis

The application of Arabidopsis genetics to research into the responses of plants to light has enabled rapid recent advances in this field. The plant photoreceptor phytochrome mediates well-defined responses that can be exploited to provide elegant and specific genetic screens. By this means, not only have mutants affecting the phytochromes themselves been isolated, but also mutants affecting the transduction of phytochrome signals. The genes involved in these processes have now begun to be characterized by using this genetic approach to isolate signal transduction components. Most of the components characterized so far are capable of being translocated to the cell nucleus, and they may help to define a new system of regulation of gene expression. This review summarises the ongoing contribution made by genetics to our understanding of light perception and signal transduction by the phytochrome system.     

Floral transition mutants in Arabidopsis

An inventory of genetic differences in flowering time in Arabidopsis is presented and discussed. Many genes influence the transition to flowering in a quantitative way. Two groups of mutants and natural variants can be distinguished: those that are responsive to environmental factors and those that are less responsive or unresponsive. It is possible that all late/early-flowering mutants isolated to date carry a mutation with an effect, either promotive or repressive, on a floral repressor. The interaction between light perception and flowering has been studied by analysis of phytochrome- and cryptochrome-deficient mutants, which showed that phyA and probably also cryptochrome have a promotive role in flowering, whereas phyB and other stable phytochromes have an inhibitory role. A circadian rhythm is important in establishing daylength sensitivity, as was shown by the phenotype of the elf 3 mutants.     

Dual effect of phytochrome A on hypocotyl growth under continuous red light

The role of phytochrome A in the control of hypocotyl growth under continuous red light (Rc) was investigated using phyA and phyB mutants of Arabidopsis thaliana, which lack phytochrome A (phyA) or phytochrome B (phyB), respectively, and transgenic seedlings of Nicotiana tabacum overexpressing Avena phyA, compared to the corresponding wild type (WT). In WT seedlings of A. thaliana, hypocotyl growth inhibition showed a biphasic response to the fluence rate of Rc, with a brake at 10^?2μmol m^?2 s^?1. At equal total fluence rate, hourly pulses of red light caused slightly more inhibition than Rc. The response to very low fluences of continuous or pulsed red light was absent in the phyA and phyA phyB mutants and present in the phyB mutant. The second part of the response was steeper in the phyA mutant than in the WT but was absent in the phyB mutant. In WT tobacco the response to Rc was biphasic. Overexpression of Avena phyA enhanced the response only at very low fluence rates of Rc (< 10^?2μmol m^?2 s^?1). In both species, the effect of hourly pulses of far-red light was similar to the maximum inhibition observed in the first phase of the response to Rc. Using reciprocity failure (i.e. higher inhibition under continuous than pulsed light) as the operational criterion, a ‘true’ high-irradiance reaction occurred under continuous far-red light but not under Rc or red plus far-red light mixtures. Native and overexpressed phyA are proposed to mediate very low fluence responses under Rc. In WT A. thaliana, this effect is counteracted by a negative action of phyA on phyB-mediated low-fluence responses.     

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.     

The effect of daylength on the transition to flowering in phytochrome-deficient, late-flowering and double mutants of Arabidopsis thaliana

The effect of daylength on flowering was investigated in the following mutants of Arabidopsis thaliana : phytochrome B deficient ( hy3=phyB ); phytochrome chromophore deficient ( hy2 ); late-flowering ( co, gi. fca and fwa ); the hy2 and hy3 , late-flowering double mutants and the hy2, hy3 , late-flowering triple mutants. The hy mutants flower with fewer rosette leaves than the Landsberg erecta wild type under both long day and short day conditions and express this effect to a different degree in all late-flowering mutant backgrounds and under both daylengths, with the exception of fca under short days. The number of cauline leaves and days to flowering is less affected by the hy genotype. The hy2, hy3 double mutants flower with even fewer rosette leaves than the hy2 and hy3 monogenic mutants, suggesting an inhibitory role for phytochrome B and other stable phytochromes on flowering. The complex interaction between phytochrome, daylength and the effect of the late-flowering genes on the various parameters that describe the transition to flowering in Arabidopsis is discussed.     

Integrated metabolite and gene expression profiling revealing phytochrome A regulation of polyamine biosynthesis of Arabidopsis thaliana

In this study, metabolite profiling was demonstrated as a usefultool to plot a specific metabolic pathway, which is regulatedby phytochrome A (phyA). Etiolated Arabidopsis wild-type (WT)and phyA mutant seedlings were irradiated with either far-redlight (FR) or white light (W). Primary metabolites of the irradiatedseedlings were profiled by gas chromatography time-of-flightmass spectrometry (GC/TOF-MS) to obtain new insights on phyA-regulatedmetabolic pathways. Comparison of metabolite profiles in phyAand WT seedlings grown under FR revealed a number of metabolitesthat contribute to the differences between phyA and the WT.Several metabolites, including some amino acids, organic acids,and major sugars, as well as putrescine, were found in smalleramounts in WT compared with the content in phyA seedlings grownunder FR. There were also significant differences between metaboliteprofiles of WT and phyA seedlings during de-etiolation underW. The polyamine biosynthetic pathway was investigated further,because putrescine, one of the polyamines existing in a widevariety of living organisms, was found to be present in loweramounts in WT than in phyA under both light conditions. Theexpression levels of polyamine biosynthesis-related genes wereinvestigated by quantitative real-time RT-PCR. The gene expressionprofiles revealed that the arginine decarboxylase 2 (ADC2) genewas transcribed less in the WT than in phyA seedlings underboth light conditions. This finding suggests that ADC2 is negativelyregulated by phyA during photomorphogenesis. In addition, S-adenosylmethioninedecarboxylase 2 and 4 (SAMDC2 and SAMDC4) were found to be regulatedby phyA but in a different manner from the regulation of ADC2. Key words: Arabidopsis thaliana, gene expression profiling, metabolite profiling, phytochrome A, polyamine biosynthesis Received 19 October 2007; Revised 17 January 2008 Accepted 18 January 2008     

Both phytochrome A and phytochrome B are required for the normal expression of phototropism in Arabidopsis thaliana seedlings

The role of phytochrome A (phyA) and phytochrome B (phyB) in phototropism was investigated by using the phytochrome-deficient mutants phyA-101 , phyB-1 and a phyA/phyB double mutant. The red-light-induced enhancement of phototropism, which is normally observed in wild-type seedlings, could not be detected in the phyA/phyB mutant at fluences of red light between 0.1 and 19 000 μmol m⁻². The loss of phyB has been shown to have no apparent effect on enhancement, while the loss of phyA resulted in a loss of enhancement only in the low fluence range (Janoudi et al. 1997). The conclusions of the aforementioned study can now be modified based on the current results which indicate that phototropic enhancement in the high fluence range is mediated by either phyA or phyB, and that other phytochromes have no role in enhancement. First positive phototropism was unaffected in phyA-101 and phyB-1 However, the magnitude of first positive phototropism in the phyA/phyB mutant was significantly lower than that of the wild-type Landsberg parent. Thus, the presence of either phyA or phyB is required for normal expression of first positive phototropism. The time threshold for second positive phototropism is unaltered in the phyA-101 and phyB mutants. However, the time threshold in the phyA/phyB mutant is about 2 h, approximately six times that of the wild type. Finally, the magnitude of second positive phototropism in both phyA-101 and phyB-1 is diminished in comparison with the wild-type response. Thus, phyA and phyB, acting independently or in combination, regulate the magnitude of phototropic curvature and the time threshold for second positive phototropism. We conclude that the presence of phyA and phyB is required, but not sufficient, for the expression of normal phototropism.     

Flowering responses to light-breaks in photomorphogenic mutants of Arabidopsis thaliana, a long-day plant

Flowering response and plant form of photomorphogenic mutants (hy1, hy2, hy3, hy4 and hy5) of Arabidopsis thaliana (L.), a long-day plant, were examined in long and short days. There were only slight differences among genotypes including Landsberg wild type with respect to the flowering time under long days. The effect of 1 h light-(night)-breaks of far-red, red, blue and white light given in the middle of the dark period of plants grown under short days, was studied. Effects of far-red light applied at the end or the beginning of the main photoperiod on flowering and plant form were also examined. The light-breaks with all the above mentioned light qualities promoted floral initiation of all the genotypes including the wild type in terms of both the flowering time and the number of rosette leaves. In general, far-red light was most effective. It is possible to classify the hy-mutants into 3 groups by their responses to light-breaks under short day conditions: (a) Mutants hy2 and hy3, which have a reduced number of rosette leaves, and flower early. Red light is as effective as far-red light. The wavelength of light-breaks is relatively unimportant for flowering response. (b) Mutants hy4, hy5 and Landsberg wild type, which have a greater number of rosette leaves, and flower relatively late. The effectiveness of light-breaks is in the following order, far-red, blue, and red light, which is in reverse order to the transformation of phytochrome to the P_fr form. (c) Mutant hy1, which behaves anomalously with respect to relations between flowering time and number of rosette leaves; late flowering with reduced number of rosette leaves. Red, blue and far-red light are effective, but white light is ineffective for reducing the number of rosette leaves. When far-red light was given in the middle of the night or at the end of the main photoperiod, it markedly reduced the number of rosette leaves compared to those grown under short days for all the genotypes, while when applied at the beginning of the main photoperiod far-red light did not affect the number of rosette leaves. Different effects on the plant form dependent on the time of treatment with far-red light-breaks are also discussed.     

Degradation of phytochrome A and the high irradiance response in Arabidopsis: a kinetic analysis

The ability to respond to far‐red‐rich light is essential for seedlings germinating below dense canopies. Physiological and genetic studies have demonstrated that phytochrome A is the only photoreceptor mediating responses to far‐red light. However, all phytochromes including phytochrome A are believed to be activated by red light and to be inactivated by far‐red light. To address the fundamental question of why phytochrome A has its highest physiological activity at presumably inactivating wavelengths, we analysed light‐induced degradation of phytochrome A in Arabidopsis. Rate constants were obtained for all reaction events in a two‐step model of degradation. Based on biochemical data, the model includes a tagging mechanism preceding degradation. The parameterized model describes Pr accumulation, wavelength dependencies of degradation kinetics and steady‐state levels as well as Pfr‐induced Pr degradation. Subsequently, experimentally derived fluence rate response curves, action spectrum and response curves to dichromatic irradiation were compared to simulations based on the model of degradation. Two kinetically defined phytochrome subspecies, untagged Pfr and tagged Pr, have steady‐state levels closely matching the physiological response curves. Therefore, sensing of far‐red light by phytochrome A can be quantitatively explained based exclusively on regulated protein degradation.     

Arabidopsis mutants deregulated in RCI2A expression reveal new signaling pathways in abiotic stress responses

To uncover new pathways involved in low-temperature signal transduction, we screened for mutants altered in cold-induced expression of RCI2A, an Arabidopsis gene that is not a member of the CBF/DREB1 regulon and is induced not only by low temperature but also by abscisic acid (ABA), dehydration (DH) and NaCl. This was accomplished by generating a line of Arabidopsis carrying a transgene consisting of the RCI2A promoter fused to the firefly luciferase coding sequence. A number of mutants showing low or high RCI2A expression in response to low temperature were identified. These mutants also displayed deregulated RCI2A expression in response to ABA, DH or NaCl. Interestingly, however, they were not altered in stress-induced expression of RD29A, a CBF/DREB1-target gene, suggesting that the mutations affect signaling intermediates of CBF/DREB1-independent regulatory pathways. Several mutants showed alterations in their tolerance to freezing, DH or salt stress, as well as in their ABA sensitivity, which indicates that the signaling intermediates defined by the corresponding mutations play an important role in Arabidopsis tolerance to abiotic stresses. Based on the mutants identified, we discuss the involvement of CBF/DREB1-independent pathways in modulating stress signaling.     

Phytochrome A, phytochrome B and HY4 are involved in hypocotyl growth responses to natural radiation in Arabidopsis: weak de-etiolation of the phyA mutant under dense canopies

The roles of phytochrome A (phyA), phytochrome B (phyB) and a putative blue-light (BL) photoreceptor (HY4) in the control of hypocotyl growth by natural radiation were investigated using phyA, phyB and hy4 mutants of Arabidopsis thaliana. Full sunlight inhibited hypocotyl growth to a larger extent in wild-type (WT) than in phyA, phyB and, particularly, hy4 seedlings. In WT seedlings, hypocotyl growth was promoted by selectively lowering BL irradiance, lowering red-light (R) plus far-red-light (FR) irradiance or lowering the R/FR ratio (which was achieved either by increasing FR or by reducing R). The effects of lowering BL were reduced in hy4 and exaggerated in phyA seedlings. The effects of lowering R+FR were reduced in phyA and exaggerated in hy4 seedlings. Neither phyB nor hy4 mutants responded to low R/FR ratios. Neighbouring plants reflecting FR without shading caused subtle reductions of the R/FR ratio. This signal promoted hypocotyl growth in WT but not in phyA, phyB or hy4 seedlings. Intermediate canopy shade produced similar effects in all genotypes. Under deep shade, de-etiolation was severely impaired in phyA seedlings, which died prematurely. Thus, the FR ‘high-irradiance reaction’ mediated by phyA could be important for seedling survival under dense canopies.     

The nuclear localization signal and the C-terminal region of FHY1 are required for transmission of phytochrome A signals

Plants use the family of phytochrome photoreceptors to sense their light environment in the red/far-red region of the spectrum. Phytochrome A (phyA) is the primary photoreceptor that regulates germination and early seedling development. This phytochrome mediates seedling de-etiolation for the developmental transition from heterotrophic to photoauxotrophic growth. High intensity far-red light provides a way to specifically assess the role of phyA in this process and was used to isolate phyA-signaling intermediates. fhy1 and pat3 (renamed fhy1-3) are independently isolated alleles of a gene encoding a phyA signal transduction component. FHY1 is a small 24 kDa protein that shows no homology to known functional motifs, besides a small conserved septin-related domain at the C-terminus, a putative nuclear localization signal (NLS) and a putative nuclear exclusion signal (NES). Here we demonstrate that the septin-related domain is important for FHY1 to transmit phyA signals. Moreover, the putative NLS and NES of FHY1 are indeed involved in its nuclear localization and exclusion. Nuclear localization of FHY1 is needed for it to execute responses downstream of phyA. Together with the results from global expression analysis, our findings point to an important role of FHY1 in phyA signaling through its nuclear translocation and induction of gene expression.     

Analysis of leaf proteins in late flowering mutants of Arabidopsis thaliana

Late flowering monogenic mutants of Arabidopsis thaliana (L.) Heynh. at the loci co, gi, fca, fve, fwa, fha, fpa, fy and their corresponding wild type, Landsberg erecta , were analysed by two-dimensional gel electrophoresis. All plants were grown under continuous light and proteins were extracted from leaves of the same age (20-day-old). The polypeptide patterns of the mutants at the loci co, gi, fca, fve, fwa, fha, fpa , and Landsberg erecta were identical. The mutant at the fy locus showed a qualitative difference with Landsberg erecta . Crosses were made between this line and the wild type Landsberg erecta . F2 plants, resulting from autopollination of the hybrid, were analysed and showed no cosegregation between the observed protein and the flowering phenotype, indicating that these two lines differ by more than a single mutation.     

Mutational analysis of blue-light sensing in Arabidopsis

Blue light regulates many physiological and developmental processes in higher plants through the action of multiple photosensory systems. The analysis of photomorphogenic mutants is leading to a better understanding of how the different photosensory systems mediate the wide range of responses observed in blue light. A review of the current literature on photomorphogenic mutants makes it apparent that redundancies exist in photoreceptor function. For example, many blue-light responses that have been shown to be regulated by a blue-light photosensory system are also under phytochrome control. The study of various light-response mutants suggests that a complex sensory network regulates light-mediated responses. This article attempts to piece together information regarding the sensory systems responsible for blue-light-regulated responses.     

Late embryo-defective mutants of Arabidopsis

The embryo-defective (emb) mutants of Arabidopsis constitute a large and diverse group of mutants disrupted in a broad range of embryonic processes, including morphogonesis, cell differentiation, and maturation programs. This report describes a subset of these mutants, the late embryo defectives, which develop beyond the globular stage of embryogenesis but fail to complete normal morphogenesis. A representative sample of 12 late mutants was chosen for this study, patterns of morphogenesis were characterized, the germination potential of mutant seeds was investigated, and additional mutant alleles within the collection were identified. Morphological defects in mutant embryos became apparent during the heart stage of development, when embryos normally begin the rapid cell division and expansion required for the completion of morphogenesis. Despite their morphological abnormalities, mutant embryos often germinated from dry seed, demonstrating that genetic programs required for the establishment of desiccation tolerance remained intact. Mutant seedlings displayed a wide range of developmental abnormalities, including altered morphology, lack of pigmentation, dwarfism, and disorganized vegetative growth. One late mutant was found to be allelic to an early embryo defective that arrests at the globular stage. These results suggest that a number of late EMB genes encode basic cellular and metabolic functions needed for cell division, enlargement, and embryonic growth. The rapid growth and metabolic changes that occur at the heart stage may present a barrier to normal development in the late mutants, resulting in altered embryo morphology and other developmental defects. It is proposed that many Arabidopsis mutants with abnormal embryo and seedling morphology are not defective in the regulation of pattern formation or morphogenesis, but rather in fundamental physiological and cellular processes required for the completion of normal growth and development. © 1995 Wiley-Liss, Inc.     

Phytochrome A, phytochrome B and other phytochrome(s) regulate ATHB-2 gene expression in etiolated and green Arabidopsis plants

The expression of the Arabidopsis ATHB-2 gene is light-regulated both in seedlings and in adult plants. The gene is expressed at high levels in rapidly elongating etiolated seedlings and is down-regulated by a pulse of red light (R) through the action of a phytochrome other than phytochrome A or B, or by a pulse of far-red light (FR) through the action of phytochrome A. In green plants, the expression of the ATHB-2 gene is rapidly and strongly enhanced by lowering the R:FR ratio perceived by a phytochrome other than A or B. Returning the plant to a high R:FR ratio results in an equally rapid decrease of the ATHB-2 mRNA. Consistently, plants overproducing ATHB-2 show developmental phenotypes characteristic of plants grown in low R:FR: elongated petioles, reduced leaf area, early flowering, and reduced number of rosette leaves. Taken together, the data strongly suggest a direct involvement of ATHB-2 in light-regulated growth phenomena throughout Arabidopsis development.     

Supplementary ultraviolet-B irradiation reveals differences in stress responses between Arabidopsis thaliana ecotypes

Irradiation of Arabidopsis thaliana ecotypes C24, Wassilewskija (Ws) and Columbia-0 (Col-0) with supplementary ultraviolet-A+B (UV-A+B) radiation revealed ecotype-specific differences in expression of the gene for the pathogenesis-related protein PR-5. C24 showed an increased expression level of PR-5 (5- and 20-fold higher compared with Col-0 and Ws, respectively). Expression of other molecular markers such as CHS (encoding chalcone synthase), MEB5.2 [encoding a gene strongly up-regulated by ultraviolet-B (UV-B)] and PYROA [encoding a pyridoxine (Vitamin B6) biosynthesis enzyme] only showed slight differences between ecotypes. Oxidative stress during UVA+B exposure was monitored by staining for H2O2. This analysis also revealed important ecotype-specific differences. 'H2O2 hot spots' were found in C24, whereas an even distribution of H2O2 was found in Ws and Col-0. Necrotic lesions also appeared on C24 leaves after prolonged UV-B exposure. There was a reverse correlation between the H2O2 steady-state concentration and the PR-5 gene expression; Ws showed the highest level of H2O2 accumulation but the lowest expression level of the PR-5 gene. Furthermore, application of paraquat on the rosettes led to similar PR-5 expression and H2O2 accumulation patterns as were found after UV-A+B irradiation. The observed ecotypic differences were also reflected in a statistically significant UV-B-dependent decrease in biomass, rosette size and leaf area for Ws, but not for C24 and Col-0. Our results show that a significant ecotype-specific genetic variability in general UV-B responses in Arabidopsis exists. Moreover, the signal transduction or gene regulation pathway for PR-5 differs from the other molecular markers used in this study.