Phytochromes and photomorphogenesis in Arabidopsis

Garry C. Whitelam, Samita Patel and Paul F. Devlin Biology Department, Leicester University, Leicester LE1 7RH, UK

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 di¡erent 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 ¢ve apophytochrome-encoding genes, PHYA^ PHYE. Di¡erent phytochromes have discrete biochemical and physiological properties, are di¡erentially expressed and are involved in the perception of di¡erent light signals. Photoreceptor and signal trans- duction mutants of Arabidopsis are proving to be valuable tools in the molecular dissection of photomorphogenesis. Mutants de¢cient in four of the ¢ve phytochromes have now been isolated. Their analysis indicates considerable overlap in the physiological functions of di¡erent phytochromes. In addition, mutants de¢ning components acting downstream of the phytochromes have provided evidence that di¡erent members of the family use di¡erent signalling pathways. Keywords: phytochrome; Arabidopsis; mutant; photomorphogenesis; light

1. INTRODUCTION 2. PHOTOCHROME PROPERTIES Plants possess a range of sensory systems that monitor the The phytochromes are reversibly photochromic, soluble surrounding environment so enabling them to initiate bilin-linked chromoproteins. Typically, phytochromes appropriate modi¢cations to their development. Being consist of a dimer of identical approximately 124 kDa photoautotrophic, plants are especially sensitive to the polypeptides, each of which carries a single, covalently light environment and plant morphogenesis is linked linear tetrapyrrole chromophore (phytochromo- dramatically a¡ected by variations in the intensity, bilin), attached via a thioether bond to a conserved quality, direction and duration of light. Information cysteine residue in the N-terminal globular domain of the gained about these factors is used to modulate all aspects protein (Furuya & Song 1994). The more elongated, non- of plant development, from seed germination, through chromophorylated, C-terminal domain of the protein is seedling de-etiolation and establishment, to the control of involved in dimerization (Edgerton & Jones 1992). Phyto- later events in vegetative development, the architecture of chromes can exist in either of two relatively stable the plant and the transition to reproductive development. isoforms: an R light-absorbing form, Pr, with an absorp- The various developmental responses of plants to tion maximum at about 660 nm, or an FR light- signals from the light environment are collectively absorbing form, Pfr, with an absorption maximum at referred to as photomorphogenesis. Photomorphogenesis about 730 nm. Light-induced interconversions between Pr is a complex process that depends upon the actions of and Pfr involve a Z-E isomerization of the linear tetra- several signal-transducing photoreceptor systems that pyrrole chromophore about the C15 double bond that interact with endogenous developmental programmes links the C- and D-rings of the tetrapyrrole (see Terry et and with the endogenous circadian oscillator. These al. 1993). The photoisomerization of the chromophore photoreceptor systems include the phytochromes, which leads to reversible conformational changes throughout the absorb mainly in the red (R) and far-red (FR) regions protein moiety of phytochrome (Quail 1991). The altered of the spectrum, the cryptochromes, which absorb in protein conformations stabilize the chromophore isomers. the blue^UV-A region of the spectrum, and distinct Based on physiological and genetic studies, the Pfr form UV-A and UV-B light-absorbing photoreceptors. The of phytochrome is generally considered to be biologically phytochromes are the most extensively characterized of active, and Pr is considered to be inactive. It is assumed these photoreceptors. Recently, some of the blue^UV-A that the light-induced protein conformational changes photoreceptors have been identi¢ed and characterized at account for the di¡erences in activity between Pr and Pfr. the molecular level (Cashmore 1997). The identity of The absorption spectra of Pr and Pfr show considerable any of the UV-A or UV-B photoreceptors remains overlap throughout the visible light spectrum. As a elusive. consequence, the phytochromes are present in an

Phil. Trans. R. Soc. Lond. B (1998) 353, 1445^1453 1445 & 1998 The Royal Society 1446 G. C.Whitelam and others Phytochromes and photomorphogenesis equilibrium mixture of the two forms under almost all irradiation conditions. All higher plants studied so far, as well as some lower plants, possess multiple discrete phytochrome species which make up a family of closely related photoreceptors, the apoproteins of which are encoded by a small family of divergent genes (Quail 1994). The size of the phyto- chrome family varies among di¡erent plant species. In Arabidopsis thaliana, ¢ve apophytochrome encoding genes (PHYA^PHYE) have been characterized (Sharrock & Quail 1989; Clack et al. 1994). The PHYA gene has been demonstrated to encode the apoprotein of the well- characterized, light-labile phytochrome that predomi- nates in etiolated seedlings, phytochrome A (phyA). Phytochrome A is rapidly depleted when etiolated tissues are exposed to light as a consequence of Pfr-induced proteolysis (Quail 1994). Light exposure also induces a reduction in PHYA transcript abundance (Quail 1994). The other PHY (B^E) genes encode the apoproteins of lower abundance, which appear to be more light-stable phytochromes, previously referred to as type 2 phyto- chromes (see Kendrick & Kronenberg 1994). The Arabidopsis PHYB and PHYD polypeptides are about 80% identical (Mathews & Sharrock 1997) and are some- what more related to PHYE than to either PHYA or PHYC (about 50% identity). Furthermore, the PHYB, PHYD and PHYE polypeptides are the most recently evolved members of the phytochrome family. Counterparts of PHYA, PHYB and other PHY genes are present in most, if not all, higher plants (Mathews & Sharrock 1997). The spatial and temporal expression patterns of the Arabidopsis PHYA, PHYB, PHYD and PHYE genes have been studied by introducing into Arabidopsis fusions Figure 1. Phytochrome photoreceptor mutants of Arabidopsis. between PHY promoter regions and the Escherichia coli Wild-type (WT), phyA and phyB seedlings were grown in the GUS gene. Staining for GUS indicated detectable activity dark (D), under continuous red light (R), continuous far-red of all of the promoters throughout seedling development light (FR), or continuous blue light (B) for four days. Scale bar, 1 cm. (Somers & Quail 1995; Goosey et al. 1997). The PHYA and PHYB promoters showed similar spatial patterns of activity, being expressed in most parts of the plant. Thus, Arabidopsis has been a focus for both approaches. Dark- di¡erences in photoreceptor apoprotein gene expression grown Arabidopsis seedlings display a typical etiolated cannot account for the di¡erent physiological roles of phenotype, characterized by extreme hypocotyl these two phytochromes (see following paragraphs). The elongation growth, a hypocotyl hook, small folded PHYD and PHYE promoters were observed to drive much cotyledons, absence of chlorophyll and lack of plastid lower GUS expression than the PHYA or PHYB promoter development. In response to R, FR or blue-light signals, a and were found to show a more restricted pattern of series of growth and developmental changes are initiated expression, notably in cotyledons, leaves and sepals that lead to de-etiolation and photomorphogenic seedling (Goosey et al. 1997). In the shoots of young seedlings, the development (see ¢gure 1). Genetic screens for mutants activities of the PHYA, PHYB and PHYD promoters were that are insensitive to light with regard to photomorpho- found to be higher for dark-grown seedlings than for genic seedling development have led to the identi¢cation light-grown seedlings, whereas the opposite was the case of mutations in the PHYA and PHYB genes, as well as in for PHYE (Goosey et al. 1997). The PHYA promoter shows two genes (HY1 and HY2) that encode proteins involved the greatest down-regulation by light (Somers & Quail in tetrapyrrole chromophore synthesis. This screen also 1995). identi¢ed mutations in cryptochrome apoprotein. The physiological characterization of photoreceptor mutants and transgenic plants is enabling us to form a picture of 3. PHYTOCHROME FUNCTIONS the roles of, and interactions among, the phytochromes. The most revealing approaches to the identi¢cation of Arabidopsis mutants de¢cient in phyA (phyA) were the roles of individual photoreceptors have been those isolated independently by several laboratories in screens involving the analysis of mutants that are de¢cient in one for a long hypocotyl phenotype following seedling or more photoreceptors. Additional insights into photo- growth of prolonged FR light (Nagatani et al. 1993; receptor functions have also come from the study of trans- Parks & Quail 1993; Whitelam et al. 1993). Seedlings that genic plants that overexpress photoreceptor genes. are null for phyA display a complete insensitivity to

Phil. Trans. R. Soc. Lond. B (1998) Phytochromes and photomorphogenesis G. C.Whitelam and others 1447 prolonged FR light with respect to all aspects of seedling day plant, and £owering is promoted as daylength photomorphogenesis, including inhibition of hypocotyl increases. As expected, the £owering of wild-type seed- elongation, the opening and expansion of cotyledons lings is very markedly promoted when short photoperiods (¢gure 1), the synthesis of anthocyanin and the are extended to 16 h by low £uence rate incandescent regulation of expression of several genes (see, for light. However, the £owering of phyA mutants is hardly example, Whitelam et al. 1993; Johnson et al. 1994; Barnes promoted by this type of day extension (Johnson et al. et al. 1996). As other apophytochrome mutants are not 1994). Furthermore, for seedlings grown under long days impaired in their responses to prolonged FR light, phyA in the greenhouse, phyA mutants typically £ower signi¢- is implicated as the sole photoreceptor mediating seed- cantly later than wild-type seedlings (G. C. Whitelam, ling responses to this waveband (see Quail et al. 1995). unpublished observations). The phytochrome A-de¢cient This view is supported by the ¢nding that transgenic fun mutant of another long day plant, garden pea (Pisum overexpression of an oat PHYA cDNA in Arabidopsis leads sativum), also has an impaired ability to perceive long days to exaggerated light responses, particularly responses to and is late £owering under such conditions (Weller et al. FR light (Boylan & Quail 1991; Whitelam et al. 1992). 1997). In contrast to the late £owering of phyA mutants, Sensitivity to R light in phyA seedlings is more or less transgenic Arabidopsis seedlings that overexpress an oat normal, indicating that phyA plays only a small role in PHYA cDNA are markedly early £owering, particularly seedling responses to this waveband. following growth under short photoperiods (Bagnall et al. There are two distinct modes of phytochrome action 1995). Thus, the transgenic overexpressors grown under which have been recognized for the responses of etiolated short photoperiods, respond in a manner similar to that seedlings to FR light (Smith & Whitelam 1990). In the of wild-type seedlings grown under long photoperiods. A FR high irradiance response (FR-HIR), prolonged similar phenomenon has recently been reported for trans- irradiation with continuous FR light of a relatively high genic hybrid aspen overexpressing oat PHYA cDNA £uence rate (0.1^50 mmol m72 s71) is required for display (Olsen et al. 1997). In wild-type aspen trees, short-day of responses, and the extent of the HIR is dependent on conditions induce apical growth cessation and cold accli- the £uence rate of FR light (see Smith & Whitelam matization; transgenic trees show a marked insensitivity 1990). In contrast, very low £uence responses (VLFR) to short photoperiods. are initiated by £uences of light as low as 1079 mol m72 As well as playing a key role in seedling establishment and are fully saturated by very low concentrations of Pfr. and daylength perception, phyA is also involved in the As even a single pulse of broad band FR light can photocontrol of Arabidopsis seed germination (Johnson et saturate the VLFR, this response mode does not obviously al. 1994; Reed et al. 1994; Shinomura et al. 1994; Botto et display prototypical R^FR reversibility (see Smith & al. 1996; Whitelam & Devlin 1996). For dormant wild- Whitelam 1990). Both HIR and VLFR (e.g. for the type seeds, germination is signi¢cantly promoted by brief inhibition of hypocotyl elongation and the promotion of or prolonged exposure to broad band FR light. This e¡ect cotyledon expansion) modes are absent in phyA mutant of FR light is not seen in seeds of the phyA mutant seedlings (Casal et al. 1997). (Johnson et al. 1994). The germination promoting e¡ect of Although seedlings growing in the natural environment single pulses of FR light indicates that phyA is operating would never be exposed to prolonged FR light, there is in the VLFR mode (Shinomura et al. 1996). Botto et al. evidence that FR-HIR is of fundamental importance in (1996) have shown that, in the ¢eld, the promotion of seedling establishment under natural canopy shade seed germination by single pulses of deep canopy shade^ conditions. Yanovsky and co-workers (1995) have shown light also re£ects a phyA-mediated VLFR. that, for Arabidopsis seedlings growing under dense Phytochrome B-de¢cient Arabidopsis mutants were vegetational shade, an FR light-rich light environment, isolated many years ago on the basis of their long hypo- phyA mutants display severely impaired de-etiolation and cotyl phenotype following growth under white light extreme hypocotyl elongation. As a consequence, (Koornneef et al. 1980; Reed et al. 1993). Etiolated phyB signi¢cant numbers of phyA seedlings fail to become seedlings display a marked insensitivity to brief or established and die. Wild-type seedlings are less elongated prolonged R light with respect to almost all aspects of under these conditions and are so more able to become seedling de-etiolation, including inhibition of the hypo- established. cotyl elongation, cotyledon expansion and chlorophyll When grown in white light, or under non-shaded synthesis. However, phyB seedlings de-etiolate normally conditions in the greenhouse, phyA mutants develop following growth under prolonged FR or blue light normally and display a vegetative phenotype that is (Koornneef et al. 1980; Reed et al. 1993). This indicates indistinguishable from that of wild-type seedlings that phyB plays a principal role in seedling responses to (Whitelam et al. 1993). This might be taken to indicate R light but plays no role in responses to prolonged FR that phyA plays only a restricted or specialized role in light. Thus, phyB appears to control seedling de-etiolation early seedling photomorphogenesis. However, despite under R light and phyA controls the same processes being light-labile, phyA is one of the most abundant under FR light. phytochromes in the fully de-etiolated light-grown plant Phytochrome B also plays a principal role in the photo- (Clack et al. 1994) and there is abundant evidence that regulation of Arabidopsis seed germination (Shinomura et phyA continue to regulate development throughout the al. 1994, 1996; Reed et al. 1994). The germination of life of the plant. For example, Johnson and co-workers dormant wild-type seeds is markedly promoted by a (1994) showed that Arabidopsis phyA mutant seedlings single brief pulse of R light. Furthermore, the e¡ect of the £ower later than wild-type seedlings following growth R light pulse can be nulli¢ed by a subsequent pulse of FR under 8-h photoperiods. Arabidopsis is a facultative long- light. This R^FR reversible response, the classical

Phil. Trans. R. Soc. Lond. B (1998) 1448 G. C.Whitelam and others Phytochromes and photomorphogenesis hallmark of phytochrome action, re£ects the low £uence encodes a novel type of homeodomain protein, and the response mode of phytochrome action, and is largely CAB gene, which encodes a structural component of the (although not completely) absent in seeds from phyB-null light-harvesting complex, are light-regulated in an R^ mutants (Shinomura et al. 1994, 1996; Reed et al. 1994). FR reversible manner in phyAphyB mutants (Carabelli et The Pfr form of phyB, present in the dry seed, can also al. 1996; Hamazato et al. 1997). promote the germination of seeds that have been allowed A phytochrome D-de¢cient mutant has recently been to imbibe in darkness (McCormac et al. 1993). obtained following the discovery that certain accessions Phytochrome B plays a predominant role in controlling of the Ws ecotype of Arabidopsis carry a 14-bp deletion, the shade avoidance syndrome of responses (Smith 1995; beginning at amino acid 29, of the coding region of the Smith & Whitelam 1997). The perception of alterations in PHYD gene (Aukerman et al. 1997). The deletion leads to the relative amounts of R and FR light represents one of translation termination at a nonsense codon 138 nucleo- the most important aspects of photomorphogenesis in tides downstream of the end point of the deletion. This nature. Light that is re£ected from, or transmitted phyD mutation has been introgressed into the La er through, vegetation is depleted in the R region relative to ecotype and has been combined with other phy mutations. the FR region as a consequence of absorption by chloro- Analyses of the various mutants indicates that phyD plays phyll. This reduced ratio of R to FR light (R:FR ratio) is a role similar to that played by phyB in initiating shade perceived by the phytochrome system as a change in the avoidance responses. In both the Ws and La er ecotypes, relative amounts of Pr and Pfr, and provides the plant phyD mutant seedlings grown under R light have very with an unambiguous signal that potential competitors slightly elongated hypocotyls compared with wild-type are nearby (see Smith 1995). Many plants, including seedlings (Aukerman et al. 1997). This e¡ect of phyD-de¢- Arabidopsis, in an attempt to avoid being shaded, respond ciency is more marked in a phyB mutant background. to these low R:FR ratio signals by increasing elongation Furthermore, phyD-de¢ciency eliminates the small EOD of internodes and/or petioles, reducing leaf growth, FR light-induced hypocotyl elongation response displayed increasing apical dominance and accelerating £owering by Ws phyB seedlings. Phytochrome D-de¢ciency causes (Smith 1995; Smith & Whitelam 1997). This shade avoid- several defects in adult plant development, some of which ance phenotype in constitutively displayed by phyB are only readily detected in a phyB background. In mutants (see, for example, Whitelam & Smith 1991; particular, in both the Ws and La er ecotypes, mutants Devlin et al. 1996). Furthermore, seedlings that are null de¢cient in both phyB and phyD £ower earlier, and have for phyB show greatly attenuated (although not comple- noticeably longer petioles than mutants lacking only phyB tely absent) responses to low R:FR ratio signals (see, for (Aukerman et al. 1997; see ¢gure 2). example, Whitelam & Smith 1991; Halliday et al. 1994; The overlapping functions of the closely related phyB Devlin et al. 1996). In wild-type plants, the shade avoid- and phyD indicates that there is signi¢cant redundancy ance syndrome of responses can be quite e¡ectively within the phytochrome family with regard to R:FR ratio induced by pulses of FR light given at the end of each signal perception and the initiation of shade avoidance photoperiod. These end-of-day (EOD) responses are also reactions. This idea is further reinforced by our recent greatly attenuated in phyB-null seedlings (Nagatani et al. isolation and analysis of a phyE mutant in Arabidopsis 1991; Devlin et al. 1996). Taken together, these ¢ndings which suggests that phyE also plays a role in mediating implicate phyB as a main contributor to shade avoidance. shade avoidance responses. The phyE mutant was isolated

The early £owering of phyB mutants is displayed in a screen involving M2 seedlings derived from muta- following seedling growth under either long or short genesis of the phyAphyB mutant. Previously, Devlin and photoperiods (see, for example, Goto et al. 1991; Halliday co-workers (1996) had shown that phyAphyB seedlings et al. 1994). This presumably re£ects the constitutive shade respond to EOD FR light treatments by elongation of the avoidance phenotype. Nevertheless, phyB mutants are still internodes between `rosette' leaves and by early £owering responsive to daylength, £owering much earlier under (see ¢gure 2). Signi¢cantly, both of these responses are long photoperiods than under short photoperiods (see, for also displayed by phyAphyBphyD triple mutants (¢gure 2), example, Goto et al. 1991; Bagnall et al. 1995). This suggests suggesting that either phyC and/or phyE mediates these that in Arabidopsis phyB may not be playing a signi¢cant responses. In the screen, M2 seedlings were grown for role in the detection of daylength. several weeks under white light and elongated^early- The retention of responses to R and FR light by £owering individuals were selected. The selected plants mutants that are doubly null for phyA and phyB has were selfed and the progeny re-screened. Several provided some clues to the physiological functions of elongated and/or early £owering mutants were isolated. other members of the phytochrome family. For example, One of the mutants displaying both elongated rosette it has been shown that phyAphyB double mutants respond internodes and early £owering was found to have a 1-bp to low R:FR ratio signals and EOD FR light treatments deletion within the coding region of the PHYE gene at by a promotion of elongation growth and earlier position equivalent to amino acid 784. The resulting £owering (Halliday et al. 1994; Devlin et al. 1996). The frame shift creates a stop codon 42-bp downstream of the responses of phyAphyB seedlings to EOD FR light pulses deletion. The deletion in PHYE disrupts a HinfI restric- show R^FR reversibility, clearly implicating the action of tion enzyme site which provided a diagnostic test used in one or more other phytochromes (Devlin et al. 1996). the isolation of the phyE mutant following backcrosses to Similarly, seeds of the phyAphyB double mutant show an phyAphyB to the wild-type. One of the most striking R^FR reversible promotion of germination (Whitelam & features of the phyAphyBphyE triple mutant is that, in Devlin 1996; Poppe & Scha« fer 1997). Also, the steady- contrast with phyAphyB or phyAphyBphyD seedlings, this state level of transcripts of the ATHB-2 gene, which mutant shows little or no response to EOD FR light

Phil. Trans. R. Soc. Lond. B (1998) Phytochromes and photomorphogenesis G. C.Whitelam and others 1449

Figure 2. Shade avoidance responses of phytochrome- de¢cient mutants. Plants were grown for 60 days under 8 h light : 16 h dark photoperiods (control), or under the same conditions with a 10 min pulse of FR light given immediately prior to transfer to darkness (+EOD FR). Scale bar, 5 cm.

treatments (¢gure 2). Monogenic phyE mutant seedlings However, the size of the deletion (at least 14-kbp) appear indistinguishable from wild-type seedlings at both precludes attempts to assign aspects of the phenotype to the seedling and rosette stage. However, phyE-de¢ciency phyC-de¢ciency. The introduction of a PHYC transgene, is detectable in a phyB background, where the phyBphyE under the control of the cauli£ower mosaic virus 35S double mutant has more elongated petioles and is earlier promoter, into gn7 failed to rescue any features of the gn7 £owering than the monogenic phyB mutant (G. C. mutant phenotype (G. C. Whitelam, unpublished data). Whitelam, unpublished observations). This is similar to Some clues to the possible photoregulatory roles of the situation observed for the phyD mutant (Aukerman et phyC have come from experiments in which PHYC has al. 1997; ¢gure 2). been overexpressed in wild-type plants (Qin et al. 1997; Mutants that are de¢cient in phyC have so far proved Halliday et al. 1997). Arabidopsis plants overexpressing elusive. We have isolated a mutant, tentatively designated Arabidopsis PHYC display a slightly enhanced hypocotyl gn7, that carries a large deletion (spanning PHYC) on the elongation inhibition response to prolonged R light and top arm of chromosome 5 (G. C. Whitelam, unpublished an increase in primary leaf expansion following growth data). The mutant is diminutive with small leaves and in white light (Qin et al. 1997). The overexpression of reduced elongation growth, shows reduced apical domi- Arabidopsis PHYC in tobacco plants does not alter respon- nance, aberrant £ower development and is male sterile. sivity of the hypocotyls to R light, but it does lead to

Phil. Trans. R. Soc. Lond. B (1998) 1450 G. C.Whitelam and others Phytochromes and photomorphogenesis signi¢cant increases in both cotyledon and leaf expansion, mediating an interaction with a component of the signal- as well as increasing overall plant size (Halliday et al. ling pathway. The observation that overexpression of 1997). The e¡ects on leaf expansion are speci¢c to PHYC PHYB leads to a dominant negative interference with the overexpression because in both Arabidopsis and tobacco, action of endogenous phyA in the FR-HIR controlling the leaf expansion is not enhanced by the overexpression of inhibition of hypocotyl elongation provides additional either PHYA or PHYB (Qin et al. 1997; Halliday et al. circumstantial evidence that phyA and phyB share a 1997). This may indicate that phyC has discrete functions, common reaction partner (Wagner et al. 1996b). To not directly comparable to the functions of the other reconcile this proposal with the speci¢city of signal members of the family. perception determined by the N-terminal domains of phyA and phyB, a two-point contact model for phyto- chrome action has been proposed (Quail 1997; Wagner et 4. PHYTOCHROME SIGNAL TRANSDUCTION al. 1997). In this model, the N-terminal domains of phyA It is obvious that to bring about photomorphogenesis, and phyB carry determinants that allow each to recognize the absorption of light by the phytochromes has to be its own, discrete cognate reaction partner. The common, coupled to the modulation of gene expression or other C-terminal determinants, required for regulatory activity, changes in cell physiology. Genetic and transgenic are then proposed to mediate an identical modi¢cation of approaches are being used to elucidate the pathways of the reaction partners, for example, a phosphorylation of a phytochrome signal transduction in Arabidopsis. Because key residue. In this model, the subsequent events in the of their importance in seedling de-etiolation, particularly signalling pathway could converge immediately or the photoregulation of hypocotyl elongation, and the remain separate until the then `end points' (see Quail obvious phenotype associated with their absence, phyto- 1997). This latter idea is supported by the genetic analysis chrome A and phytochrome B have been the main focus of phyA and phyB signal transduction (see this paper). of these studies. It has been suggested that phyA and phyB are di¡eren- In seedling de-etiolation, phytochromes A and B tially localized within the cell (Sakamoto & Nagatani control a large number of common responses, including 1996) and so may have discrete primary actions. inhibition of hypocotyl growth, promotion of hypocotyl Immunochemical methods have previously established hook opening and cotyledon expansion, and the photo- that phyA is a soluble, cytoplasmic protein. Sakamoto & induction of CAB gene expression. Of course, although Nagatani (1996) present evidence suggesting that the `outputs' of phyA and phyB action may be common, Arabidopsis phyB is a nuclear protein. Following the these two photoreceptors display discrete photosensory expression of translational fusions between the non- speci¢cities (see here; Quail 1997). Furthermore, phyA chromophorylated C-terminus of PHYB and GUS in and phyB appear to have quite discrete regulatory transgenic Arabidopsis plants, GUS activity was observed functions in the adult plants. Nevertheless, in to be localized to the nucleus in both cotyledon proto- de-etiolating seedlings it seems likely that the phyA and plasts and epidermal peels. In addition, PHYB protein phyB signalling pathways share some common was detected immunochemically in preparations of components. This notion is supported by the ¢nding that isolated nuclei and anti-PHYB monoclonal antibodies these phytochromes have a common spatial pattern of were found to stain nuclei in immunocytochemical tests expression and so both will be present in the same cells. (Sakamoto & Nagatani 1996). The derived amino-acid Circumstantial evidence from the analysis of transgenic sequence of PHYB (and other apophytochromes), is plants that overexpress PHY gene sequences provides known to contain sequences with some homology to support for the idea of shared intermediates in phyA and nuclear localization signals. However, these proposals are phyB signalling. Wagner et al. (1996a) have done not very readily reconciled with observations on the reciprocal domain swap experiments in which the activities of chimeric phytochromes and with the chromophore-bearing N-terminal domain of phyA fused dominant negative e¡ects of transgenic phyB on with the C-terminal domain of phyB (phyA^B), and the endogenous phyA activity (Wagner et al. 1996a,b). converse fusion (phyB^A), have been overexpressed in Genetic approaches to the dissection phytochrome transgenic plants. Overexpression of either chimera leads signal transduction pathways in Arabidopsis have tended to to a light-hypersensitive phenotype. Analyses of the focus on the identi¢cation of mutants that show gross photoresponses of transformants to R and FR light defects in de-etiolation. Almost all mutants isolated on showed that the phyA^B chimera mimicked the e¡ects of the basis of an insensitivity to light, for example, long phyA overexpression, whereas the phyB^A chimera hypocotyl mutants, have de¢ned genes encoding the mimicked the e¡ects of phyB overexpression. This apoproteins of photoreceptors or proteins necessary for indicates that the photosensory speci¢cities of phyA and photoreceptor chromophore synthesis. However, there are phyB reside in the N-terminal domains (Wagner et al. a small number of exceptions, and the genes de¢ned by 1996a). Furthermore, because the overexpression of either these mutants are thought to identify positive regulators N-terminal domain alone does not cause light-hypersensi- of phytochrome signalling. tivity, it seems that the C-terminal domain provides at The analysis of putative signal transduction mutants least some of the determinants necessary for the trans- tends to indicate that the phyA and phyB signalling mission of the light signal. The interchangeability of the pathways are discrete. Mutants selectively impaired in C-terminal domains suggests that these determinants are de-etiolation responses to prolonged FR light have been common to phyA and phyB (Wagner et al. 1996a). described (Whitelam et al. 1993). These mutants de¢ne Although the role of the C-terminal domain may be components speci¢c to the phyA signalling pathway. The structural, it is possible that it may be involved in fhy1 and fhy3 mutants resemble phyA mutants in their

Phil. Trans. R. Soc. Lond. B (1998) Phytochromes and photomorphogenesis G. C.Whitelam and others 1451 insensitivity to prolonged FR light and a normal both R and FR light, and as such possibly de¢ne sensitivity to prolonged R light. However, neither of the common components of the phyA and phyB signalling mutations is linked to the PHYA gene and both mutants pathways. The hy5 mutant displays a light-insensitive have normal levels of immunochemically and spectrally phenotype characterized by a long hypocotyl for seed- detectable phyA (Whitelam et al. 1993). Additional physio- lings grown under R, FR, blue and UV-A light condi- logical and molecular analyses of the fhy1 mutant have tions, but not UV-B conditions (Koornneef et al. 1980). revealed that it is defective in a subset of phyA-mediated This suggests that the HY5 gene product is required as a responses, suggesting that the phyA signalling pathway positive regulator in the transduction of light signals may be branched (Johnson et al. 1994; Barnes et al. 1996). perceived by phyA, phyB and one or more of the crypto- Thus, although the inhibition of hypocotyl elongation chromes. The HY5 gene has recently been cloned and under FR irradiation is impaired in fhy1, the mutant shown to encode a transcription factor of the b-zip class displays normal germination responses to FR light (see Barnes et al. 1997). The pef1 mutant also displays an (Johnson et al. 1994). Similarly, whereas the FR light- elongated hypocotyl following growth under either R or mediated induction of CHS gene expression is de¢cient in FR light (Ahmad & Cashmore 1996). fhy1, the induction of CAB and NR gene expression is A large number of mutants that display photomorpho- relatively una¡ected by the mutation (Barnes et al. 1996). genic development in the absence of light, and so may The induction of all of these genes by FR light is de¢cient de¢ne suppressers of photomorphogenesis, have been in phyA seedlings. The CHS and CAB genes had previously identi¢ed. These include the recessive det^cop^fus mutants been proposed to de¢ne distinct branches of the phyA (see von Arnim & Deng 1996). Severe alleles of ten of the signalling pathway based on biochemical analyses done in det^cop^fus mutants lead to extremely pleiotropic photo- microinjected tomato hypocotyl cells (see Barnes et al. morphogenic phenotypes and seedling lethality. This 1997). suggests that the products of these DET^COP^FUS genes Several mutants that show a selective defect in de- are involved in essential cellular processes both in the etiolation responses to R light, but which are unlinked to light and the dark and not simply in repressing photomor- PHYB, have been isolated. For example, the pef2 and pef3 phogenesis in the dark (see Castle & Meinke 1994; Mayer mutants, which were selected on the basis of their early et al. 1996). The DET1, COP1, COP9 and FUS6 genes have £owering, display a long hypocotyl phenotype following been cloned and shown to encode novel nuclear-localized growth under R light, but a normal inhibition of hypo- proteins (Deng et al. 1992; Pepper et al. 1994; Wei et al. cotyl elongation in response to prolonged FR light 1994; Castle & Meinke 1994). The COP1 polypeptide has (Ahmad & Cashmore 1996). As phyB mutants are some homology with several regulatory proteins involved characterized by a long hypocotyl phenotype selectively in the repression of transcription (see von Arnim & Deng under R light and by early £owering, it is possible that 1996). Light has been reported to in£uence the sub- PEF2 and PEF3 may encode proteins that act early in the cellular location of the COP1 protein (von Arnim & phyB signalling pathway. The red1 mutant also displays a Deng 1994). In etiolated tissues, COP1 appears to be selective defect in seedling responses to R light (Wagner et predominantly nuclear, and light treatment is reported to al. 1997). This mutant was selected from the M2 cause a shift to a predominantly cytoplasmic localization. population derived following mutagenesis of a transgenic Furthermore, the nuclear localization of COP1 in the Arabidopsis line that overexpresses PHYB and so is dark is reported to be dependent on its interaction with a hypersensitive to R light. The red1 mutation segregates nuclear complex of other COP^FUS proteins, the COP9 independently of the PHYB transgene and the mutant complex (Chamovitz & Deng 1996). It is proposed that phenotype is displayed in non-transgenic background. In COP1 interacts with the COP9 complex in the dark to addition to their long hypocotyls, red1 mutants show repress photomorphogenesis and that light causes the attenuated responses to EOD FR light treatments dissociation of COP1 from the complex, followed by its (Wagner et al. 1997). This suggests that RED1 encodes a export from the nucleus, so relieving the repression protein that functions as an intermediate in a phyB (Chamovitz & Deng 1996). signalling pathway that regulates elongation growth. It has been argued that the DET^COP^FUS gene Signi¢cantly, Wagner et al. (1997) have reported that red1 products may be general repressors of gene expression seedlings do not show early £owering, suggesting that and development, rather than playing a speci¢c role in elongation growth regulation and £owering time repre- photomorphogenesis. That severe alleles of several of the sent separate branches of phyB signalling. mutants are lethal argues that the products of the wild- The chlorate-resistant mutant, cr88, also displays a type genes are necessary for general development in both long hypocotyl phenotype selectively under R light (Lin the light and the dark (Castle & Meinke 1994). Further- & Cheng 1997). Chlorate-resistance results from a defect more, weak alleles of cop1, det1 and cop9 lead to inap- in the light-dependent induction of expression of NR2, propriate expression of several sets of genes, not only one of the two Arabidopsis genes that encode nitrate those that are normally light-regulated (Mayer et al. reductase. Light-mediated induction of expression of the 1996). CAB and RBCS genes is also impaired in cr88, although the inductions of NR1 and NiR expression are normal. These observations suggest that CR88 may encode a 5. CONCLUSIONS signalling component involved in a subset of phyB- Our understanding of the perception and transduction mediated responses. of regulatory light signals has advanced signi¢cantly in There are at least two putative phytochrome signalling recent years, principally through the application of mutants that display a long hypocotyl phenotype under genetic and transgenic methods. Arabidopsis mutants that

Phil. Trans. R. Soc. Lond. B (1998) 1452 G. C.Whitelam and others Phytochromes and photomorphogenesis de¢ne four of the ¢ve phytochrome structural genes have Chamovitz, D. A. & Deng, X.-W. 1996 Light signalling in been identi¢ed and are being characterized. Although plants. Crit. Rev. Pl. Sci. 15, 455^478. phytochromes A and B play the predominant role in seed Clack, T., Mathews, S. & Sharrock, R. A. 1994 The phyto- germination and seedling de-etiolation, they are involved chrome apoprotein family in Arabidopsis is encoded by 5 in the perception of quite discrete light signals. In the genesöthe sequences and expression of PHYD and PHYE. Pl. Molec. Biol. 25, 413 427. established plant, phyA is involved in the detection of ^ Deng, X.-W., Matsui, M., Wei, N., Wagner, D., Chu, A. M., daylength, whereas phytochromes B, D and E all play a Feldmann, K. A. & Quail, P. H. 1992 COP1, an Arabidopsis role in the perception of R:FR ratio signals. regulatory gene, encodes a protein with both a zinc-binding Several of the components that act downstream of the motif and a Gb homologous domain. Cell 71, 791^801. phytochromes are also being identi¢ed. Several mutants, Devlin, P. F., Halliday, K. J., Harberd, N. P. & Whitelam, G. C. such as fhy1^fhy3, pef2^pef3 and red1 de¢ne signalling 1996 The rosette habit of Arabidopsis thaliana is dependent components that are speci¢c to the actions of particular upon phytochrome action: novel phytochromes control inter- phytochromes. The cloning of the genes de¢ned by these node elongation and £owering time. Pl. J. 10, 1127^1134. mutations will be crucial in dissecting the early steps of Edgerton, M. D. & Jones, A. M. 1992 Localization of protein^ light signalling. To date, there have been few genetic protein interactions between the subunits of phytochrome. Pl. studies of the transduction of R:FR ratio signals. 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