phytochrome signaling mechanism · an emerging integrated picture of phytochrome signaling in...

The Arabidopsis Book ©2002 American Society of Plant Biologists

As sessile organisms, plants are unable to move activelytowards favorable or away from unfavorable environmentalconditions. Therefore, through their evolution, plants haveadapted a high degree of developmental plasticity to opti-mize their growth and reproduction in response to theirambient environments. Light is one of the major environ-mental signals that influence plant growth and develop-ment. Not only is light the primary energy source for plants,but it also provides them with positional information tomodulate their developmental processes such as seedgermination, seedling de-etiolation, gravitropism and pho-totropism, chloroplast movement, shade avoidance, circa-dian rhythms, and flowering time. Plants can detect almostall facets of light such as direction, duration, quantity, andwavelength by using three major classes of photorecep-tors: the red (R)/far-red (FR) light (600-750 nm) absorbingphytochromes (phys), the blue (B)/UV-A (320-500 nm)absorbing cryptochromes (crys) and phototropins (phots),and the UV-B (282-320 nm) sensing UV-B receptors(Kendrick and Kronenberg, 1994; Briggs and Olney, 2001;Briggs et al., 2001). These photoreceptors perceive, inter-pret, and transduce light signals, via distinct intracellularsignaling pathways, to photoresponsive nuclear genes,which modulates plant growth and development.

The phenotypic changes associated with the seedlingphotomorphogenic development are among the most dra-matic events mediated by these photoreceptors. Seedlings

grown in the dark undergo skotomorphogenesis (etiolation)and are characterized by long hypocotyls, closed cotyle-dons and apical hooks, and development of the proplastidsinto etioplasts. Light-grown seedlings undergo photomor-phogenesis (de-etiolation) and are characterized by shorthypocotyls, open and expanded cotyledons, and develop-ment of the proplastids into green mature chloroplasts (thusa process considered “de-etiolation” of the etioplasts,McNellis and Deng, 1995, Figure 1). The past decade hasseen dramatic advances in our knowledge of plant pho-toreceptors and in our understanding of their signal trans-duction pathways that lead to various physiologicalresponses. Here, we briefly review the most recentprogress that has provided new insights into constructingan emerging integrated picture of phytochrome signaling inArabidopsis, the model plant for molecular genetic studies.However, results derived from other model organisms orplant species which provide unique insights into phy-tochrome signaling mechanism are also briefly discussed inthis review where deemed appropriate. The interestedreaders are referred to the accompanying reviews on othersubjects related to phytochrome signaling, such as photo-morphogenesis (reviewed by Joanne Chory), blue light sig-naling (reviewed by Winslow Briggs), circadian rhythms(reviewed by C Robertson McClung and Steve Kay), pho-totropism (reviewed by Mannie Liscum), flowering(reviewed by George Coupland, Caroline Dean and Detlef

Phytochrome Signaling Mechanism

Haiyang Wang and Xing Wang Deng

Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT, 06520-8104, USA

Key Words: Arabidopsis, phytochrome, light signaling, photomorphogenesis

All correspondence should be addressed to:Professor Xing Wang DengDepartment of Molecular Cellular and Developmental BiologyYale University, P.O.Box 208104165 Prospect Street, OML 352ANew Haven, Connecticut 06520-8104Phone 1-203-432-8908; Fax 1-203-432-5726e-mail: [email protected]

INTRODUCTION

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Weigel) and chapters of phytohormones (such as auxin,brassinosteroids, and ethylene) and sugar sensing(reviewed by Sue Gibson and Ian Graham).

THE DISCOVERY AND ACTION MODES OFPHYTOCHROMES

Early in the twentieth century it was shown that a pigment,separable from the activity of photosynthesis, wasinvolved in photoperiod detection and floral induction(Garner and Allard, 1920). The nature of this pigment wasnot discovered until 30 years later. In the 1950’s, phy-tochromes were characterized as the proteinous pigmentthat mediates the reversible control of night-break of shortday flowering plants (such as tobacco and soybean) andlettuce (c.v. Grand rapids) seed germination by red and far-red light (Borthwick et al., 1952). In the lettuce seed germi-nation case, red light stimulates seed germination, but thisinduction can be inhibited by subsequent exposure to far-red light. The seeds can be repeately treated by sequentialred or far-red light, and the final germination response is

determined by the last light treatment. This characteristicphotoreversibility of responses aided researchers to purifyand characterize the responsible dichromic photoreceptorthat was later termed phytochrome for “plant color”.Another distinguishing feature of this response is its con-formity to the Bunsen-Roscoe Reciprocity Law, whichstates that a response should be dependent only on thetotal amount of photons received irrespective of the dura-tion of the exposure. Thus, the red/far-red reversibility andreciprocity constitute the hallmarks of the classical phy-tochrome responses. This class of phytochrome respons-es is now defined as the low fluence responses (LFRs,Mancinelli, 1994).

In addition to the control of lettuce seed germination,LFRs also induce other transient responses, such aschanges in ion flux, leaf movement, and chloroplast rota-tion (Haupt and Hader, 1994; Roux, 1994). LFRs alsoinduce changes in gene expression during de-etiolation,stem elongation, leaf expansion, and the transition to flow-ering (Vince-Prue, 1994). Besides the R/FR reversibleLFRs, two more modes of phytochrome action have sincebeen discovered: the very-low-fluence responses (VLFRs)which are activated by extremely low light intensities, suchas the expression of the LHCB gene, and the high-irradi-ance responses (HIRs) which depend on prolonged expo-sure to relatively high light intensities. HIRs are prevalentprimarily in the control of the de-etiolation process (e.g.inhibition of hypocotyl elongation) under all light qualities(Mustilli and Bowler, 1997; Batschauer, 1998; Table 1).

THE PHYTOCHROME GENE FAMILY AND THECHROMOPHORE

Two Reversible Forms of Phytochromes

The realization that phytochrome is enriched in dark-grownseedlings allowed its purification with relative ease due tothe lack of photosynthetic pigments in dark-grownseedlings (Butler et al., 1959, 1964). Based on variousphysiological evidence, it was predicted that phy-tochromes exist in vivo in two photoreversible forms. Thepurification of phytochrome confirmed this view andshowed that in dark-grown plants, phytochrome is presentin the Pr form. On exposure to red light, the Pr form is con-verted to the Pfr form, which is considered as the biologi-cally active form. The Pfr form is converted back to the Prform on absorption of far-red light. This photoconversionof phytochrome is correlated with changes in the absorp-tion maxima of these two forms: the purified phytochrome

Figure 1. The contrasting phenotypes of dark- vs. light-grown Arabidopsis seedlings.Dark-grown seedlings undergo a skotomorphogenicdevelopment program (etiolation), which is characterizedby elongated hypocotyls, closed cotyledons and apicalhooks. Light-grown seedlings undergo photomorphogen-esis and are characterized by short hypocotyls, open andexpanded green cotyledons.

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in the Pr form is blue in color and absorbs maximally at 666nm, whereas the Pfr form is olive-green in color andabsorbs maximally at 730 nm (Quail, 1997, Figure 2).

Classification of Phytochromes and The Gene Family

Purified phytochrome from etiolated seedlings was foundto be a soluble homodimer, with each apoproteinmonomer bearing a covalently attached linear tetrapyrrolechromophore. The molecular mass of the phytochromeapoprotein is approximately 120 kDa. The chromophore isattached via a thioether linkage to an invariant cysteine ina well-conserved domain among all phytochromes. In the1980’s, spectrophotometric studies indicated that thereare at least two distinct pools of phytochromes, Type I(light labile) and type II (light stable). The light-labile pooldegrades fairly rapidly upon exposure to red or white light.In Arabidopsis, there are five phytochromes (termed phyA-phyE) encoded by five distinct members of the phy-tochrome gene family (Sharrock and Quail, 1989). phyA isa type I phytochrome, and phyB-phyE are all type II phy-tochromes. PHYB and PHYD polypeptides are about 80%identical and are somewhat more related to PHYE thanthey are to either PHYA or PHYC (about 50% identity). ThePHYB, PHYD and PHYE polypeptides are the most recent-ly evolved members of the phytochrome family (Figure 3A;Table 2). Counterparts of PHYA, PHYB and other PHYgenes are present in most, if not all, higher plants (Clack etal., 1994; Sharrock and Quail, 1989; Mathews andSharrock, 1997).

All five phytochromes are expressed throughout theplant with only minor differences in their expression pat-terns (Somer and Quail 1995; Goosey et al., 1997), howev-er, their abundance and stability differ dramatically. phyA ismost abundant in dark-grown seedlings and its level dropup to 100 fold after exposure to light. The degradation of

Figure 2. Absorption spectra of phytochromes and theirdual physiological functions.(A) Absorption spectra of the two forms (Pr and Pfr) ofphytochromes. The Pr form absorbs maximally at 660nm, while the Pfr form absorbs maximally at 730 nm.(B) Dual roles (sensory and regulatory) of the phy-tochrome molecules. Phytochromes sense the light envi-ronment (shown on the left are the principal parametersof the light signals), undergo a photoconversion from theinactive Pr form to the active Pfr form, and transduce thesignals through distinct signaling pathways, which ulti-mately leads to regulated gene expression and appropri-ate morphogenesis. Shown on the right are the majorfacets of plant growth and development controlled byphytochrome actions (adapted from Quail, 1997).


phyA is light dependent and requires selective recognitionand ubiquitination of Pfr (Clough et al., 1999; Hennig et al.,1999). PHYA gene expression is also repressed at thetranscriptional level by light treatment (Somers and Quail,1995; Canton and Quail, 1999). Therefore, the regulation ofphyA protein level is the result of a coordinated transcrip-tional and post-transcriptional regulation. In light-grownplants, phyB is the most abundant phytochrome, whereasphyC-phyE are the less abundant type II phytochromes(Clark et al., 1994; Hirschfeld et al., 1998).

General Structure of Phytochromes

Studies with biochemically purified phyA holoproteins froma number of plant species indicated that the phytochromemolecule consists of two structural domains: a photosen-sory, globular N-terminal chromophore-binding domainwhich is sufficient for light absorption and photoreversibil-ity (~ 70 kDa), and a regulatory, conformationally moreextended C-terminal domain which is important for dimer-ization and downstream signaling (~ 55 kDa). These twodomains are connected via a flexible hinge region. The C-terminal domain contains several conserved subdo-mains/motifs including the regulatory core sequence (Quailbox), the dimerization motif, and the histidine kinase-relat-ed domain (HKRD). A pair of Per-Arnt-Sim (PAS) motifoverlaps with the Quail box (Figure3B). PAS domains havediverse function; they can be used either as protein-proteininteraction platforms or as response modules to small lig-ands or changes in light conditions, oxygen levels, andredox potentials (Quail, 1997; Neff et al., 2000). Most pointmutations in the PRD domain of both phyA and phyB donot affect photoreversibility but eliminate the biologicalactivity (Quail et al., 1995; Quail, 1997).

Chromophore Biosynthesis

As mentioned above, functional phytochrome holoproteinsrequire the covalent attachment of a chromophore to eachphytochrome apoprotein monomer. The structure of thephytochrome chromophore was investigated initially bydegradation approaches and determined to be a lineartetrapyrrole, phytochromobilin (PΦB). The ligation site ofthe chromophore was investigated by proton NMR spec-troscopy of phytochromopeptides isolated from sequentialpepsin-thermolysin digestion of oat phytochrome in the Pr

form. PΦB was shown to ligate via the A-ring to a cysteineresidue located in the N-terminal half apoprotein of PHYA(Lagarias and Rapoport, 1980).

It is interesting to mention that each pyrrole ring of thelinear tetrapyrrole chromophore may play a different role inchromophore assembly and the photochromic propertiesof phytochromes. A recent study found that the A-ring actsmainly as the anchor for ligation to PHYB. The side chains

Figure 3. The Arabidopsis phytochrome family and thedomain structure map of a generic phytochrome mole-cule.(A) The phylogenetic distance tree of the five phy-tochrome species from Arabidopsis thaliana. PHYB andPHYD share ~ 80% amino acid sequence identity, andare the products of a recent gene duplication. They arealso more closely related to PHYE (~55% identity) than toother phytochromes, and these three genes are consid-ered to form a subgroup of the Arabidopsis phytochromegene family. PHYA and PHYC form the other branch ofthe family evolution tree (adapted from Clack et al.,1994).(B) The domain structure map of a generic phytochromemolecule. The coordinates indicate positions of the con-sensus sequence derived from the alignment of multiplefull-length phytochrome polypeptide sequences byMathews et al. (1995). The N-terminal photosensorydomain (CBD, for chromophore binding domain) and theC-terminal regulatory domain are joined by a flexiblehinge region (H). The chromophore binding site (C374) islocated in the N-terminal photosensory domain. The C-terminal domain contains several conserved motifs,including the regulatory core sequence (Quail box), twodimerization motifs (D1 and D2), two PAS domains (P1and P2), and the histidine kinase-related domain (HKRD).The positions for the junction site and these individualmotifs are indicated (adapted from Quail, 1997).


of the B- and C-rings are crucial to position the chro-mophore properly in the chromophore pocket of PHYBand for photoreversible spectral changes. The side chainof the D-ring is required for the reversible spectral changeof the adducts (Hanzawa et al., 2001).

The synthesis of PΦB is directed by an enzymatic cas-cade in the plastid that begins with 5-aminolevulinic acid.The early steps in the PΦB pathway are shared with thoserequired to synthesize chlorophyll and heme. The commit-ted step is the oxidative cleavage of a portion of the hemepool by a ferredoxin-dependent heme oxygenase (HO) to

form biliverdin IX (BV). BV is then converted into 3Z-PΦBby the ferredoxin-dependent bilin reductase PΦB syn-thase. Both 3Z-PΦB and its isomerized form 3E-PΦB canserve as functional precursors of the phytochrome chro-mophore. PΦB is then exported to the cytoplasm where itbinds to the newly synthesized apo-phys (Terry et al.,1997, Figure 4 A and B). Absorption of red light triggers a“Z” to ‘E” isomerization in the C-15 double bond betweenthe C and D rings of the linear tetrapyrrole, resulting in thefar-red light absorbing form Pfr. This Pr-to-Pfr transition isaccompanied by rearrangement of the protein backbone.


Pfr can be converted to Pr either by a slow non-photoin-duced reaction (dark reversion) or much faster uponabsorption of far-red light (Quail, 1997; Fankhauser, 2001;Figure 4C).

Arabidopsis photomorphogenic mutants defective in thePΦB-synthetic pathway have been isolated. Thesemutants (hy1 and hy2) have dramatically reduced levels ofPΦB and consequently functional holo-phys, and thusexhibit severely impaired photomorphogenesis (Parks andQuail, 1991). The Arabidopsis HY1 locus encodes a HO

(designated AtHO1) responsible for much of PΦB synthe-sis in Arabidopsis (Davis et al., 1999; Muramoto et al.,1999; Table 2). Three additional HO genes (AtHO2-4) existin the Arabidopsis genome, and these additional HOs mayprovide alternative pathways for making BV (Davis et al.,2001). The Arabidopsis HY2 locus, which appears to be aunique gene in the Arabidopsis genome, encodes the phy-tochromobilin synthase (Kohchi et al., 2001; Table 2).

It is generally assumed that all phys have the same chro-mophore. Since type II phytochrome species are present in

Figure 4. Arabidopsis phytochrome chromophore.(A) The biosynthesis pathway of Arabidopsis phytochrome chromophore (adapted from Kohchi et al., 2001). (B) Chemical structures of heme, BV, PF**B, PCB, and PEB. Heme oxygenase converts heme to BV by an oxidativecleavage between rings A and D at the position marked (arrow).(C) Red light (R) triggers a “Z” to “E” isomerization in the C-15 double bond between the C and D rings of the lineartetrapyrrole, which is accompanied by rearrangement of the apoprotein backbone. This results in the photoconversion ofphytochromes from the Pr form to the Pfr form. Far-red (FR) light converts the Pfr form back to the Pr form.


very low abundance in plants, these have not been purifiedto homogeneity using conventional biochemical techniques,except phyB. The phyB purified from transgenicArabidopsis shows spectral properties similar to phyA (Elichand Chory, 1997). It should be pointed out that besidesPΦB, phycocyanobilin (PCB), the chromophore of the light-harvesting pigment phycocyanin, also can bind phy-tochrome resulting in Pr and Pfr spectra that are slightly blueshifted compared with the PΦB adducts. PΦB differs fromPCB only by substitution of the D-ring ethyl group with avinyl group (Lagarias and Rapoport, 1980, Figure 4B). Thisfinding allowed the constitution of photoreversible phy-tochromes by expressing recombinant phytochrome pro-teins in yeast and assembling them in vitro. Analysis ofreconstituted recombinant phyA, phyB, phyC and phyErevealed that they have similar but not identical spectralproperties. For example, the yeast-assembled phyC has661/725 nm and phyE has 670/724 nm as the red/far-redabsorption maxima (Kunkel et al., 1996; Remberg et al.,1998; Eichenberg et al., 2000). Thus it is likely that all phy-tochromes possess similar spectral properties.

Consistent with this notion, overexpression of the mam-malian biliverdin reductase in Arabidopsis was found tocause the loss of multiple phytochrome activities bydegrading phytochromobilin in vivo and constituted a newclass of chromophore mutants which is phenotypicallystronger than the hy1 or hy2 mutants. Many of the trans-genic plants were highly chlorotic and did not survive, sug-gesting an essential role for phytochromes in light-mediat-

ed plant growth and development (Lagarias et al., 1997).

ACTIONS AND INTERACTIONS OF PHYTOCHROMEFAMILY MEMBERS

Phytochromes regulate a variety of developmental process-es throughout the life cycle of plants. In most instances, theroles of individual phytochromes are studied in the contextof specific responses and/or developmental stages.

Seed Germination and Seedlings De-etiolation

The isolation and construction of genetic mutants lackingone or more of these photoreceptors as well as overex-pression studies of individual phytochromes have nowmade it possible to assess the role of each individual phy-tochrome in plant development. These studies revealedthat different phytochromes play both distinct and over-lapping roles within the spectrum of plant photomorpho-genesis (Quail., 1998; Table 3). For example, analysis ofphyA and phyB single and double mutants has shown thatthese two phytochromes affect a number of identical


processes in response to different fluences or wavelengthsof light. Both phyA and phyB affect germination, however,phyA is responsible for the photoirreversible VLFRresponses triggered by a broad spectrum of irradiations(ultraviolet, visible and far-red light), while phyB controlsthe R/FR photoreversible effects of low fluence response(Reed et al., 1994; Botto et al., 1996; Shinomura et al.,1996).

The regulation of hypocotyl elongation by light duringseedling de-etiolation is another example of the complexinterplay among these photoreceptors. Under far-red light,phyA is probably the only active photoreceptor, as illus-trated by the quasi-complete lack of de-etiolation of nullalleles of phyA (Nagatani et al., 1993; Whitelam et al.,1993). In white or red light, phyB plays a major role, buteven null mutants do not have a hypocotyl as long as thatof dark-grown plants. The long hypocotyl and reducedcotyledon expansion phenotype of phyB null mutants isenhanced in double mutants with phyA or phyD, whichindicates that it is the co-action of multiple photoreceptorsthat senses white light during de-etiolation (Neff & VanVolkenburgh, 1994; Johnson et al., 1994; Chory et al.,1996). Further, a recent high-resolution kinetic analysis ofthe growth of Arabidopsis seedlings revealed that the redlight inhibition of hypocotyl elongation is controlled by asequential and coordinated action of phyA and phyB. phyAcontributes to the initial hypocotyl growth inhibition (first 3hr of irradiation), while phyB functions in the later phase(Parks and Spalding, 1999).

Vegetative Development

It should be emphasized that in reality it is the nature of theincident light signal and the informational homeostasis pro-vided by the action and interaction of multiple signalingpathways of these photoreceptors that determines the ulti-mate cellular responses. For example, by sensing the lowR/FR ratio of light in their surroundings, plants initiate theshade-avoidance response by increasing the elongationgrowth of petioles and stems, the length-to-width ratio ofleaves, and accelerating flowering (Smith and Whitelam,1997). This response is adapted to an enrichment of far-redlight under a leaf canopy or to reflected light from nearbyleaves, and is a mechanism for neighbor detection.Accelerated flowering under shade, in which there is morefar-red light, may allow plants to complete their life cyclebefore the canopy of other plants becomes too dense. Thismanifestation of light quality monitoring by phytochromescan be phenocopied by end-of-day far-red (EOD-FR) treat-ments. phyB-deficient plants, which have a constitutive

elongated-petiole and early-flowering phenotype, do notdisplay a petiole elongation response to EOD-FR, but theydo respond to EOD-FR by earlier flowering (Devlin et al.,1996), indicating that phyB plays a major role in the percep-tion of low R/FR signals. In contrast, phyA mutants show afairly normal shade avoidance response but are impaired intheir perception of daylength (Bagnall et al., 1995; Casal etal., 1998). However, an antagonism between the two phy-tochromes can be detected upon overexpression of phyA inlight-grown plants. The resulting transgenic lines no longerdisplay the shade-avoidance response, which apparently isthe manifestation of the opposing effects of phyA and phyBin response to elevated levels of far-red light (Smith, 1995).This antagonism is minimal in wild-type light-grown plantsowing to low phyA levels.

The phyAphyB double mutants still respond to EOD-FRtreatments by flowering early, suggesting the operation ofother phytochromes. The recent identification of a natural-ly occurring mutation in the PHYD gene in the ArabidopsisWassilewskija (WS) ecotype (Aukerman et al., 1997) hasprovided evidence that phyD performs a similar role ofphyB. The monogenic phyD mutant plants have no obvi-ous phenotypic abnormality, whereas plants impaired inboth the PHYB and the PHYD genes display significantlylonger hypocotyls under either R or white light and flowerearlier than the phyB monogenic mutants (Devlin et al.,1999). Moreover, the triple mutant phyAphyBphyD stillretains the ability to respond to EOD-FR treatments bydeveloping elongated rosette internodes and acceleratedflowering responses (Smith and Whitelam, 1997; Whitelamand Devlin, 1997), implicating the actions of phyC and/orphyE in these responses. The isolation of the phyE mutantconfirmed this hypothesis. The phyE mutants show nophenotypic alteration unless it is in the phyB mutant back-ground and the phyBphyE double mutants flower muchearlier than the phyB monogenic mutants (Devlin et al.,1998). These studies show that phyB, phyD and phyE con-trol shade avoidance responses and inhibit flowering in aconditional redundant manner.

No mutation for phyC has been reported yet, but over-expression studies suggest a role in primary leaf expan-sion (Qin et al., 1997). ). Analysis of thephyAphyBphyDphyE quadruple mutant revealed that phyCappears to play no role in response to low R/FR ratio,although phyC does seem to be involved in the regulationof gene expression (e.g. ATHB-2 is still induced by R,Whitelam, 2001). The analysis of phyC null mutants will bevaluable in more precisely assessing the function of phyC.

Recently, it was found that phyE also plays a role in thecontrol of seed germination by FR light. Either phyE isdirectly involved in the photoperception of FR for thisresponse or the action of phyA in mediating seed germi-nation requires the presence of phyE. Other VLFRs andHIRs for seedling de-etiolation are normal in phyE mutants.


On the other hand, phyA mutants flower later than wild-type plants under long days, whereas phyE mutants flowerslightly early. Plants doubly null for both phyA and phyEflower at the same time as phyE single mutant plants, indi-cating that the phyE mutation is epistatic to phyA withrespect to flowering time (Whitelam, 2001).

Interestingly, the observed early flowering phenotype ofphyB mutants grown under 210C is diminished whengrown under 160C. Further, a substantial delay in floweringtime under 160C compared with 210C was observed forevery genotype carrying the phyB mutation (phyB,phyAphyB, phyBphyD, phyBphyE, phyAphyBphyD,phyAphyBphyE, and phyAphyBphyDphyE), indicating thatat 210C phyB negatively regulates flowering and at 160C,the effects of phyB are severely attenuated (dampened).However, an early flowering phenotype is still observed forthe phyE mutant at 160C, suggesting that phyE may con-trol flowering time over a broader temperature range thanphyB (Halliday et al, 2001).

Structural Basis for the Differential Functions ofPhytochromes

The underlying mechanism for the observed functional dif-ferences between different phytochromes and their actionmodes has begun to be unraveled. Previous experiments bydeleting regions, random mutagenesis of the full-lengthclone, or domain swapping between phyA and phyB haveprovided some useful information regarding the function ofvarious parts of phytochromes. For example, the N-terminal70 amino acids (the 6-kD domain) of oat phyA is required forcorrect chromophore/apoprotein interactions and under-goes a substantial conformational change upon photocon-version of Pr to Pfr. In this region, one domain (residues 13-62) is necessary for conformational stability and another one(residues 6-12) is involved in attenuating phytochromeresponses (Jordan et al., 1996, 1997). An oat phyA deletionmutant lacking amino acids 7 through 69 is inactive in trans-genic tobacco (Nicotiana tabacum, Cherry et al., 1992),whereas deletion of amino acids 1-52 of oat phyA causes adominant negative interference in Arabidopsis (Boylan et al.,1994). Clough et al (1999) showed that both the N-terminaland C-terminal halves of phyA are essential for Pfr degrada-tion. The N-terminal region provides important selectiverecognition signals for ubiquitin conjugation, and an intactC-terminal domain is essential for phyA breakdown. Further,a domain swapping and deletion analysis suggests that theN terminus of phytochrome is essential for its specific pho-tosensory properties and that the C termini of phyA andphyB are interchangeable (Wagner et al., 1996). It is impor-

tant to note, however, the domain swapping experimentwhere fusion proteins between oat phyA and rice phyB wereectopically expressed in wild-type Arabidopsis. It has beendocumented that the dark reversion rates and the light labil-ity of monocot and dicot phyAs are quite different (Neff etal., 2000). Therefore, these results should be interpretedwith caution.

A recent study demonstrated that the continuous FRlight treatment could be replaced by intermittent FR lightpulses to induce the FR-HIR responses. Analysis of theseaction spectra suggests that neither phyA in its Pr formsynthesized in the dark nor in its photoconverted Pfr formis active in inducing the signal. Instead the short-lived sig-nal was produced during phototransformation from Pfr toPr (Shinomura et al., 2000). This is in sharp contrast withthe case of phyB. Alternative irradiation with R and FR lightphotoreversibly switches on or off the phyB responses,indicating that Pfr is the active form from which the R-induced signal is derived.

At the molecular level, it has been demonstrated thatexpression of the nuclear photosynthetic gene LHCB inresponse to red light depends on both phyA and phyB(Reed et al., 1994; Cerdan et al., 1997). However, phyA andphyB respond to light of different wavelengths and flu-ences (phyA is responsible for VLFRs, and phyB is respon-sible for LFRs, Hamazato et al., 1997). Further, it has beensuggested that phyA mediates the activity of the LHCBpromoter in response to VLFRs and HIRs by targeting dis-tinct regions of the same LHCB1*2 promoter (Cerdan et al.,2000), suggesting that these different action modes ofphytochromes may entail distinct signaling pathways. Thisnotion is consistent with the observations that VLF1 andVLF2 are distinct components of the VLFR pathway ofphyA response (Yanovsky et al., 1997), whereas FHY3 pri-marily acts in the HIR response pathway of phyA(Yanovsky et al., 2000).

Since phyB, phyD and phyE are most similar to eachother and possess partially redundant functions, a recentstudy was carried out to explore the functional determinantsof these photoreceptors. Interestingly, neither PHYD norPHYE coding regions expressed under the control of thePHYB promoter efficiently complements a phyB null muta-tion, with phyE being partially active and phyD completelyinactive. In agreement with this result, overexpression ofphyD under the 35S promoter has a negligible effect on thehypocotyl elongation response to red or white light, where-as overexpression phyE can suppress the phyB mutantphenotype to certain degree. These results indicate thatphyE can interact with the phyB signaling components inthe cell, but not very efficiently, and phyD appears to not sig-nal through the phyB pathway to any significant extent.Further, chimeric coding sequences in which the ends andthe central region of PHYB and PHYD were exchanged andexpressed under the 35S promoter. It was found that most


of the red hypersensitivity of the chimeras was attributableto the central third of the molecule, with the amino-terminaland carboxy-terminal regions being effectively interchange-able, suggesting that the differential activities of phyB andphyD are primarily determined by their central domains(Sharrock et al., 2001).

PHYTOCHROME SIGNALING AND THE CIRCADIANCLOCK

Plants make use of an array of photoreceptors (includingphytochromes) in gathering information about the lightenvironment for setting the clock to oscillate with a periodof about 24 hours (i.e. entrainment of the circadian clock).It has been shown that mutations of photoreceptor genesPHYA and PHYB, cause the circadian rhythm of CAB2 pro-moter activity to oscillate at a pace slower (with a longerperiod length) than that of the wild type under various lightconditions (Somers et al., 1998). This study revealed thatin the regulation of the Arabidopsis circadian clock, phyAacts in low intensities of red light and blue light, while phyBfunctions in high-intensity red light. Recently it was shownthat phyD and phyE also act as photoreceptors in red lightinput to the clock, and that phyA and phyB act additivelyin red light input to the clock (Devlin and Kay, 2000). On theother hand, the action of the circadian clock governs, atany given time, the effect of a photoreceptor (or a plant’sresponsiveness to the light signal) on floral initiation, whichoften exhibits the photoperiodic response rhythm. Such aregulation of the signal transduction of photoreceptors bythe circadian clock has been referred to as gating (Millarand Kay, 1996; Anderson et al., 1997). One mechanism toachieve such a gating effect would be clock regulation ofcomponents of the light input pathway. Indeed, the mRNAabundance and transcriptional levels of PHYA, PHYB andPHYC have been shown to be robustly rhythmic, whereasPHYD and PHYE expression is, at most, weakly rhythmicin Arabidopsis (Bognar et al., 1999; McClung, 2001).

Besides the photoreceptors themselves, severalArabidopsis flowering-time genes have been recently iso-lated and shown to be associated with the function of thecircadian clock. Interestingly, these clock-related genesaffect both flowering time and hypocotyl elongation(Dowson-Day and Miller, 1999; Somer, 1999), and theyinclude ELF3, TOC1, CCA1, LHY, ADO1/ZTL/LKP1,ADO2/LKP2, ADO3/FKF1, and GI (Hicks et al., 1996; Wangand Tobin, 1998; Schaffer etal., 1998; Fowler et al., 1999;Park et al., 1999; Nelson, et al., 2000; Somer et al., 2000;Strayer et al., 2000; Table 2). It has been proposed thatELF3 and GI are most likely components of the light input

pathway to the circadian clock, whereas TOC1, CCA1 andLHY are likely to be components of the central oscillator.Like phyB mutants, both the gi and the elf3 mutants dis-play elongated hypocotyls in red light. However, the gimutants are late flowering, which is in contrast with theearly flowering phenotype of the phyB and elf3 mutants.This clearly suggests that ELF3 and GI play different rolesor use different mechanisms in controlling hypocotyl elon-gation and flowering responses. Both ELF3 and GI arenuclear proteins and are most likely involved in regulatingthe expression of flowering-time genes (Huq et al., 2000a;Liu et al., 2001). Intriguingly, both ELF3 (a novel nuclearprotein) and ADO1/ZTL/LKP1 (a protein with an amino-ter-minus PAS domain, multiple kelch repeats, and an F-box)are capable of directly interacting with phyB (Liu et al.,2001; Jarillo et al., 2001). The implications of these physi-cal interactions are not yet clear.

It has been shown that CCA1 binds in a circadian fash-ion to a short element of the LHCB1*1 (CAB2) promoter,which is sufficient to confer phytochrome responsivenessand circadian transcription (Wang and Tobin, 1997).Interestingly, the expression of CCA1 and LHY itself isunder the control of phytochrome signaling (Martinez-Garcia et al., 2000), suggesting a mechanism for phy-tochrome input to the clock.

The interactions and feedback regulations between phy-tochrome signaling and the circadian clock make it difficultto determine whether the abnormalities in flowering timeobserved in the phytochrome mutants are the conse-quence of a malfunction of the circadian clock, a manifes-tation of the direct action of the respective photoreceptor,or both. Mutations in the PHYA and PHYB genes affect thecircadian clock in a similar manner (they all cause longerperiod length for the circadian expression of the CAB2promoter), yet their effects on flowering time are opposite(e.g. phyA mutants flower late but phyB mutants flowerearly). These findings suggest that the observed alter-ations in flowering time of the phyA and phyB mutants areunlikely to be the direct consequence of a malfunction ofthe circadian clock. Instead, these photoreceptors maydirectly affect the floral initiation process, but the signaltransduction of photoreceptors may be gated (rather thanexecuted) by the circadian clock. This notion gained sup-port from a recent genetic study which showed that ELF3requires phyB function in early morphogenesis but not forthe regulation of flowering time, suggesting that ELF3 andphyB control flowering via independent signal transductionpathways (Liu et al., 2001).

PHYTOCHROMES AS LIGHT-REGULATED KINASES


It is clear now that phytochromes function as light-regulat-ed switches for a number of developmental processes, buthow do phytochromes initiate their signal transductionupon photoconversion? A long-standing hypothesis is thatphytochromes act as light-regulated kinases. This view issupported by the detected kinase activity in purified phy-tochrome preparations by the Lagarias group (Wong et al.,1986). However, this finding was contested by otherresearchers who could not detect a kinase activity in phy-tochrome preparations (Kim et al., 1989). Recently, the dis-covery of phytochrome-like photoreceptors in bacteria,collectively called bacteriophytochromes (BphPs), hasdramatically expanded our understanding of the originsand modes of action of phytochromes in plants. Thus, abrief summary of the most relevant information on BphPshere is essential.

Bacterial Phytochromes

Bacteria constantly regulate their physiology and behaviorto respond and adapt to their external environment using atypical “two-component” system which consists of a sen-sor protein and a response regulator protein. The sensorprotein detects a change in the external environment andcommunicates this information to the response regulatorprotein, which in turn either controls the expression of spe-cific genes or initiates other appropriate cellular functionsto respond to the environmental stimuli. The communica-tion between these two components occurs via phospho-rylation-dephosphorylation steps. It is well known thatthese sensor proteins act as histidine kinases, autophos-phorylate themselves and the phosphate group is thentransferred to a regulator molecule, leading to a cascade ofevents that modulates gene expression. Early physiologi-cal studies revealed that cyanobacteria display photore-versible effects analogous to those of plant phytochromesto optimize their photosynthesis depending on light condi-tions (Vierstra and Davis, 2000).

The most intensively studied effect is complementarychromatic adaptation (CCA), a process in which certaincyanobacteria can regulate the composition of their lightharvesting complexes (phycobilisome). In the cyanobac-terium Fremyella diplosiphon, shifts in the ratio of red togreen light lead to transcriptional changes and altered syn-thesis of several phycobilisome components. These twocolors have opposite effects: red light activates cpcB2A2,an operon encoding the biliprotein phycocyanin (PC), andinactivates the cpeBA operon, which encodes phycoery-thrin (PE), whereas green light activates PE synthesis andshuts down PC synthesis. The effects of red and green

light on the transcription of these light-harvesting genesare photoreversible. The study of CCA in the cyanobac-terium Fremyella diplosiphon led to the identification ofRcaE, for response to chromatic adapation E. Mutations inthis gene cause defects in CCA and abolish the responsesto red or green light. A region in the amino terminal portionof the protein (74 kDa) shows limited homology to the CBDdomain of the plant phytochrome. Within this region, RcaEalso contains a cysteine residue (C) at position 198, and itwas recently demonstrated that RcaE can covalentlyattach a tetrapyrrole chromophore in vivo and in vitro,dependent on the presence of C198. The carboxy terminalportion of RcaE has the signature (all four kinase motifsnecessary for catalysis H, N, D/F, and G) of a typical bac-terial histidine kinase domain (HKD) present in the two-component system, including the active-site histidine(Kehoe, 1996). Both its proposed position in the CCA sig-nal-transduction chain and its relation to phytochromessuggest that RcaE controls CCA by acting as a photore-ceptor. In addition, a response regulator (RcaF) for RcaEhas been identified, which is located in the genome direct-ly downstream of RcaE (Kehoe, 1997).

Phytochrome-like sequences were also identified by theKazusa sequencing project of the cyanobacteriumSynechocystis sp. PCC6803. Four genes with varyingdegree of relatedness to RcaE and higher plant phy-tochromes were uncovered (Hughes and Lamparter, 1999;Wu and Lagarias, 2000). One of them, Cph1 for cyanobac-terial phytochrome 1, was shown to be able to bind to theplant phytochrome chromophore (phytochromobilin PΦBor phycocyanobilin PCB) autocatalytically and to displayabsorption spectra with photoreversible red (Pr) or far-red(Pfr) absorption maxima typical of plant phytochromes.Further, Cph1 was shown to be a light-regulated histidinekinase. Both autophosphorylation of Cph1 and transphos-phorylation of Rcp1 (the response regulator for Cph1) areinhibited by red light and stimulated by far-red light (Yeh etal., 1997; Lamparter et al., 1997). Although the in vivo rel-evance of the protein kinase activity of Cph1 in light sig-naling is still under investigation, these studies clearlyestablish that Cph1 is a bona fide cyanobacterial phy-tochrome. Indeed, the discovery of Cph1 created anexcitement in the phytochrome research field becausewith the ability to exploit bacterial genetics, these BphPsnow offer simple models to help unravel the biochemicaland biophysical events that initiate phy signal transmis-sion. In particular, the efficiency with which highly solublerecombinant phytochrome can be prepared from E. colioverexpressors offers fresh hope that the three-dimen-sional structure of this class of photoreceptors could beresolved via NMR and X-ray diffraction analysis of phy-tochrome crystals (Hughes and Lamparter, 1999).

Later, phytochrome related photoreceptors were alsoidentified in both the purple photosynthetic bacterium


Rhodospirillum and non-photosynthetic bacteria such asDeinococcus radiodurans, Pseudomononas putida andPseudomonas aeruginosa (Jiang et al., 1999; Davis et al.,1999). Together, these studies of phytochromes in prokary-otes have provided compelling evidence that these phy-tochrome-like photoreceptors can perceive the light sig-nals and relay the information via a His kinase signaling

cascade, most likely through a response regulator, such asRcp1. The organization and distribution of BphPs supportthe view that phytochromes first appeared in eubacteria,were modified by photosynthetic bacteria to better quanti-tatively mediate the shade avoidance response, and thenwere transferred to plants from cyanobacteria during theendosymbiotic formation of chloroplasts (Fankhauser,2000; Vierstra and Davis, 2000).

It should be mentioned that the identities of the chro-mophores used by these BphPs have not been explicitlydetermined. For cyanobacteria, it is likely that PCB (and/or possibly PEB, phycoerythrobilin, Figure 4B) is thechromophore since these species make copious amountsof this bilin as an accessory pigment in photosynthesis.The nature of the BphP chromophore(s) for the non-pho-tosynthetic bacteria is even less clear since these speciesare not known to produce linear bilins. Interestingly, puta-tive heme oxygenase genes (HOs) in D. radiodurans, P.aeruginosa and P. putida have been identified, indicatingthat these species have the capacity to produce lineartetrapyrrole biliverdin (BV). Remarkably, the D. radiodu-rans HO is encoded by a single open reading frame (des-ignated Bph0) that is immediately upstream (4 bp overlap)of the BphP in an apparent operon. In this way, chro-mophore and apoprotein synthesis appears to be con-nected transcriptionally. Further, this BphP can assemblewith BV to produce a stable photochromic adduct, where-as neither higher-plant phytochromes nor SynechocystisCph1 can bind BV. Thus it appears that BV might be thechromophore specific to the D. radiodurans BphP(Vierstra and Davis, 2000).

Higher Plants Phytochromes

The discovery of BphPs led to more careful sequenceanalysis of higher plants phytochromes and similaritybetween the C-termini of phytochromes and BphPs wasidentified (Schneider-Poetsch, 1992, Figure 5A).However, plant phytochromes have two additionaldomains: a serine-rich N-terminal extension domain(NTE) and a PAS repeatdomain (PRD) located betweenthe CBD and the HKRD. In addition, several criticalresidues required for activity in the majority of bacterialsensor kinases are not conserved in all plant phy-tochromes. Moreover, mutating some of the remainingcritical residues for His kinase activity does not affect theactivity. Therefore, it appears that plant phytochromesare not active His kinases (Vierstra and Davis, 2000). Totackle this question, Clark Lagarias’s group developed arecombinant system to express and purify plant phy-

Figure 5. Arabidopsis phytochromes function as light-regulated kinases.(A) Domain conservation between a cyanobacterial phy-tochrome (Cph1) and Arabidopsis phytochromes. Theconserved cysteine residue for chromophore binding isindicated (*). HKD: histidine kinase domain; PRD: PASrelated domain; HKRD: histidine kinase related domain.(H) highlights the conserved histidine on the HKD domainof Cph1. The percent amino acid identities between theHKD of Cph1 and both PRD and HKRD of Arabidopsisphytochromes are indicated. Also note that Arabidopsisphytochromes have distinct N-terminal extensions (NTE).(B) Proposed roles of Arabidopsis phytochrome kinaseactivity. Light regulated kinase activity may auto-phos-phorylate the phytochrome molecules themselves, ortrans-phosphorylate their interacting partners (PIFs). Inturn, these phosphorylation events could affect the stabil-ity of photoreceptor in case of phyA, the subcellular local-ization of phytochromes, their ability to interact with PIFs,and the activities of other signaling intermediates. Theamino acid numbers correspond to the oat phyA (adapt-ed from Neff et al., 2000).


tochromes in yeast. Purified oat phyA expressed in S.cerevisiae and the green alga Mesotaenium caldariorumphytochrome expressed in P. pastoris were assembledwith PCB in vitro. These phytochromes display theexpected spectroscopic properties and protein kinaseactivity in a light and chromophore-regulated manner. Inaddition, the purified oat phyA can phosphorylate histoneH1 and Rcp1, the response regulator substrate of Cph1.However, unlike their cyanobacterial counterparts, theyauto-phosphorylate on Ser/Thr rather than His/Asp.These studies provided strong indication that the kinaseactivity is an intrinsic property of plant phytochromes andnot an artifact due to co-purification of another proteinkinase (Yeh and Lagarias, 1998).

Physiological Roles of Phytochrome Kinase activity

The claim that higher plant phytochromes function asprotein kinases triggered many questƒions such as whatis the biological role of the kinase activity? Which form ismore active? Pr or Pfr? Answers to these questions havejust begun to be unraveled. For example, recombinantoat phyA is a light and chromophore-modulated proteinkinase with Pfr being more active than Pr (Yeh andLagarias, 1998). Two phosphorylation sites have beenmapped for oat phyA. The Ser-7 is phosphorylated in vivoin both the Pr and Pfr forms, and mutagenesis studiessuggest that this residue is implicated in down-regulationof phyA signaling (Stockhaus et al., 1992). The Ser-17 isphosphorylated by protein kinase A in vitro only in the Prform. Mutation of the first 10 Ser of phyA to Ala (all con-tained within the first 20 aa) or deletion of this regionresults in a mutant that is hypersensitive to light(Stockhaus et al., 1992; Jordan et al., 1996, 1997). Takentogether, these results suggest a desensitization mecha-nism via these serines.

Another serine residue, Ser-598, is preferentially phos-phorylated in the Pfr form in vivo. The importance of thisresidue has been demonstrated in vitro because a S598Kmutant loses light-regulated kinase activity (Fankhauser etal., 1999). Consistent with this notion, when an oat PHYAcDNA with a Ser-598 to Ala substitution was expressed inthe phyA mutants, the transgenic plants exhibited hyper-sensitivity to far-red light, suggesting that the Ser-598phosphorylation may serve as a desensitizing mechanismof the Pfr activity by disrupting the interactions betweenphytochromes with their downstream signaling partners(Park et al., 2000, Figure 5B).

A complication on this issue arised from a recent studywhich showed that although point mutations in the HKRD

region of phyB cause strong phenotypes in hypocotyllength and flowering time, indicating that this domain isimportant for phyB signaling. Quite surprisingly, deletion ofthis domain resulted in a milder phenotype, suggestingthat this domain is dispensable (Krall and Reed, 2000).

Do higher plants phytochromes initiate a two-compo-nent phosphorelay cascade similar to that of RcaE? Thereis no affirmative answer to this question, but a recent studyhints that phytochromes could be the target of a two-com-ponent signaling system operating in plants. A responseregulator from Arabidopsis (ARR4) was identified as beingpredominantly expressed in response to red light, sug-gesting that ARR4 may be involved in phyB signaling.Further, ARR4 specifically interacts with the extreme N-ter-minus of phyB both in vivo and in vitro. Interaction of ARR4with phyB results in the stabilization of the active Pfr formof the photoreceptor as determined by inhibition of Pfr—Prdark reversion in vivo. Accordingly, transgenic Arabidopsisplants overexpressing ARR4 display hypersensitivity to redlight with respect hypocotyl and root growth as well asflowering time. Further, the Asp residue involved in phos-photransfer in the receiver domain of ARR4 was identified,and transgenic plants overexpressing a mutated form ofARR4 in which the Asp was substituted by a Asn residue,revealed a hyposensitive phenotype regarding all phyB-dependent light responses. These data indicate that phyBis the target of a novel two-component phosphorelay sys-tem that modulates red-light-dependent signaling by

Figure 6. A summary of identified phytochrome-interact-ing partners (PIFs).Some PIFs interact with both phyA and phyB, whereasothers specifically interact with either phyA only or phyBonly. Note that most of these physical interactions weredetected with the yeast two-hybrid assay and/or in vitrobinding assay.


direct interaction of its response regulator (ARR4) with thephotoreceptor (Sweere et al., 2001; Table 2). Further stud-ies should reveal how a two-component system is func-tionally linked to red light signaling and how other endoge-nous or environmental signals modulate phyB activity. Forexample, the expression ARR4 has been demonstrated tobe rapidly induced by cytokinin (Brandstatter and Kieber,1998), suggesting a possible link between cytokinin sig-naling and red light response mediated by phyB.

PHYTOCHROME-INTERACTING SIGNALINGPARTNERS

Phytochrome-interacting Partners (PIFs)

Protein-protein interactions are necessary for many signaltransduction cascades. It is reasonable to expect that phy-tochrome also interact with some partner protein(s) to relayinformation about the light environment in the cells.Identification of the molecular components responsible forintracellular photosignal transduction is currently an area ofintense research effort. Both general screenings for phy-tochrome interacting partners and targeted protein-proteininteraction studies have identified a number of phy-tochrome-interacting factors (PIFs). Those include PIF3 (Niet al., 1998), PKS1 (Fankhauser et al., 1999), NDPK2 (Choiet al; 1999), cryptochromes (both CRY1 and CRY2) and theAUX/IAA proteins (Ahmad et al., 1998; Colón-Carmona etal., 2000; Mas et al., 2000; Reed, 2001; Figure 6; Table 2).The physiological roles for some of these factors in phy-tochrome signaling have been substantiated by recentmolecular genetic studies. PIF3 is a nuclear localized basichelix-loop-helix (bHLH) protein. Transgenic Arabidopsisseedlings with antisense-imposed reductions in PIF3 levelsexhibited strongly reduced responsiveness to light signalsperceived by phyB, and partially reduced responsiveness tosignals perceived by phyA. These data indicate that PIF3 isfunctionally active in both phyA and phyB signaling path-ways in the plant cell, consistent with its binding to bothphotoreceptors. Further, a T-DNA tagged pif3 mutant (des-ignated poc1) also exhibits enhanced responsiveness to redlight (Halliday et al., 1999). This exaggerated response ofthe poc1 mutants to red light is caused by a T-DNA inser-tion into the promoter of the PIF3 gene, and thus likely torepresent a gain-of–function phenotype. The phyB mutationis epistatic to this mutant, indicating that PIF3 is an authen-tic phyB signaling component. PKS1 is a basic, soluble,cytoplasmic protein and has been proven to be a substrate

for light-regulated phytochrome serine/threonine kinaseactivity, indicating that protein phosphorylation is involvedin phytochrome signaling, and which might modulate phy-tochrome kinase activity or their subcellular localization.PKS1 overexpressing plants display less sensitivity to redlight, suggesting that it acts as an inhibitor of phyB signal-ing (Fankhauser et al., 1999). In contrast, NDPK2 (nucleo-side diphosphate kinase 2) appears to be a positive regula-tor of both phyA and phyB signaling. Although hypocotylelongation is not obviously affected by this locus, its loss offunction alleles have a small but significant reduction incotyledon greening and opening of the hypocotyl/cotyledonhook during de-etiolation (Choi et al; 1999).

Recently, using the C-terminal 300 amino acids ofArabidopsis phyB as a bait, two additional phytochrome-interacting proteins, PRP1 and PAB1 were isolated. PRP1(phytochrome related phosphatase 1) shares 30% identityto protein phosphatase 2Cs (PP2C) and has been shownto possess the activity of a serine/threonine phosphatase.Overexpression of the catalytic domain of PRP1 inArabidopsis plants leads to a reduced sensitivity to redlight. In contrast, a knockout of PRP1 (prp1 mutant) ismore sensitive to red light and less sensitive to far-red lightthan wild-type plants, suggesting that PRP1 negativelyregulates phyB signaling and positively regulates phyA sig-naling, possibly by dephosphorylating phytochromes orother signaling components. PAB1 (phytochrome actinbinding protein 1) contains six kelch repeats and binds toactin filaments in vitro. The pab1 mutant is less sensitive tored light and has reduced leaf expansion after a certaindevelopmental stage, leading to smaller rosettes in matureplants, indicating that PAB1 might play a fundamental rolein cell expansion. PAB1-GFP localizes to a web-like pat-tern (possibly ER) in the cytoplasm of hypocotyl cells(Chen et al., 2001).

Functional Domains and Intra-molecular Signaling ofPhytochromes

It should be pointed out that these proteins interact withdifferent structural motifs in the C-terminal domain of phy-tochromes and that their interactions are differentially reg-ulated by light. For example, NDPK2 binds to the Quail boxpreferentially in the Pfr form in a GTP-dependent manner(Choi et al., 1999; Park et al., 2000). PIF3 also binds to theQuail box preferentially in the Pfr form. However, it is dif-ferent from NDPK2 in that both the N-terminal and the C-terminal domains are apparently required for full activity (Niet al., 1999; Zhu et al., 2000). On the other hand, PKS1


binds to the Ser/Thr kinase motif equally well in both the Prand Pfr forms, indicating that this motif is exposed in bothspectral forms. However, PKS1 phosphorylation and phy-tochrome autophosphorylation are stimulated by a factorof 2 to 2.5 in the Pfr form (Fankhauser et al., 1999). Thus itis probable that phosphorylation is an important regulato-ry event for the phytochrome-PKS1 interaction. Thesestudies suggest that different inter-domain crosstalks acti-vate a specific motif in the C-terminal domain for recogni-tion by different factors and contribute to the spectralintegrity of phytochromes. Direct evidence for such anotion has come from chemical cross-linking experimentswhich detected a R/FR-dependent interaction between theN-terminal peptide and the distal C-terminal peptide (Parket al., 2000). Consistent with this, a mutation in the CBDdomain of phyB (phyB-401) causes a defect in the pho-toreversibility and enhanced light sensitivity (Kretsch et al.,2000). Similarly, another phyB missense mutation, phyB-101, is in the second PAS repeats (Bradley et al., 1996).This mutation affects spectral properties of the pigment,causing accelerated dark reversion from Pfr to Pr, andalters the EOD-FR response in seedlings (Elich and Chory,1997). These results also support the notion of inter-domain cross-talk within the phytochrome molecules.

In vitro kinase assays have identified other substrates ofphytochromes, including the blue light photoreceptors

CRY1 and IAA proteins (Ahmad et al., 1998; Colón-Carmona et al., 2000). Although the phosphorylation ofCRY1 is not light-dependent in an in vitro experiment, invivo analysis shows that cry1 phosphorylation is stimulat-ed by red light. Moreover, the identification of the shy2mutant as a suppressor of phyB and of phytochrome chro-mophore mutants and the gene encodes IAA3, one of theearly auxin-inducible genes (Tian and Reed, 1999), sug-gesting that the interactions between phytochromes andthe AUX/IAA proteins and phosphorylation events are like-ly to be biologically relevant.

GENETICALLY IDENTIFIED EARLY INTERMEDIATESOF PHYTOCHROME SIGNALING

Genetic screening for Arabidopsis mutants potentiallydefective in signaling intermediates either specific to phyAor phyB, or shared by both pathways has identified a num-ber of candidate loci (Figure 7).

Phytochrome A-specific Signaling Components

Mutants affected in phyA-specific signaling process werescreened under a continuous far-red light (FRc) conditionand a number of potential signaling components specificto this pathway have been identified, including FHY1,FHY3 (Whitelam et al., 1993), FIN2 (Soh et al., 1998), SPA1(Hoecker et al., 1998), FAR1 (Hudson et al., 1999), FIN219(Hsieh et al., 2000), PAT1 (Bolle et al., 2000), EID1 (Bucheet al., 2000), HFR1/RSF1/REP1 (Fairchild et al., 2000;Fankhauser and Chory, 2000; Soh et al., 2000) and LAF1(Ballesteros et al. 2001). The fhy1, fhy3, fin2, fin219, far1,laf1, laf6, and hfr1/rep1/rsf1 mutants show less sensitivityin continuous far-red light, indicating that their respectivegenes encode positive regulators of the phyA signalingpathway. On the other hand, mutations in the SPA1 andEID1 genes cause increased sensitivity to the FR light sig-nal, and it is most likely that they act as negative regulatorsof the signaling cascade.

Among the positive regulators of phyA signaling identi-fied, their loss-of-function mutants (mostly null mutationalleles) only exhibit partial defects with different spectra andstrength in phyA signaling, suggesting that phyA signalinginvolves multiple branches or parallel pathways controllingoverlapping yet distinctive sets of far-red light responses(hypocotyl growth, apical hook and cotyledon opening,

Figure 7. A simplified genetic model for phytochrome-mediated signaling pathways. phyA and phyB signalingentails specific as well as shared components. These locipresumably act upstream of the COP/DET/FUS genes,thus controlling HY5 and the degree of photomor-phogenic development. Arrows indicate a positive action,and the bars indicate a repressive effect.


anthocyanin accumulation, far-red light pre-conditionedblocking of greening, gravitropic response, light-regulatedgene expression etc., Barnes et al., 1996; Hudson, 2000;Wang and Deng, 2001). Moreover, the finding that the dou-ble mutants fhy3-1/far1-2, fhy3-1/fhy1-1 and far1-2/fhy1-1all display an additive effect, whereas the fhy3-1/spa1-3double mutant has an intermediate hypocotyl-length phe-notype (Wang and Deng, 2001), also indicates that there isno simple downstream/upstream relationship among thesephyA signaling components. Further, complex genetic rela-tionships such as non-allelic non-complementationbetween fin2 and fhy3-1 as well as between fin219 and fhy1have been reported (Soh et al., 1998; Hsieh et al., 2000),suggesting that their gene products may directly interact orengage in extensive cross-talk.

Several phyA signaling intermediates have been charac-terized at the molecular level (Table 2). LAF6 is a plastid-localized ATP-binding-cassette protein involved in coordi-nating intercompartmental communication between plas-tids and the nucleus (MØller et al., 2000). PAT1 is a newmember of the GRAS family (Bolle et al., 2000), whereasFIN219 is a GH3-like protein whose expression is rapidlyinducible by auxin (Hsieh et al., 2000). Both PAT1 andFIN219 are cytoplasmically localized proteins. FAR1,FHY3, SPA1, HFR1, LAF1 and EID1 are all nuclear local-ized factors. Interestingly, FAR1 and FHY3 encode twoclosely related proteins that constitute one branch of alarge gene family (Hudson et al., 1999; Wang and Deng,2001). HFR1 is an atypical bHLH transcription factor close-ly related to PIF3 (Fairchild et al., 2000) and LAF1 is a MYBtype transcription activator (Ballesteros et al. 2001). SPA1possesses a C-terminal WD-40 repeat domain that is mostclosely related to that of COP1 (Hoecker et al., 1999),whereas EID1 is a novel F-box protein most probablyinvolved in ubiquitin-dependent proteolysis (Dieterle et al.,2001). The biochemical functions of PAT1, FIN219, FAR1,FHY3 and SPA1 remain largely unknown.

The findings that fhy3 and far1 mutants display similaryet distinct phenotypes and that FHY3 and FAR1 encodetwo homologous proteins are particularly interesting. Morestrikingly, overexpression of FAR1 or FHY3 can suppressthe phenotype of each other’s loss-of-function mutations.It is also of interest to note that overexpression of partialfragments of FHY3 in a wild-type background causesreduced sensitivity to FRc in a dosage-dependent manner.Especially, Arabidopsis seedlings homozygous for thetransgene overexpressing the C-terminal portion of FHY3(C473-839), which contains a Coil-coil domain, display anapparent complete loss of FRc responses, remarkablysimilar to phyA null mutants. This result indicates that theC-terminal fragment of FHY3 may interact with other inter-mediates of phyA signaling and that non-productive bind-ing of this truncated FHY3 protein with its interactive part-ners could shut down the entire phyA signaling by a dom-

inant-negative interference. This interference is substan-tially stronger than the effect of an fhy3 null mutation.Direct evidence for this view is provided by the demon-stration that FHY3 and FAR1 directly interact with eachother in a yeast two-hybrid assay and an in vivo co-immunoprecipitation assay (Wang and Deng, 2001). It isconceivable that through interactions with FAR1 and otherinteractive partners of FHY3 and FAR1, FHY3 could exertits effect on a large number of phyA signaling intermedi-ates in mediating FRc responses. Therefore, FHY3, togeth-er with FAR1, could constitute a central regulatory knot inthe phyA signaling network. Determining the protein-pro-tein interactions among these phyA signaling intermedi-ates and identifying their novel interactive partners shouldenhance our understanding of the phyA signaling pathway.

Phytochrome B-specific Signaling Components

Putative phyB-specific signaling mutants have also beenidentified, including red1, pef2 and pef3. They share anumber of features with phyB mutants, such as a longhypocotyl phenotype specifically under red light, earlyflowering in short days, and elongated petioles, suggestingthat these loci positively regulate phyB signaling (Reed etal., 1993; Ahmad and Cashmore, 1996; Wagner et al.,1997). On the other hand, the srl1 mutants show enhancedresponsiveness to red light, suggesting that SRL1 is a neg-atively acting component specific to phyB signaling (Huqet al., 2000b). The molecular identities of these genes arecurrently unknown.

Signaling Components Shared by Both phyA andphyB

Additional mutants, pef1 and psi2, affect responses frommultiple photoreceptors. The pef1 mutants show attenuat-ed red and far-red responses, whereas the psi2 mutant ishypersensitive to red and far-red light, and has necroticlesions in light-grown plants (Ahmad and Cashmore 1996;Genoud et al., 1998), suggesting that these two loci areshared by both the phyA and phyB signaling pathways.The cloning of these genes and characterization of theirfunctions should greatly enhance our understandingregarding how these two pathways converge to regulatephotomorphogenesis.


G proteins, Calcium-binding Protein and Ion Flux inPhytochrome Signaling

It should be pointed out that the multiplicity of phy-tochrome-regulated responses signifies that this diversityresults from operation of multiple signal transduction path-ways triggered by the phytochrome. Previous microinjec-tion and pharmacological studies have suggested theinvolvement of G-proteins, cGMP and Ca2+/calmodulin inphytochrome signaling (Bowler et al., 1994; Neuhaus et al.,1997). Particularly, heterotrimeric G-proteins have beenimplicated in several processes during plant growth anddevelopment, and they transduce extracellular signals tothe cell. In general, heterotrimeric G-proteins consist ofthree subunits: a, b, and g. Analysis of the completegenome sequence of Arabidopsis indicated that theArabidopsis genome contains only a single Ga gene, pre-viously designated ATGPA1 (Ma et al., 1990), and a singleGb gene, designated AGB1 (Weiss et al., 1994). Recentlythe Arabidopsis Gg subunit was identified by a yeast two-hybrid screen using Gb as a bait (Mason and Botella, 2001;Table 2) and it was shown that Gg also is encoded by asingle copy gene in Arabidopsis.

Transgenic Arabidopsis plants conditionally overex-pressing the Ga subunit of the heterotrimeric G-proteinunder the control of a glucocorticoid-inducible promoterexhibited a light-dependent hypersensitive response as aresult of reduced hypocotyl cell elongation. Further, thisenhanced response in far-red and red light requires func-tional phyA and phyB, respectively. Interestingly, theresponse to far-red light depends on functional FHY1 butnot on FIN219 and FHY3, suggesting that the ArabidopsisGa protein may act only on a discrete branch of the phyAsignaling pathway (Okamoto et al., 2001). However, a sep-arate study reported that loss-of-function gpa1 mutantsdisplay partial de-etiolation in the dark, with shorthypocotyls and open apical hooks typical of light-irradiat-ed seedlings. The short hypocotyl of gpa1 seedlings isreportedly due to a defect in cell division, not cell elonga-tion (Ullah et al., 2001). The reason for the discrepancyregarding GPA1 in photomorphogenesis reported in thesetwo studies is currently unknown. Regardless, these stud-ies provide genetic support for the involvement of het-erotrimeric G proteins in light control of plant photomor-phogenesis.

Genetic studies have also provided supporting evidencefor the involvement of Ca2+ in phytochrome signaling. Thesub1 mutant exhibits hypersensitive responses to both far-red light and blue light. The SUB1 gene was found toencode a Ca2+-binding protein. Genetic interaction stud-ies suggest that SUB1 is a component of a cryptochromesignaling pathway and is a modulator of the phyA signal-

ing pathway. Further, SUB1 negatively regulates HY5, abZIP transcription factor and a positive regulator of photo-morphogenesis (Guo et al., 2001; Table 2).

Some early studies also suggested that phytochromeexerts its effects by first altering the permeability of theplasma membrane to ions (Kendrick and Bossem, 1987).This is most likely to be true for some light-regulatedresponses in certain plant species, such as the bud induc-tion process of the moss Physcomitrella patens(Ermolayeva et al., 1997), and the unrolling of the primaryleaf wrapped within the oat coleoptiles (Viner et al., 1988).However, there have been no reports of changes in cyto-plasmic ion (such as Ca2+) concentration in Arabidopsishypocotyl cells in response to light, neither is there anyelectrophysiological evidence that phytochrome signalingin hypocotyls involves changes in ion fluxes (Parks andSpalding, 1999; Spalding, 2000). Therefore, the role of ionflux and membrane depolarization in phytochrome controlof Arabidopsis photomorphogenesis remains elusive.

LIGHT-REGULATED CELLULAR LOCALIZATION OFPHYTOCHROMES

Phytochrome apoproteins are synthesized within thecytosol and assembled autocatalytically with the plastid-derived chromophore. For years, there has been a debateabout the intracellular localization of phytochrome. Earlystudies using an immunohistological approach and cellfractionation assay supported the notion that phy-tochromes are predominantly localized outside the nucle-us (Pratt, 1994). Recently, it was shown that upon photo-conversion of Pr to Pfr, both phyA and phyB tagged withGUS or green fluorescent protein (GFP) can translocatefrom cytoplasm into the nucleus where they form intranu-clear spots (Sakamoto and Nagatani, 1996; Yamaguchi etal. 1999; Kircher et al., 1999; Nagy et al., 2000). In etiolat-ed seedlings and dark-adapted plants the phyB:GFPfusion protein is localized in the cytosol. The nucleartranslocation and spot formation of phyB:GFP is inducedby continuous red light treatment, or multiple red lightpulses which are reversible by subsequent far-red treat-ment, indicating that the nuclear import of phyB is mediat-ed by a low-fluence response (LFR) of phytochrome. It fol-lows that the nuclear import of phyB is regulated by phyBitself, and/or by some other red-light absorbing photore-ceptor(s). On the other hand, brief irradiation with red, far-red, or blue light can induce rapid nuclear import andintranuclear spot formation of phyA:GFP (one magnitudeof order faster than that of phyB), preceded by an evenfaster cytosolic spot formation of the fusion protein, a phe-


nomenon reminiscent of SAP (sequestered area of phy-tochrome) formation. Besides, nuclear translocation ofphyA is also induced by continuous far-red but not by con-tinuous red light treatment. Thus, the nuclear translocationof phyA is mediated by both the very low fluence response(VLFR) and the far-red high irradiance response (HIR), indi-cating that phyA regulates its own nuclear import inde-pendent of any other types of phytochromes. It should bepointed out that the transport seems to be not complete,i.e. a significant portion of phyA:GFP remains cytosolic(Nagy et al., 2000).

Full-length phyC-E have also been fused to GFP andexpressed in transgenic Arabidopsis. Surprisingly, thenucleocytoplasmic partitioning of these phytochromespecies seems not to be regulated by light. They aredetected in the cytosol and the nucleus in etiolatedseedlings. However, these fusion proteins are similar tophyA or phyB:GFP in the formation of nuclear speckles ina light-dependent manner (Nagy et al., 2000). Interestingly,the nuclear import of all phytochrome species is regulatedby the circadian clock and displays oscillations under con-stant conditions, suggesting a new regulatory loop whichcould modulate the gating of phytochrome signaling by thecircadian clock and resetting of the circadian clock bythese photoreceptors (Nagy, 2001).

At the current stage, very little is known about themolecular machinery and factors modulating the nucleo-cytoplasmic partitioning of phytochromes. It has beenspeculated that the Pr conformers of phytochromes areanchored/retained in the cytosol and the Pfr conformersdo not interact with the anchoring proteins and are thussubject to nuclear import. The autophosphorylation pat-tern of phytochromes and phosphorylation of other pro-teins by the kinase activity of phytochromes (such asPKS1) may play a role in modulating the retention/releasestatus of phytochromes, thus attributing to the control oftheir nuclear import (Nagy et al., 2000). In addition, the var-ious phytochrome signaling intermediates describedabove and other signaling cascades (such as phytohor-mones) could also affect the intracellular distribution ofphytochromes, thus modulating the amount of phy-tochromes available for interaction with other componentsin the nucleus to regulate light-responsive gene transcrip-tion. Further studies aimed to map the possible nuclearlocalization signal (NLS) and nuclear export signal (NES)and to determine the role of phosphorylation and dephos-phorylation in regulating phytochrome subcellular translo-cation may help to resolve these issues. Particularly, thebiological significance of the nuclear translocation eventsfor phytochromes remains to be substantiated.

PHYTOCHROME SIGNALING AND THE

DOWNSTREAM COP/DET/FUS PROTEINS

The fact that distinct photoreceptor-triggered signalingprocesses can lead to similar photomorphogenic develop-ment implies that these signaling pathways converge toregulate developmentally important genes through a set ofcommon late signaling intermediates. Indeed, aside fromthese phytochrome-specific signaling intermediates,genetic screens have also identified eleven pleiotropicCOP/DET/FUS loci whose gene products act as negativeregulators of photomorphogenesis and function down-stream of multiple photoreceptors, including phyA andphyB (Wei and Deng, 1996, Figure 8). Among theseCOP/DET/FUS proteins, COP1 is a RING-finger proteinwith WD-40 repeats (Deng et al., 1992). Studies with afunctional GUS-COP1 fusion protein indicated that COP1acts within the nucleus to suppress photomorphogenicdevelopment in darkness, and that inactivation of COP1 bylight was accompanied by reduced COP1 abundance in

Figure 8. The phenotype of cop1 (constitutive photomor-phogenic) mutants and the proposed roles of theCOP/DET/FUS proteins in photomorphogenesis.(A) Dark-grown cop1 mutant seedlings phenotypicallymimic light-grown wild-type seedlings. (B) A total of eleven pleiotropic COP/DET/FUS loci func-tion as repressors of photomorphogenesis. Light signalsperceived by multiple photoreceptors are transduced toinactivate these COP/DET/FUS proteins, and to turn onphotomorphogenic development.


the nucleus (von Arnim and Deng, 1994). In the dark, COP1directly interacts with and targets HY5, a bZIP transcrip-tion factor which acts to promote photomorphogenicdevelopment, for degradation via a 26S proteasome-medi-ated process (Figure 9). Thus, COP1 presumably functionsas a putative ubiquitin ligase (Oyama et al., 1997; Ang etal., 1998; Osterlund et al., 2000). Eight otherCOP/DET/FUS proteins are subunits (CSN1-CSN8) of aprotein complex, the COP9 signalosome (Castle andMeinke, 1994; Peng et al., 2001a, Peng et al., 2001b;Karniol et al., 1999; Kwok et al., 1998; Serino et al., 1999;

Wei et al., 1994; Table 2). Its subunit-by-subunit similarityto the lid subcomplex of the 26S proteasome and thedemonstrated interaction between these protein complex-es suggest that the COP9 signalosome may be involved inregulated proteolysis (Kwok et al., 1999; Peng et al.,2001c, Schwechheimer and Deng, 2000). This notion isfurther supported by a recent demonstration that theCOP9 signalosome physically interacts with the SCFTIR1

E3 ubiquitin ligase and is required for efficient degradationof a candidate substrate of SCFTIR1 (Schwechheimer etal., 2001). Based on the pleiotropic nature of thecop/det/fus mutant phenotype, it is likely that theCOP/DET/FUS proteins repress the activities of multipletranscription factors or transcriptional regulators.Consistent with this notion, multiple COP1-interactingtranscriptional factors, besides HY5, have been identified(Osterlund et al., 1999; Holm et al., 2001).

It is generally assumed that light-induced photomor-phogenic development requires the inactivation of theseCOP/DET/FUS proteins. However, little is known as to howthe light-activated photoreceptors (including phy-tochromes) regulate the activities of those downstreamCOP/DET/FUS proteins to bring about the physiologicalresponses. At least one of the mechanisms through whichphytochrome signaling regulates the downstreamCOP/DET/FUS protein activities is achieved by triggeringthe nuclear depletion of COP1 under their respective light-responsive regimes (FR and R for phyA and phyB, respec-tively) (Osterlund and Deng, 1998). The finding that thekinetics of GUS-COP1 nuclear depletion by FRc is alsoaffected by other phyA-specific signaling mutations(including phyA, fhy3, fhy1, far1, and fin219) suggests thatthose phyA-specific signaling components functionupstream of COP1 and are involved in regulating thenucleo-cytoplasmic partitioning of COP1. As a result, theaccumulation of the bZIP transcription factor HY5 and, inturn, the degrees of photomorphogenic development ofthese mutants are affected (Wang and Deng, 2001).However, it should be pointed out that there is evidencethat these loci entail additional signaling pathways besidessignaling through COP1 (Wang and Deng, 2001).

PHYTOCHROME REGULATION OF NUCLEAR GENEEXPRESSION

Transcriptional regulation of gene expression representsan important step in the control of various processes ofplant growth and development. The photoregulation ofgene expression in higher plants has been extensivelystudied during the past two decades and a number of pho-

Figure 9. The cellular basis and proposed biochemicalmode of COP1 function.(A) Light-regulated nucleocytoplasmic partitioning ofCOP1. In darkness, COP1 is enriched in the nucleus tosuppress photomorphogenic development. Light signals(perceived by phytochromes and other photoreceptors)trigger the nuclear depletion of COP1, thus abrogatingthe repressive effect of COP1. Note that the COP9 sig-nalosome may contribute to the control of nuclear local-ization of COP1 or the stability of COP1 in the nucleus.The roles of DET1 and COP10 in this cellular processhave not been determined.(B) A putative role of COP1 as an E3 ubiquitin ligase.COP1 mediates the ubiquitination of HY5 and its subse-quent degradation via the proteasome by recruiting an E2and HY5 through distinct interacting domains (RING-fin-ger and WD-40 repeat domain, respectively, adaptedfrom Osterlund et al., 2000).


toregulated genes have been identified from several plantspecies. For example, the transcript level of both theLHCB genes for light-harvesting chlorophyll a/b-bindingprotein of photosystem II and the RBCS genes for ribu-lose-1, 5-biphosphate carboxylase/oxygenase small sub-unit increases by light illumination, and this light-induciblegene expression is mediated by phytochromes.Consequently, the promoter region of LHCB and RBCSgenes have been isolated and analyzed to identify variouscis-regulatory elements responsible for light- or phy-tochrome regulation, such as the GT-1 boxes of RBCS, G-box of tomato RBCS-3A, GATA motif, 3AF binding sitesand AT-1 binding site. In addition, trans-acting factorswhich bind to light-responsive cis-regulatory elements(LREs) such as GT-1, GBF, GAF1, 3AF-1 and AT-1 were iso-lated, and some of which have been shown to be involvedin phytochrome-mediated light responsiveness (von Arnimand Deng, 1996; Terzaghi and Cashmore, 1995; Menkenset al., 1995; Kuno et al., 2000; Kuno and Furuya, 2000).Further, it was suggested that the combinatorial interactionof multiple LREs is the key determinant for mediating lightcontrol of promoter activity (Puente et al., 1996). However,these traditional studies only revealed limited informationon the role of individual phytochromes in the photoregula-tion of gene expression.

Recently, the newly developed gene chip technology(Richmond and Somerville, 2000) has been applied tostudy light regulation of gene expression and to define theroles of some individual phytochromes in gene regulationunder their respective light regimes (Ma et al., 2001). It wasrevealed that a large number of genes, possibly over onethird of the genome, are coordinatedly regulated by vari-ous light signals, including the red and far-red lights thatare primarily perceived by phytochromes. Utilization of thephyA and phyB null mutants under specific light conditionsconfirmed the roles of phyA and phyB in mediating the far-red and red light regulation of genome expression. phyAseems to be the primary photoreceptor for mediating far-red light reglation of gene expression, whereas phyB isonly one of the phytochromes mediating red light regula-tion of genome expression (Ma et al., 2001).

The genome profiling study revealed an interesting fea-ture of light-regulated gene expression: many cellularmetabolic and regulatory pathways are found to be coor-dinately regulated by light. Some of them (such as all pho-tosynthetic genes, glycolysis and the TCA cycle etc.) areactivated by light, whereas others (such as cell wall-loos-ening enzymes and water channel protein aquaporins) arerepressed by light (Ma et al., 2001). These results substan-tiate the notion that light-regulated plant developmentinvolves a coordinated regulation of different pathways.Similar conclusions have also been drawn from studies oncircadian clock control of gene expression (Harmer et al.,2000; Schaffer et al., 2001).

AN EMERGING INTEGRATED PICTURE OFPHYTOCHROME SIGNALING

Among these fascinating achievements on phytochromeresearch, two key discoveries have dramatically advancedour understanding of phytochrome signaling. First, it hasbeen demonstrated that upon photoconversion of Pr toPfr, phytochromes (both phyA and phyB) can translocatefrom cytoplasm into the nucleus (Kircher et al., 1999; Nagyet al., 2000). Second, the Pfr form of phytochromes hasbeen demonstrated to interact with a light-responsive ele-ment (G box)-bound bHLH protein, PIF3 (Ni et al., 1999;Martínez-Garcia et al., 2000). These findings suggest anemerging model for phytochrome signaling, whereby phy-tochromes perceive light, enter the nucleus, interact withtranscriptional regulators, and thus regulate gene tran-scription. It is conceivable that the direct targeting of lightsignals to a promoter-element bound transcription factorwould allow plants to continuously monitor their light envi-ronments and to react to changes in light availability byconcomitant changes in light-regulated gene expression.However, it should be pointed out that the biochemicalbasis for the regulation of gene expression brought aboutby the interaction of phytochrome with PIF3 is not yetknown. By binding to the G-box-bound PIF3, phytochromecould regulate transcription either: (a) directly, by function-ing as a transcriptional coactivator or corepressor involvedin recruitment or modulation of the pre-initiation complex,or (b) indirectly, by biochemically or allosterically alteringthe intrinsic transcriptional regulatory activity of PIF3(Quail, 2000). Particularly, with the knowledge that theactivities of a number of transcription factors are regulatedby phosphorylation (Hardtke et al., 2000) and that phy-tochrome is a light-regulated kinase, it will be interesting totest whether PIF3 is a substrate of phytochrome kinaseactivity and how it may modulate the function of PIF3.

A Class of bHLH Proteins Operate in PhytochromeSignaling

It should be noted that PIF3 could bind both phyA andphyB, although it binds phyB with a higher apparent affin-ity (Zhu et al., 2000). Also, transgenic Arabidopsis plantswith altered PIF3 levels have a markly greater effect onphyB- than on phyA-regulated photoresponses (Ni et al.,1998; Halliday et al., 1999). These results suggest thatPIF3 has a dominant role in phyB signaling, but a moreminor role in phyA signaling. The finding that PIF3 is a


member of the bHLH family of transcription factors alsosuggests that phytochromes might directly regulate adiverse set of genes through an integrated transcriptionalnetwork orchestrated by an array of interacting bHLH pro-teins. This is suggested by the extensive size of this pro-tein family in Arabidopsis and the known nature of bHLHproteins that are able to form homodimers and/or het-erodimers. Heterodimers can have DNA-binding sitespecificity different from that of their respective homod-imers, and they could also function as convergence pointsfor integrating upstream signal inputs specific to each ofbHLH proteins present in the heterodimers (Quail, 2000,Figure 10A). Such a transcriptional network generated bycombinatorial heterodimeric interactions of different mem-bers of the bHLH family should explain the distinct yet

overlapping transcriptional profiles mediated by phyA andphyB, respectively (Ma et al., 2001). Recent studies in Dr.Quail’s group have provided new genetic and molecularevidence to support such a model. They found two addi-tional bHLH proteins involved in phytochrome signaling.One of them, PIF4, was isolated as a PIF3 interacting pro-tein and it also interacts specifically with the Pfr form ofphyB, but not phyA. PIF4 could form homodimer with itselfor heterodimer with PIF3, and both forms can bind to thePIF3-binding promoter element. Missense mutants ofphyB that are impaired in signaling show reduced bindingto PIF4, suggesting a biologically relevant interaction.Overexpressing PIF4 in transgenic Arabidopsis causes ahyposensitive phenotype specific to red light. Conversely,antisense PIF4 plants are hypersensitive to red light.

Figure 10. A molecular model depicting phytochromecontrol of gene expression.(A) An illustration of the transcriptional diversity generat-ed by combinatorial heterodimeric interactions of thebHLH family members and their selective interactionswith different phytochrome members. As shown, bHLH1interacts with phyA only, whereas bHLH2 binds phyBonly. The homodimeric and heterodimeric combinationsof the bHLH family members may target different G-boxvariants in the promoters of a variety of light-responsivegenes and regulate their expression (adapted from Quail,2000).(B) An emerging integrated picture of phytochrome sig-naling. Upon photoconversion to the Pfr form, phy-tochromes translocate into the nucleus, where they inter-act with the G-box-bound PIF3 and activate the expres-sion of the primary target genes (such as the MYB classtranscription factors CCA1 and LHY). The encoded pri-mary target gene products (many of them are transcrip-tional regulators) in turn are responsible for orchestratingthe expression of the secondary target gene expression(such as the induction of LHCB expression by CCA1),thus generating a transcriptional network controlling dif-ferent aspects of phytochrome physiology. Other phy-tochrome-interacting partners (PIFs) and their signalingintermediates may function as modifiers to participate insuch a direct light signaling pathway. Moreover, COP1and the COP9 signalosome could also interact (directly orindirectly) with the transcriptional machinery, PIFs orother signaling intermediates, and regulate their abun-dance through a light-regulated proteolysis process. Notethat far-red light converts the active Pfr form to the inac-tive Pr form, abolishing its interaction with PIF3 and turn-ing off the transcriptional cascade. R: red light; FR: far-red light; Pr: inactive, Pr conformer of phytochromes; Pfr:active, Pfr conformer of phytochromes; PIC: pre-initiationcomplex; TATA: TATA box.


Further, a T-DNA knockout mutant of PIF4, srl2, was inde-pendently identified from a genetic screen as a hypersen-sitive mutant under red light. Together, these studies pro-vided compelling evidence that PIF4 is an authentic phyBsignaling component and that it acts early in the signalingpathway. The mechanism for PIF4 attenuation of phyB sig-naling is not yet elucidated. One possibility, however, isthat the PIF3/PIF4 heterodimer might possess reducedtranscriptional activity compared to the PIF3 homodimer.Alternatively, the PIF4 homodimer may compete with PIF3in binding the biologically active Pfr form of phyB in thenucleoplasm, thus titrating out the available phyB for inter-acting with PIF3 (Huq and Quail, 2001).

On the other hand, HFR1, another bHLH protein, isgenetically identified as a positive regulator specific tophyA signaling, and it is capable of forming a homodimeras well as heterodimerizing with PIF

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