The Phytochromes, a Family of Red/Far-red Absorbing Photoreceptors*

Plants Possess Multiple Photoreceptors Because plants are photo-auxotrophic they are particularly sensitive to their light environment. To fine-tune their development according to light intensity, direction, spectral quality, and periodicity they possess a multiplicity of light sensors (1). In Arabidopsis there are eight identified photoreceptors, but this list is still incomplete. It includes three UV-A/blue light receptors (phototropin, a photoreceptor to sense light direction, and two cryptochromes that mediate many photomorphogenic responses (2, 3)) and five phytochromes (phy) named phyA–phyE that absorb mainly red/far-red light, with phyA also responding to broad-spectrum light (UV-A to far-red) of very low intensity (4). All these photoreceptors bind to a chromophore, which for the phytochromes is a linear tetrapyrrole (phytochromobilin) (5). Because many light effects are induced by the co-action of several photoreceptors and because some photoreceptors regulate multiple aspects of photomorphogenesis, a genetic approach was instrumental for dissecting the specific roles of individual photoreceptors (1). As a consequence, research has concentrated on a few species that are particularly well suited for molecular genetic studies, in particular Arabidopsis (6).

signaling. The analysis of these mutants highlighted the role of phytochromes in sensing light quality, intensity, and the duration of the light cycle and revealed that type I and type II phy have distinct modes of photoperception (14,15).
Light-stable phy are responsible for the classical red/far-red reversible phy responses. In Arabidopsis phyB plays the most prominent role; it is the major red light receptor for seedling de-etiolation, and it affects many light-regulated cell elongation responses, shade avoidance, and the regulation of flowering time by day length (20). phyD and phyE mutants have more subtle phenotypes that are only revealed in double or triple mutant combinations (21)(22)(23). Because certain phytohormone mutants also display similar phenotypes, a subset of phy responses might be mediated by light-regulated hormonal signaling (14, 24 -26).
phyA, the only type I phy in Arabidopsis, plays a major role in gene expression and germination in response to very low fluences of broad spectrum light as well as in sensing day-length extension (27)(28)(29)(30). phyA is also essential for de-etiolation in far-red enriched light (31)(32)(33). Such conditions are found when a young seedling develops under a dense canopy of plants. This is a particularly interesting phy function because, contrary to most phy responses, it is induced by far-red light and inhibited by red light (see above) (34). This high irradiance response to far-red light identifies a novel form of active phy, Pr, that has been cycled through Pfr, which will be referred to as Pr*. Pr* has acquired novel properties that are distinct from Pr and Pfr, but the molecular nature of the distinction between Pr* and Pr is unknown (34). As illustrated above, type I and type II phy play distinct roles; however, it must be pointed out that depending on the responses their role can be overlapping, coordinated, or even antagonistic (35)(36)(37)(38)(39).

Molecular Properties of Phytochromes and
Bacteriophytochromes Phytochromes bind phytochromobilin (P⌽B) via a thioether linkage to a cysteine residue in the most conserved domain among phy (Fig. 1). The first committed step in chromophore biosynthesis is the cleavage of the tetrapyrrole ring of heme (Fig. 1A). This reaction is catalyzed by a heme oxygenase encoded by the HY1 gene in Arabidopsis (40,41). Hy2 mutants are most probably defective in the P⌽B synthase enzyme; this step is followed by an isomerization in the C-3 double bond of P⌽B (42). The nature of the P⌽B isomerase is still unclear, but phy itself is capable of catalyzing this reaction (42). phy chromophore mutants can be mimicked by overexpression of a mammalian biliverdin reductase (43). phy apoprotein binds to the 3E-P⌽B in the cytoplasm to yield the Pr form of the photoreceptor. This reaction requires the bilin lyase domain (BLD) of the photoreceptor. Absorption of red light triggers a "Z" to "E" isomerization in the C-15 double bond between the C and D rings of the linear tetrapyrrole, resulting in the far-red light-absorbing form Pfr (44) (Fig. 1A). Conformational changes in the protein backbone are required to maintain this high energy state of the photoreceptor (45). Pfr can be converted to Pr either by a slow non-photoinduced reaction (dark reversion) or much faster upon absorption of far-red light. It is generally assumed that all phy have the same chromophore. Because of the very low levels of type II phy this has not been verified in vivo. Analysis of reconstituted recombinant phyA, phyB, phyC, and phyE reveals that they have similar but not identical spectral properties (46 -48).
Phytochromes are soluble homodimers composed of two functional domains: an N-terminal light-sensing domain and a C-terminal signaling domain (Fig. 1). The N-terminal portion is necessary and sufficient for photoperception and possesses the bilin lyase activity allowing attachment of the chromophore to the apoprotein (42). The minimal BLD is actually less than 200 amino acids long (49). The first 70 amino acids of the protein are dispensable for chromophore binding; they constitute the N-terminal extension (ATE). The ATE is poorly conserved, possibly accounting for some functional differences among phy. Structure function analysis has revealed that in phyA, the ATE is composed of two subdomains (50,51). The ATE might be implicated in stabilization of the Pfr form of the photoreceptor, which is particularly interesting in view of the large structural changes observed in this part of the protein upon Pr to Pfr phototransformation (45).
The importance of the C-terminal half of plant phytochromes is highlighted by the numerous missense mutations affecting this portion of the protein (4,52). This signaling domain is composed of a PAS (Per/Arndt/Sim)-related domain (PRD) and a histidine ki-nase-related domain (HKRD) (Fig. 1) (53,54). PAS domains have diverse functions; they can be used either as protein-protein interaction platforms or as co-factor binding domains (55). Interestingly such modules are used to bind the chromophore in various blue light receptors (2). In phytochromes, the PRD domain is required for interaction with phy signaling partners but might also play a role in stabilization of the Pfr form of phyB (4,47,56,57). Interestingly a mutation in the BLD domain of phyB has the opposite effect, leading to a phyB protein locked in the Pfr conformation (58).
The discovery of phytochromes in prokaryotes had two important consequences: they provided a phylogenetic origin for plant phy and suggested a biochemical mechanism for phy signaling (59 -61). Cyanobacterial phytochrome 1 (Cph1) is composed of a BLD and a histidine kinase domain (Fig. 1B). Cph1 autophosphorylates and phosphorylates the Rcp1 response regulator in a lightregulated fashion ( Fig. 2A) (61). It is noteworthy that in several cases response regulators are encoded in the same operon as the photoreceptor, suggesting that light-regulated protein phosphorylation is a common theme in bacteriophytochrome signaling. More complex structures have been found in the purple bacteria Rhodospirillum centenum where the photoreceptor has an additional photoactive yellow protein (PYP) domain, which is highly related to the PAS domains present in plant phy (62). Bacteriophytochromes have been discovered in non-photosynthetic organisms as well (63). Their biological function is not always understood, but they play rather diverse roles depending on the species, including chromatic adaptation, resetting of the circadian clock, and regulation of pigment biosynthesis (59,(62)(63)(64). Interestingly, some of these functions have been conserved in plant phy (38,65).
The HKRD domain of plant phytochromes is only distantly related to bacterial histidine kinases, and several residues essential for kinase activity are absent in plant phy (66). In fact, work over the past 20 years has indicated that oat phyA might be a Ser/Thr kinase, and this has been confirmed convincingly quite recently (19,54). Recombinant oat phyA is a light and chromophore-modulated protein kinase with Pfr being a more active form than Pr (Fig.  2C) (54). Oat phyA is a phosphoprotein in vivo; two phosphorylation sites have been mapped, and interestingly they correspond to residues phosphorylated after in vitro kinase assays (67). Ser-7 is constitutively phosphorylated, and mutagenesis studies suggest that phosphorylation of this residue is implicated in down-regulation of phyA signaling (50). Ser-599 phosphorylation is only observed in phyA extracted from light-treated plants (67). Phosphorylation of this residue might therefore be a molecular tag distinguishing between different forms of phy. The importance of this residue has been demonstrated in vitro because a S599K mutant loses light-regulated kinase activity (68). The cryptochromes and PKS1 (phytochrome kinase substrate 1) are also substrates of phyA as a protein kinase, but the role of phosphorylation during phy-mediated light signaling remains to be determined in vivo (68,69). Based on reverse genetic studies it has been proposed that PKS1 acts as a negative regulator of phyB and phyA signaling (68). 2 The phyA-cryptochrome interaction might be the molecular basis for the co-action between those photoreceptors (69). These studies suggest a role for phy-mediated phosphorylation; quite surprisingly, however, deletion of the HKRD domain of phyB has a milder phenotype than certain point mutations in the HKRD (52).
All five members of the phy family are widely expressed (70,71). PHYA transcription is negatively regulated by light through a negative feedback loop dependent on both phyA and phyB (72). phyB mRNA is under circadian regulation; however, this regulated gene expression has only a minor impact on the steady state level of the protein (73). The stability of the phyA protein is greatly dependent on the light conditions. PfrA is selectively ubiquitinated leading to proteolytic degradation, phyA protein levels being therefore about 100-fold lower in the light than in the dark (Fig. 2C) (74). Interestingly PrA* (PrA which has been cycled through Pfr) has a half-life similar to the one of PfrA. Ubiquitination of PrA* might therefore be the molecular tag distinguishing it from PrA. It is noteworthy that the PrA* form of phy (just like the signal gener-2 J. Casal, J. Chory, and C. Fankhauser, unpublished data.

FIG. 1. Structural domains and spectral properties of phytochromes.
A, biosynthetic pathway of the phytochrome chromophore. Enzymes are indicated in red, and the Arabidopsis genes are indicated. Note that chromophore binding is autocatalytic; it requires the bilin lyase domain of the phytochrome; phytochromes also possess PB isomerase activity. The arrow indicates the site of chromophore attachment to a cysteine in the bilin lyase domain. Absorption of red light triggers a "Z" to "E" isomerization in the C-15 double bond between the C and D rings (indicated in bold). The absorption spectra of Pr and Pfr are indicated. BVR, biliverdin reductase. B, structural domains of prokaryotic and plant phytochromes. PAS is the acronym from the founding members of this protein domain (Per/Arndt/Sim). Cph1, cyanobacterial phytochrome 1 from Synechocystis sp. PCC6803; RcaE, regulator of chromatic adaptation E from Fremyella diplosiphon; BphP, bacteriophytochrome 1 from Deinococcus radiodurans; Ppr, PYP-phytochrome-related from Rhodospirillum centenum. ated by PrA*) is very short lived (34,74,75).
Subcellular localization is probably a major level of regulation for plant phytochrome action. Both phyA and phyB are cytoplasmic in the dark, and appropriate light treatments trigger their translocation into the nucleus (Fig. 2B) (12). This relocation takes several hours for phyB in contrast with the more rapid translocation of phyA. This differential behavior correlates well with the sequential action of those phy during light-induced inhibition of hypocotyl growth (39). The slow nuclear translocation of phyB implies that PfrB will be present in the cytoplasm where it could also play a role. Several rapid phy effects, such as ion fluxes at the plasma membrane, have been reported; they could be induced by the cytoplasmic pool of PfrB (76).

Signaling, What Happens after Photoperception?
Pharmacological studies using microinjection of a tomato phy mutant have identified heterotrimeric G proteins, cGMP and Ca 2ϩ , as second messengers in phy signaling (77). Genetic screens have identified two classes of signaling components, those acting downstream of a single photoreceptor and those acting downstream of multiple photoreceptors (Fig. 3). This presumably reflects the fact that light signals perceived by different photoreceptors must be integrated (14,15). The latter class (light signal integration, see Fig. 3) includes both positively acting factors (i.e. HY5) and a large group of negative regulators of photomorphogenesis (DET/COP/ FUS) (16,17). Mutants with phenotypes under specific light conditions (i.e. only red light) are considered as acting early in the cascade. The study of such mutants and of phy interacting proteins reveal a complex signaling web (Fig. 3) (14, 15, 26, 78 -82). These loci can be classified into three groups: those acting specifically downstream of phyA, downstream of phyB, or downstream of both. FIG. 2. Light regulation of phytochrome functions. A, lightregulated His kinase activity initiates phytochrome-mediated light signaling in prokaryotes. The cyanobacterial phytochrome 1 (Cph1) has light-modulated protein kinase activity. Autophosphorylation is attenuated by red light and increased by far-red light. Phosphorylated Pr could trigger a light signal. The phosphorylated Pr transfers its phosphate to the Rcp response regulator; this could in turn initiate light signaling. Phosphorylated Rcp could transfer this phosphate to a yet to be determined acceptor (X). Note that for most plant phytochrome responses Pfr is the active isoform. B, nuclear events in plant phytochrome signaling. Phytochrome B only translocates into the nucleus in its far-red light absorbing form (PfrB). In the nucleus PfrB interacts with the DNA-bound PIF3 transcription factor. Phytochrome A translocates into the nucleus in its red light absorbing form, Pr* (Pr that has been cycled through Pfr; this occurs preferentially in far-red rich light). Translocation also occurs for Pfr (the far-red absorbing form); however, irradiation of plants with red light triggers rapid phyA degradation. In the nucleus phyA presumably affects RSF1-mediated gene activation. PKS1 is a negative regulator of phytochrome signaling; it has been proposed that PKS1 inhibits nuclear translocation of phytochromes. C, other light-regulated properties of plant phytochrome. Light affects the Ser/Thr protein kinase activity of phytochromes. Contrary to Cph1, oat phyA has higher auto-and trans-phosphorylation activity in the Pfr form. Interaction with signaling intermediates is also isoform-dependent; this is true for PIF3, which is nuclear, and for NDPK2, which is found both in the nucleus and the cytoplasm. The protein stability of phyA is very much reduced in its far-red absorbing form (Pfr). The order of action of the loci in the phyA-specific, phyB-specific, and common branch is unknown. These loci presumably act upstream of the "light signal integration" loci. This second class of genes acts downstream of multiple photoreceptors, i.e. the phytochromes (phy) and the cryptochromes (cry). RSF1/ HFR1 acts downstream of phyA but might also play a role in cry-mediated signaling. To simplify this scheme phyC, phyD, and phyE have not been indicated; however, some of these loci might be required for signaling downstream of those photoreceptors.
Interestingly both nuclear and cytoplasmic factors have been identified, and these signaling branches include positive and negative regulation (Fig. 3). Most of the cloned genes code for proteins with poorly defined biochemical functions. It is currently quite hard to propose a model that integrates all those factors, particularly because the relative position of these elements in the chain of events is still largely unknown.
Paradoxically the best understood branch of phy signaling appears to be rather simple (Fig. 2B). PfrB is selectively imported into the nucleus where it interacts with PIF3, a bHLH transcription factor. This interaction occurs specifically with PfrB but not PrB and irrespective of DNA binding by PIF3 (Fig. 2B) (83). How interaction with phyB affects PIF3 activity remains to be solved. RSF1/HFR1/REP1 is another bHLH transcription factor that is quite related to PIF3 and also plays an important role in phy signaling (82, 84 -86). RSF1/HFR1/REP1 is, however, implicated in phyA-and not phyB-mediated signaling (82, 84 -86). There is currently no data indicating a direct interaction between RSF1/HFR1/ REP1 and phyA (85). The presence of bHLH transcription factors in both phyA and phyB signaling is particularly noteworthy in view of the recently uncovered convergence of multiple phy signaling pathways on a single promoter (87). Genetic studies for both PIF3 and RSF1/HFR1/REP1 have revealed that they play important roles in phy signaling, but they also indicate that these transcription factors only account for part of the response initiated by the phy (82,83,85,86). These recent studies illustrate the potentially very short signaling chain initiated by the phytochromes, but we should keep in mind that much remains to be done to have a global view of the multiple events initiated by those photoreceptors.