Phytochromes and photomorphogenesis in Arabidopsis.

Plants have evolved exquisite sensory systems for monitoring their light environment. The intensity, quality, direction and duration of light are continuously monitored by the plant and the information gained is used to modulate all aspects of plant development. Several classes of distinct photoreceptors, sensitive to different regions of the light spectrum, mediate the developmental responses of plants to light signals. The red-far-red light-absorbing, reversibly photochromic phytochromes are perhaps the best characterized of these. Higher plants possess a family of phytochromes, the apoproteins of which are encoded by a small, divergent gene family. Arabidopsis has five apophytochrome-encoding genes, PHYA-PHYE. Different phytochromes have discrete biochemical and physiological properties, are differentially expressed and are involved in the perception of different light signals. Photoreceptor and signal transduction mutants of Arabidopsis are proving to be valuable tools in the molecular dissection of photomorphogenesis. Mutants deficient in four of the five phytochromes have now been isolated. Their analysis indicates considerable overlap in the physiological functions of different phytochromes. In addition, mutants defining components acting downstream of the phytochromes have provided evidence that different members of the family use different signalling pathways.     

Overexpression of rice phytochrome A partially complements phytochrome B deficiency in Arabidopsis

The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile, phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light, as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants. Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB seedlings. Received: 12 July 1998 / Accepted: 13 August 1998     

Mutant analyses define multiple roles for phytochrome C in Arabidopsis photomorphogenesis

The analysis of Arabidopsis mutants deficient in the A, B, D, and E phytochromes has revealed that each of these phytochrome isoforms has both distinct and overlapping roles throughout plant photomorphogenesis. Although overexpression studies of phytochrome C (phyC) have suggested photomorphogenic roles for this receptor, conclusive evidence of function has been lacking as a result of the absence of mutants in the PHYC locus. Here, we describe the isolation of a T-DNA insertion mutant of phyC (phyC-1), the subsequent creation of mutant lines deficient in multiple phytochrome combinations, and the physiological characterization of these lines. In addition to operating as a weak red light sensor, phyC may perform a significant role in the modulation of other photoreceptors. phyA and phyC appear to act redundantly to modulate the phyB-mediated inhibition of hypocotyl elongation in red light and to function together to regulate rosette leaf morphology. In addition, phyC performs a significant role in the modulation of blue light sensing. Several of these phenotypes are supported by the parallel analysis of a quadruple mutant deficient in phytochromes A, B, D, and E, which thus contains only active phyC. Together, these data suggest that phyC has multiple functions throughout plant development that may include working as a coactivator with other phytochromes and the cryptochrome blue light receptors.     

The signal transducing photoreceptors of plants

Light signals are amongst the most important environmental cues regulating plant development. In addition to light quantity, plants measure the quality, direction and periodicity of incident light and use the information to optimise growth and development to the prevailing environmental conditions. Red and far-red wavelengths are perceived by the photoreversible phytochrome family of photoreceptors, whilst the detection of blue and ultraviolet (UV)-A wavelengths is conferred by the cryptochromes and phototropins. Higher plants contain multiple discrete phytochromes, the apoproteins of which are encoded by a small divergent gene family. In Arabidopsis, two cryptochrome and two phototropin family members have been identified and characterized. Photoreceptor action regulates development throughout the lifecycle of plants, from seed germination through to architecture of the mature plant and the onset of reproduction. The roles of individual photoreceptors in mediating plant development have, however, often been confounded by redundant, synergistic and in some cases mutually antagonistic mechanisms of action. The isolation of mutants null for individual photoreceptors and the construction of mutants null for multiple photoreceptors have therefore been paramount in elucidating photoreceptor functions. Photoreceptor action does not, however, operate in isolation from other signalling systems. The integration of light signals with other environmental cues enables plants to adapt their physiology to changing seasonal environments. This paper summarises current understanding of photoreceptor families and their functions throughout the lifecycle of plants. The integration of light signals with other environmental stimuli is also discussed.     

Phytochrome regulation of seed germination

Seed germination of many plant species is influenced by light. Of the various photoreceptor systems, phytochrome plays an especially important role in seed germination. The existence of at least five phytochrome genes has led to the proposal that different members of the family have different roles in the photoregulation of seed germination. Physiological analysis of seed germination ofArabidopsis thaliana (L.) Heynh. with phytochrome-deficient mutants showed for the first time that phytochrome A and phytochrome B modulate the timing of seed germination in distinct actions. Phytochrome A photo-irreversibly triggers the photoinduction of seed germination after irradiation with extremely low fluence light in a wide range of wavelengths, from UV-A, to visible, to far-red. In contrast, phytochrome B mediates the well-characterized photoreversible reaction, responding to red and far-red light of fluences four orders of magnitude higher than those to which PhyA responds. Wild plants, such asA. thaliana, survive under ground as dormant seeds for long periods, and the timing of seed germination is crucial for optimizing growth and reproduction. It therefore seems reasonable for plants to possess at least two different physiological systems for sensing the light environment over a wide spectral range with exquisite sensitivity of different phytochromes. This redundancy seems to enhance plant survival in a fluctuating environment.     

Light-independent phytochrome signaling mediated by dominant GAF domain tyrosine mutants of Arabidopsis phytochromes in transgenic plants

The photoreversibility of plant phytochromes enables continuous surveillance of the ambient light environment. Through expression of profluorescent, photoinsensitive Tyr-to-His mutant alleles of Arabidopsis thaliana phytochrome B (PHYB(Y276H)) and Arabidopsis phytochrome A (PHYA(Y242H)) in transgenic Arabidopsis plants, we demonstrate that photoconversion is not a prerequisite for phytochrome signaling. PHYB(Y276H)-expressing plants exhibit chromophore-dependent constitutive photomorphogenesis, light-independent phyB(Y276H) nuclear localization, constitutive activation of genes normally repressed in darkness, and light-insensitive seed germination. Fluence rate analyses of transgenic plants expressing PHYB(Y276H), PHYA(Y242H), and other Y(GAF) mutant alleles of PHYB demonstrate that a range of altered light-signaling activities are associated with mutation of this residue. We conclude that the universally conserved GAF domain Tyr residue, with which the bilin chromophore is intimately associated, performs a critical role in coupling light perception to signal transduction by plant phytochromes.     

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.     

Action and function of phytochrome family members revealed through the study of mutant and transgenic plants

The adaptation of plant growth and development to changes in the light environment is dependent upon photoperception by information transducing photoreceptors. The red/far-red light-absorbing phytochromes are perhaps the best characterized of these regulatory photoreceptors. Higher plants possess multiple, discrete phytochromes, the apoprotein components of which are the products of a small, divergent gene family. Different phytochromes have different biochemical and physiological properties, and are differentially expressed in the growing plant. This has led to the proposal that different phytochromes have different physiological roles. Mutations that disrupt the normal perception of light signals have proved to be a valuable resource in assigning physiological roles to different phytochromes as well as in identifying residues/domains critical for phytochrome function and in attempting to elucidate the signal transduction pathway(s) downstream of phytochromes. This article reviews some recent progress in these areas from the study of conventional and transgenic photomorphogenic mutants.     

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.     

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.     

A single red-light pulse leads to a block of greening in the high-pigment-1 mutant of tomato

A single pulse of red light (R) given to 4-d-old etiolated high-pigment-1 (hp-1) mutant tomato (Solanum lycopersicum L.) seedlings followed by a 3-d dark period is demonstrated to result in a block of greening in subsequent white light. Wild-type seedlings green normally under this regime. The block of greening in the hp-1 mutant depends on the length of the dark period before and after the R pulse and operates via the low-fluence-response mode of phytochrome action. This block of greening takes place in hp-1 double mutants lacking either phytochrome A or phytochrome B1, but is absent in the hp-1 triple mutant lacking both phytochromes A and B1. These observations enable a screen to be devised for new phytochrome B1 mutants either within the photoreceptor or mutants defective in phytochrome B1-signalling steps which result in loss of capacity to green, by mutagenising the phytochrome A-deficient hp-1, fri double mutant. Received: 20 February 1998 / Accepted: 18 June 1998     

Phytochrome gene diversity

The structures and functions of the phytochrome apoprotein genes (the PHY genes), their diversity across the plant kingdom, and their evolution are central concerns in the study of red-light sensing in plants. We summarize here recent advances in two areas relating to these topics: (1) the characteristics of the PHY gene family in Arabidopsis thaliana, the higher plant species for which the most extensive information on these genes is available, and (2) the similarity relationships, phylogeny, and evolutionary implications of PHY gene sequences and partial sequences which have been described from various plants. Together, these two areas of study, one directed at understanding in detail the phytochromes present in a single species and the other directed at a much broader understanding of PHY gene relatedness and distribution, are producing an increasingly clear picture of the diversity and evolution of plant red-light photoreceptors. Moreover, they suggest that the complexity of the phytochrome family has increased as land plants have evolved novel morphologies.     

Photomorphogenic development of the Arabidopsisshy2–1D mutation and its interaction with phytochromes in darkness

We previously reported a photomorphogenic mutation of Arabidopsis thaliana, shy2–1D, as a dominant suppressor of a hy2 mutation. Here, we report that shy2–1D confers various photo-responsive phenotypes in darkness and the dark phenotypes of the mutant are affected by phytochrome deficiency. Dark-grown seedlings of the mutant developed several photomorphogenic characteristics such as short hypocotyls, cotyledon expansion and opening, and partial differentiation of plastids. When grown further in darkness, the mutant plant underwent most of the developmental stages of a light-grown wild-type plant, including development of foliar leaves, an inflorescence stem with cauline leaves, and floral organs. In addition, two light-inducible genes, the nuclear-encoded CAB and the plastid-encoded PSBA genes, were highly expressed in the dark-grown mutant seedlings. Furthermore, reduced gravitropism, a phytochrome-modulated response, was observed in the mutant hypocotyl in darkness. Thus, shy2–1D is one of the most pleiotropic photomorphogenic mutations identified so far. The results indicate that SHY2 may be a key component regulating photomorphogenesis in Arabidopsis. Surprisingly, double mutants of the shy2–1D mutant with the phytochrome-deficient mutants hy2, hy3 (phyB-1) and fre1–1 (phyA-201) showed reduced photomorphogenic response in darkness with a longer hypocotyl, a longer inflorescence stem, and a lower level expression of the CAB gene than the shy2–1D single mutant. These results showed that phytochromes function in darkness in the shy2–1D mutant background. The implications of these results are discussed.     

Molecular and phenotypic specificity of an antisense PHYB gene in Arabidopsis

The family of phytochrome photoreceptors plays an essential role in regulating plant growth and development in response to the light environment. An antisense PHYB transgene has been introduced into wild-type Arabidopsis and shown to inhibit expression of the PHYB sense mRNA and the phyB phytochrome protein 4- to 5-fold. This inhibition is specific to phyB in that the levels of the four other phytochromes, notably the closely related phyD and phyE phytochromes, are unaffected in the antisense lines. Antisense-induced reduction in phyB causes alterations of red light effects on seedling hypocotyl elongation, rosette leaf morphology, and chlorophyll content, similar to the phenotypic changes caused by phyB null mutations. However, unlike the phyB mutants, the antisense lines do not flower early compared to the wild type. Furthermore, unlike the phyB mutants, the antisense lines do not show a reduction in phyC level compared to the wild type, making it possible to unequivocally associate several of the photomorphogenic effects seen in phyB mutants with phytochrome B alone. These results indicate that an antisense transgene approach can be used to specifically inhibit the expression and activity of a single member of the phytochrome family and to alter aspects of shade avoidance responses in a targeted manner.     

Multiple heme oxygenase family members contribute to the biosynthesis of the phytochrome chromophore in Arabidopsis

The oxidative cleavage of heme by heme oxygenases (HOs) to form biliverdin IXalpha (BV) is the committed step in the biosynthesis of the phytochrome (phy) chromophore and thus essential for proper photomorphogenesis in plants. Arabidopsis (Arabidopsis thaliana) contains four possible HO genes (HY1, HO2-4). Genetic analysis of the HY1 locus showed previously that it is the major source of BV with hy1 mutant plants displaying long hypocotyls and decreased chlorophyll accumulation consistent with a substantial deficiency in photochemically active phys. More recent analysis of HO2 suggested that it also plays a role in phy assembly and photomorphogenesis but the ho2 mutant phenotype is more subtle than that of hy1 mutants. Here, we define the functions of HO3 and HO4 in Arabidopsis. Like HY1, the HO3 and HO4 proteins have the capacity to synthesize BV from heme. Through a phenotypic analysis of T-DNA insertion mutants affecting HO3 and HO4 in combination with mutants affecting HY1 or HO2, we demonstrate that both of the encoded proteins also have roles in photomorphogenesis, especially in the absence of HY1. Disruption of HO3 and HO4 in the hy1 background further desensitizes seedlings to red and far-red light and accelerates flowering time, with the triple mutant strongly resembling seedlings deficient in the synthesis of multiple phy apoproteins. The hy1/ho3/ho4 mutant can be rescued phenotypically and for the accumulation of holo-phy by feeding seedlings BV. Taken together, we conclude that multiple members of the Arabidopsis HO family are important for synthesizing the bilin chromophore used to assemble photochemically active phys.     

The rosette habit of Arabidopsis thaliana is dependent upon phytochrome action: novel phytochromes control internode elongation and flowering time

A major function of phytochromes in light-grown plants involves the perception of changes in the relative amounts of red and far-red light (R:FR ratio) and the initiation of the shade-avoidance response. In Arabidopsis thaliana, this response is typified by increased elongation growth of petioles and accelerated flowering and can be fully induced by end-of-day far-red light (EOD FR) treatments. Phytochrome B-deficient (phyB) mutants, which have a constitutive elongated-petiole and early-flowering phenotype, do not display a petiole elongation growth response to EOD FR, but they do respond to EOD FR by earlier flowering. Seedlings deficient in both phytochrome A and phytochrome B (phyA phyB), have a greatly reduced stature compared with wild-type or either monogenic mutant. The phyA phyB double null mutants also respond to EOD FR treatments by flowering early, suggesting the operation of novel phytochromes. Contrary to the behaviour of wild-type or monogenic phyA or phyB seedlings, petiole elongation in phyA phyB seedlings is reduced in response to EOD FR treatments. This reduction in petiole elongation is accompanied by the appearance of elongated internodes such that under these conditions the plants no longer display a rosette habit.