The cell biology of phytochrome signalling … · © New Phytologist (2002) 154: 553–590 553...

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Review

Blackwell Science, Ltd

Tansley review no. 136

The cell biology of phytochrome signalling

Simon G. Møller, Patricia J. Ingles and Garry C. WhitelamDepartment of Biology, University of Leicester, University Road, Leicester, LE1 7RH, UK

Summary

Phytochrome signal transduction has in the past often been viewed as being anonspatially separated linear chain of events. However, through a combination ofmolecular, genetic and cell biological approaches, it is becoming increasingly evidentthat phytochrome signalling constitutes a highly ordered multidimensional networkof events. The discovery that some phytochromes and signalling intermediatesshow light-dependent nucleo-cytoplasmic partitioning has not only led to thesuggestion that early signalling events take place in the nucleus, but also thatsubcellular localization patterns most probably represent an important signallingcontrol point. Moreover, detailed characterization of signalling intermediates hasdemonstrated that various branches of the signalling network are spatially separatedand take place in different cellular compartments including the nucleus, cytosol,and chloroplasts. In addition, proteasome-mediated degradation of signallingintermediates most probably act in concert with subcellular partitioning events as anintegrated checkpoint. An emerging view from this is that phytochrome signalling isseparated into several subcellular organelles and that these are interconnectedin order to execute accurate responses to changes in the light environment. Byintegrating the available data, both at the cellular and subcellular level, we shouldbe able to construct a solid foundation for further dissection of phytochrome signaltransduction in plants.

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Author for correspondence:

Simon G. Møller

Tel: +44 116 252 5302

Fax: +44 116 252 3330

Email: [email protected]

Received: 17 September 2001 Accepted: 20 December 2001

Contents

Summary 553

I. Introduction 554

II. Nucleus vs cytoplasm 556

III. The nucleus 562

IV. The cytoplasm 571

V. Interactions with other signalling pathways 577

VI. Conclusions and the future 582

Acknowledgements 583

References 583


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I. Introduction

1. Light perception and signalling

Organisms respond to light in very different ways rangingfrom simple signal perception and execution in single-cellprokaryotes to complex signalling networks in multicellulareukaryotes. In plants, light plays a very important role andmoreover plants need to be extremely adaptable to light dueto their sessile nature. Light not only acts as an energy sourcefor photosynthesis, but plants have to monitor the lightquality and quantity input in order to execute the appropriatephysiological and developmental responses. To achieve thisplants have evolved a complex set of photoreceptors, includingthe blue/UV-A absorbing cryptochromes and the red/far-redlight-absorbing phytochromes, of which the latter are the bestunderstood (Furuya, 1993; Quail et al., 1995; Neff et al., 2000).

Our understanding of the roles of phytochrome action isgood, however, insight into how the perceived light signals aretransduced leading to morphological responses and alteredgene expression patterns has remained somewhat sparse.Recently, tremendous efforts have been made in both dissect-ing these intermediate signalling events and understandinghow they are integrated into the overall phytochrome signal-ling network (Møller & Chua, 1999; McCarty & Chory,2000; Neff et al., 2000). Early pharmacological approachessuggested an involvement of G-proteins, cGMP and calciumas mediators of light-regulated gene expression (Neuhauset al., 1993; Bowler et al., 1994) and these hypotheses havenow been strengthened genetically (Okamoto et al., 2001;Guo et al., 2001). More recently, the isolation of mutantsdisrupted in genes encoding phytochrome signalling inter-mediates have identified both positive and negative componentsof the phytochrome signalling pathways. In addition, usingprotein–protein interaction technology, such as yeast two-hybrid and in vitro pull-down assays, there have been excitingdevelopments in understanding the plethora of physicalinteractions that take place not only between phytochromeand early signalling intermediates but also between bona fidesignalling components. An emerging view from the availabledata is that phytochrome signal transduction is a highlyordered but yet complex network of events with differentbranches of the signalling network spatially separated intodifferent subcellular compartments. The notion that earlyphytochrome signalling events take place in the nucleuswas prompted recently by the finding that nuclear localizedtranscriptional regulators physically interact with the activeform of phyA and phyB (Ni et al., 1998; Ni et al., 1999;Fairchild et al., 2000). This was further strengthened by thedemonstration that both phyA and phyB translocate to thenucleus in a light-dependent fashion (Kircher et al., 1999a;Gil et al., 2000; Kim et al., 2000). Similarly, several othernuclear phytochrome signalling components have beenidentified clearly implying that the nucleus is a hot-spot for

early signalling events (Choi et al., 1999; Hudson et al., 1999;Büche et al., 2000; Ballesteros et al., 2001; Hicks et al., 2001).Despite this, phytochrome signalling components have alsobeen identified in the cytoplasm (Fankhauser et al., 1999;Bolle et al., 2000; Hsieh et al., 2000) and in chloroplasts(Møller et al., 2001) demonstrating that the nucleus is not theonly place for phytochrome signalling action.

We have now accumulated a large number of componentsthat we need to integrate at the whole cell level to furtherunderstand phytochrome signal transduction. It is clear thatmultiple organelles are actively involved in phytochrome sig-nalling and that these are interconnected. It is also evidentthat the subcellular partitioning of both phytochrome andsignalling intermediates provide an elegant control mecha-nism. Likewise, the finely tuned degradation of signallingcomponents by the 26S proteasome is clearly an importantcheck point. In addition, the multitude of interactions thatoccur between phytochrome signalling and other pathways isfascinating and these interactions depend on controlled sub-cellular partitioning events.

This review focuses on the cell biology of phytochrome sig-nalling. We have tried to describe the different signalling com-ponents in terms of their subcellular localization and how thisimpinges on their intrinsic function as well as their overall rolein the phytochrome signalling network. We believe that oneof the keys to further dissect phytochrome signal transductionis to use the spectrum of molecular and biochemical toolsavailable, not only to isolate new signalling components,but to integrate our pool of phytochrome knowledge in a cellbiological context.

2. Phytochrome structure and function

Phytochromes were first described by Borthwick et al. (1952)as the receptors responsible for red/far-red reversible plantresponses. It has since been shown that phytochromes belongto a closely related family of photoreceptors, the apoproteinsof which are encoded by a small family of divergent genes. InArabidopsis thaliana five discrete apophytochrome-encodinggenes, PHYA-PHYE, have been isolated and sequenced(Sharrock & Quail, 1989; Clack et al., 1994; Cowl et al.,1994). Arabidopsis PHYB and PHYD polypeptides areapprox. 80% identical (Mathews & Sharrock, 1997) and aremore closely related to PHYE than they are to either PHYAor PHYC (approx. 50% identity). Counterparts of PHYA,PHYB and other PHY genes are present in most, if not all,higher plants (Mathews & Sharrock, 1997).

All of the higher plant phytochromes appear to sharethe same basic structure, consisting of a dimer of identicalc. 124 kDa polypeptides. Each monomer carries a single cova-lently linked linear tetrapyrrole chromophore (phytochromo-bilin), attached via a thioether bond to a conserved cysteineresidue in the N-terminal globular domain of the protein. TheC-terminal domain encompasses two histidine kinase related


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domains (HRKD) and two motifs with homology to PAS(PER-ARNT-SIM) domains (Lagarias et al., 1995; Kay,1997). PAS domains are present in various signal transductionmolecules which sense environmental signals such us lightconditions, oxygen levels, and redox potential (Taylor &Zhulin, 1999). They may also mediate protein–proteininteractions. See Fig. 1 for details of domains. The amino-terminal half of phytochromes can be considered as a light-sensing domain whilst the carboxyl-terminal half can beregarded as the regulatory domain (Fig. 1).

Phytochrome can be classified into two groups based onstability in light: type I (phyA) occurs in etiolated tissues inlarge quantities and is subject to a high turnover, i.e. is lightlabile and type II (phyB-phyE), are light stable. Phytochromesundergo photoconversion between two stable states: the redlight absorbing form (Pr, synthesised in the dark) and thefar-red light absorbing form (Pfr). This Pr-to-Pfr transition isdue to a light induced double bond rotation in the chromo-phore and rearrangements of the protein backbone of theapoprotein. For most responses Pfr is considered to be thebiologically active form. The Pr forms of (at least some)phytochromes of higher plants localize to the cytoplasm,whereas a proportion of the Pfr (active) isoforms localize tothe nucleus (Kircher et al., 1999a).

Many excellent reviews have previously covered phytochrome-mediated photomorphogenesis (e.g. Whitelam & Devlin, 1997;Quail, 1998; Whitelam et al., 1998). To put phytochromesignalling in context, we will briefly describe the principalbiological roles of phytochromes.

Phytochrome A Phytochrome A (phyA) is responsibleprimarily for sensing prolonged far-red light in the far-redHigh Irradiance Response (HIR) mode of phytochromeaction. This response mode operates in the regulation of manyaspects of seedling de-etiolation, including inhibition ofhypocotyl elongation, the expansion of cotyledons, changes ingene expression and the synthesis of anthocyanin, etc. (Casalet al., 1997; Whitelam et al., 1993; Barnes et al., 1996). Theseresponses to prolonged far-red irradiation are absent inphyA null seedlings. Phytochrome A also mediates theVery Low Fluence Responses (VLFR) of etiolated seedlings.Additionally, in both young seedlings and in matureArabidopsis, phyA appears to be important for perception ofdaylength (e.g. Johnson et al., 1994).

Phytochrome B Phytochrome B (phyB) deficiency leads toimpaired de-etiolation responses in red light (Reed et al.,1993), but not in prolonged far-red, thus, it is concluded thatfor de-etiolation responses, phyA and phyB have discretephotosensory activities. Phytochrome B also plays a majorrole in the low fluence response (LFR) promotion of seedgermination, which is a red/far-red reversible response(Shinomura et al., 1996). Phytochrome B is considered to bethe main phytochrome responsible for the shade avoidanceresponse (Smith & Whitelam, 1997) as phyB-deficientmutants have the typical architecture of the mature light-grown plant displaying shade avoidance responses (elongatedgrowth habit, reduced leaf area, increased apical dominanceand early flowering, Robson et al., 1993; Halliday et al.,1994; Devlin et al., 1996). This indicates that phyB perceivesthe low red:far-red signals, which result from the far-red-richlight that is reflected from (or transmitted through) the leavesof nearby plants. However, phyB null mutants still showfurther shade avoidance responses to low red:far-red signals(Devlin et al., 1996) indicating that one or more othermembers of the phytochrome family are also involved in theperception of red:far-red signals.

Phytochromes C, D and E From analyses of various phyto-chrome mutant combinations, it is clear that both phyD andphyE are also mediators of shade avoidance responses such aspetiole elongation and flowering time, with phyE havinga specific role in regulating internode elongation (Devlinet al., 1998). Phytochrome E also plays a role in the red/far-red reversible promotion of seed germination and in thepromotion of germination by far-red light, a responsepreviously considered to be mediated solely by phyA (Henniget al., 2002). Studies of phyC function have previously reliedon analysis of transgenic plants that over-express PHYC(Halliday et al., 1997; Qin et al., 1997) and analysis of thephyAphyBphyDphyE quadruple mutant. These studies haverevealed that phyC may play a role in regulating leafexpansion (Qin et al., 1997) and in the perception ofdaylength (Halliday et al., 1997), but that phyC appears notto play a major role in responses to low red:far-red ratio.

Photoreceptors talk to each other! Analyses of Arabidopsismutants containing null alleles of one or more phytochromeshave been used to try to dissect the individual roles of

Fig. 1 Structural features of phytochrome B protein, indicating the positions of the chromophore attachment site, serine phosphorylation sites, histidine kinase-related domains (HKRD) and the two PAS domains.



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phytochromes. However, this is often complicated because aswell as having independent functions, phytochromes alsoshow redundancy of function and may modulate the actionof each other. Clearly, phytochromes also interact and coactwith other photoreceptors (Mohr, 1994)

Ahmad & Cashmore (1997) reported that cryptochromeaction, in the inhibition of hypocotyl elongation under bluelight, was dependent on the presence of phyA or phyB. How-ever, it was later shown that cry1 had biological activity in aphyAphyB null mutant background in blue light especially athigher fluence rates (Neff & Chory, 1998; Poppe et al., 1998).The reduced sensitivity of phyAphyB mutants to low fluencerate blue light was accounted for on the basis of loss a phyAcontribution to blue light perception. Cryptochrome andphytochrome also interact in phototropic curvature: priorstimulation of phytochrome by red light enhances the blue-light mediated response, and this appears to be regulatedby phyA (Parks et al., 1996). Additionally, phyB and cry2 actantagonistically in regulating flowering: phyB appears torepress whilst cry2 stimulates floral induction (Guo et al.,1998). In addition to these genetic studies indicating interac-tions between phytochrome and cryptochrome there is alsoevidence that cry1 can physically interact with phyA in yeast-two-hybrid assays (Ahmad et al., 1998) and more recentlythat cry2 can interact with phyB (Mas et al., 2000).

Phytochrome signal transduction Upon photoconversionfrom Pr to Pfr, phytochromes rapidly induce a cascade ofsignalling events. Despite half a century of research onphytochromes we are only beginning to understand how thismay be achieved. One approach relies on using a variety ofscreens to find genes acting downstream of phytochrome thatmediate signal transduction. The following table introducessome of the key players that have been identified so far andaddresses the possible subcellular compartmentalisation ofsignalling events (Table 1).

II. Nucleus vs cytoplasm

In all eukaryotic cells the nucleus is separated from thecytoplasmic compartment by the nuclear envelope. Althoughthe nucleus has a high degree of autonomy the nuclearenvelope is in intimate contact with the cytoplasm andcontains nuclear pore complexes to allow for the exchange ofmacromolecules. The nucleo-cytoplasmic exchange highwaycomprises a multitude of substrates such as histones andtranscription factors imported to the nucleus from thecytoplasm and tRNA and rRNA molecules exported from thenucleus to the cytoplasm. It is now becoming increasinglyclear that compartmentation not only aids in containmentof cellular activities but also acts as a control point for keycellular events. Indeed, it has recently been shown thatphytochromes are imported into the nucleus in a light-qualityand light-quantity dependent manner, providing an early

upstream control point for phytochrome signal transduction(Sakamoto & Nagatani, 1996; Kircher et al., 1999a; Gil et al.,2000; Kim et al., 2000; Hisada et al., 2000).

1. Where is phytochrome?

The precise intracellular localization of phytochrome wasunknown for an extended period of time and conflicting datawas painting a cloudy picture. Interest into phytochromelocalization started to flourish in the early 1970s when variousresearch groups used physiological methods to show thatphytochrome is associated with various cellular compart-ments including rough endoplasmic reticulum (Williamson &Morre, 1974), etioplasts (Welburn & Welburn, 1973), andmitochondria (Manabe & Furuya, 1974). With the adventof more sophisticated immunocytochemical and subcellularfractionation techniques it was however, becoming largelyaccepted that phytochrome (more precisely phyA) was mainlycytosolic with a small proportion possibly bound to theplasma membrane (Quail et al., 1973; Coleman & Pratt,1974; Mackenzie et al., 1975; Speth et al., 1986, 1987).Knowing that receptors are often membrane-bound uponsignal perception, the possibility of phytochrome beingassociated with cytoplasmic membrane structures seemedhighly plausible. In addition, the red light-inducedsequestering of photoactive phyA into electron dense areas,followed by reversible diffuse cytosolic distribution uponconversion back to the Pr form (Mackenzie et al., 1975;McCurdy & Pratt, 1986; Speth et al., 1986), indicated a light-dependent subcellular distribution pattern of phytochrome.Taking a slightly different approach, Mösinger et al. (1987)demonstrated that by adding oat phyA protein to isolatedbarely nuclei, the transcription rate of genes encodingchlorophyll a/b binding protein could be increased (Mösingeret al., 1987). This suggested that phyA, or at least part ofphyA, is associated with the nucleus and affects the expressiondynamics of light-regulated genes. The concept of phyA beingnuclear-associated did not receive much credit and studiesperformed by Nagatani et al. (1988) showed that phyAassociates with nuclei from dark-grown pea seedlings in anonspecific manner. The prevailing view that phyA was acytoplasmic protein were further substantiated by micro-injection and pharmacological experiments (Section IV/1).Using microinjection and pharmacological agents it wasspeculated that the cytosolic form of phyA activates aheterotrimeic G-protein either by Pfr-driven translocation tothe plasma membrane or by using a cytoplasmic intermediarymolecule to transduce the signal from Pfr to the G-protein(Neuhaus et al., 1993; Bowler et al., 1994). Although itseemed feasible that phyA may associate with the plasmamembrane, computational analysis revealed that phyA fromvarious species has no motif or structure suggestive ofmembrane insertion. These studies were challenged by in vivomicrobeam irradiation experiments in lower plants


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Table 1 Phytochrome signalling components and their subcellular localization

Mutant Phenotype of mutant Protein Interactions Reference

Nucleuspoc1 Hypersensitive for red I

nduced de-etiolationPIF3 phyA & phyB SN1

phyA & phyB in Y2HNi et al. (1998); Halliday et al. (1999); Quail (2000)Similarities to b-HLH transcription

factors; promoter insertion causing increased levels of PIF3

gi-100 Impaired response to red GIGANTEA phyB SN Huq et al. (2000b); Fowler et al. (1999)circadian clock-controlled gene that regulates photoperiodic flowering

rsf1/hfr1/rep1 rsf1:(reduced sensitivity to far-red); hfr1:(long hypocotyl in ar-red); rep1:(reduced phytochrome signaling 1)

RSF1/HFR1/REP1 phyA SN Spiegelman et al. (2000); Fankhauser & Chory (2000); Fairchild et al. (2000); Soh et al. (2000)

Similarities to b-HLH transcription factors; high sequence identity to PIF3

far1 Impaired response to far-red FAR1 phyA SN Hudson et al. (1999)Putative coiled-coil domain

spa1 Isolated as a suppressor of a weak phyA mutation

SPA1 phyA SN1 Hoecker et al. (1998, 1999, 2001)WD repeat protein COP1 in Y2H

elf3 Early flowering ELF3 phyB SN/synergistic to phyB? Liu et al. (2001); Covington et al. (2001); Hicks et al. (2001); McWatters et al. (2000)Ciracadian clock-regulated protein

hy5 Impaired responses to far-red, red and blue light

HY5 COP1 in Y2H Koorneef et al. (1980); Oyama et al. (1997); Chattopadhyay et al. (1998); Ang et al. (1998)bZIP transcription factor

eid1 Increased sensitivity to far-red EID1 phyA SN1 Büche et al. (2000); Dieterle et al. (2001)F-box protein leucine–1 zipper motif AKS1 and AKS2 in Y2H

laf1 Impaired response to far-red LAF1 phyA SN Ballesteros et al. (2001)R2R3 MYB-like transcription factor

Whitelam et al. (1993); Desnos et al. (2001)fhy1 Impaired response to far-red Novel light regulated protein phyA SN

shy2 shy2–1D and shy2–2 suppressors of the long-hypocotyl phenotype of hy2 and hy3, respectively

IAA3 Auxin-induced transcription factor

coprecipitation with phyB protein Kim et al. (1996); Reed et al. (1998); Tian & Reed (1999)

cop/det/fus Photomorphogenic phenotype in the dark

COP1*: RING finger motif, coiled-coil region and WD40 repeat domain.

Epistatic analyses suggests cop/det/fus loci act downstream

Chory et al. (1989); Deng et al. (1991);Misera et al. (1994); Pepper et al. (1994);

DET1: novel nuclear localized protein COP10COP9 signalosome: subunits CSN1-8

of phyA, phyB and CRY-1 SN.COP1 interacts with CIP1, CIP4, CIP7, CIP8 and HY5

Wei & Deng (1996); Von Arnim & Deng (1994); Deng & Quail (1999); Karniol & Chamovitz, 2000; Yamamoto et al. (2001)

ndpk2 Impaired response to red and far-red

NDPK2* phyA & phyB SN Choi et al. (1999)Nucleotide diphosphate kinase 2 phyA & phyB inY2H


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Cytosolpat1 Insensitive to far-red PAT1 phyA SN Bolle et al. (2000)

VHIID/GRAS protein

fin219 Impaired response to far-red FIN219 phyA SN Hsieh et al. (2000)Auxin-inducible GH3 protein FHY1 interaction by genetic

analysis

pks1 PKS1 overexpressor: impaired response to red; PKS1 antisense lines: no effect on plant phenotype

PKS1 phyB SN Fankhauser et al. (1999)phytochrome kinase substrate 1 phyA & phyB inY2H

CHLOROPLAST

laf6 Impaired response to far-red LAF6 PhyA SN Møller et al. (2001)ABC-like protein

gun5 Pale leaves: nuclear Lhcb1 expression in the absence of chloroplast development

GUN5: ChlH subunit of Mg-chelatase

HY1 and GUN5 metabolic interaction

Mochizuki et al. (2001); Susek et al. (1993)

UNKNOWN SUBCELLULAR LOCATION

psi2 Hypersensitive to red and far-red

Not cloned phyA & phyB SN1 Genoud et al. (1998)

pef1 Impaired response to red, and far-red

Not cloned phyA & phyB SN Ahmad & Cashmore (1996)

pef2 Impaired response to red Not cloned phyB SN Ahmad & Cashmore (1996)

pef3 Impaired response to red Not cloned phyB SN Ahmad & Cashmore (1996)

red1 Suppressor of a phyB overexpressor phenotype.

Not cloned phyB SN Wagner et al. (1997)

Impaired response to red

fhy3 Impaired response to far-red Not cloned phyA SN Whitelam et al. (1993); Yanovsky et al. (2000)

shl Hypersensitive to red, far-red, and blue

Not cloned PhyA-E, CRY Pepper et al. (2001)

fin2 Impaired response to far-red Not cloned phyA SN Soh et al. (1998)

bas1-D Suppressor of phyB mutant A cytochrome P450: activation tagging causing increased levels of CYP72B1

bas1-D is epistatic to phyB; phyA is epistatic to bas1-D; bas1-D partially suppresses a cry1-null mutation

Neff et al. (1999)

SN, signalling network; COP1*, nuclear subcellular location in the dark, excluded in the light: constitutively nuclear in root cells. SN1, determined by epistasis of phytochrome mutation NDPK2*: GFP-NDPK2 fusions show nuclear and cytoplasmic subcellular location. Y2H, yeast-two-hybrid screen.

Mutant Phenotype of mutant Protein Interactions Reference

Table 1 continued



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demonstrating a very localized response and action dichroismfor phytochrome-mediated growth responses implying thatphyA is associated with the plasma membrane (Kraml, 1994).Taken together these observations suggested that phyA ismainly cytosolic but with some of the photoactive Pfr formpossibly localized to the plasma membrane via protein–protein interactions.

2. Intracellular localization of phytochrome

A major, but surprising, breakthrough regarding phyto-chrome localization came when Sakamoto & Nagatani(1996) fused the C-terminal region (dimerisation/protein–protein interaction domain) of Arabidopsis phyB to β-glucuronidase (GUS) and showed that the fusion proteinpredominantly localizes to the nucleus in transgenic plants.This indicated for the first time that phytochrome, morespecifically the C-terminal region of phyB, contains afunctional nuclear targeting signal (NLS). To corroborate thelocalization pattern observed in transgenic plants, Sakamoto& Nagatani (1996) isolated nuclei from light-grown wild-type Arabidopsis seedlings and showed that a large amount ofthe total cellular phyB content localizes to the nucleus andthat this level decreases when seedlings are dark-adapted.It was further inferred from these studies that since theC-terminal fusion protein is constitutively nuclear localized,the spectral form of phyB must be important for the light-dependent nuclear import or cytoplasmic retention.Although the evidence for phyB nuclear localization was goodthere was concern that the nucleus may not be the only siteof phytochrome action. This was largely based on themicroinjection (Neuhaus et al., 1993; Bowler et al., 1994)and microbeam (Kraml, 1994) experiments indicating thatphyA is cytosolic and possibly membrane bound. This furtherraised the intriguing possibility that although phytochromesshare common overall structural and molecular propertiesthey may function in very different ways.

Subcellular localization of phyB In order to fully dissectthe subcellular localization pattern of phyB and to gaininsight into how this may influence photoperceptionand downstream signalling events, Nagatani and colleagues(Yamaguchi et al., 1999) generated transgenic plantsoverexpressing a fusion protein consisting of a full-lengthArabidopsis PHYB cDNA fused to GFP driven by theCaMV35S promoter. One potential problem when analysingintracellular localization patterns of fusion proteins is the riskof mislocalization and loss of functionality due to the fusionpartner itself. Nagatani and colleagues (Yamaguchi et al.,1999) addressed this elegantly by transforming the phyB/GFP fusion construct into phyB-deficient Arabidopsisseedlings (phyB-5; Reed et al. (1993)) and by doing sogenerating transgenic seedlings exhibiting a typical phyBoverexpression phenotype in response to red light irradia-

tion (Wagner et al., 1991; McCormac et al., 1993). Thisconfirmed that the phyB/GFP fusion protein is biologicallyand photochemically active and that the resulting localizationdata must therefore reflect the real in vivo situation. Asobserved with the C-terminal region of phyB (Sakamoto &Nagatani, 1996), at least some of the full-length phyB/GFPfusion protein localized to the nucleus in light treatedseedlings. However, at higher magnification it was evidentthat the phyB/GFP localizes to subnuclear foci giving rise tofluorescent speckles or spots within the nucleus. All cell typesexamined had between 5 and 10 speckles/nucleus and theywere approx. > 1 µm in size. This phenomenon has now beenexamined in more detail and it seems that the speckle numbervaries depending on the fluence rate (Gil et al., 2000). Indarkness the full-length phyB cDNA/GFP fusion proteinlocalizes diffusely to the cytoplasm corroborating the previouslocalization patterns (Sakamoto & Nagatani, 1996). In orderto further analyse the light-induced nuclear translocationof photochemically active phyB, Yamaguchi et al. (1999)examined the red-light induced translocation and speckleformation over a period of 6 h. After 2 h of red lightirradiation the phyB levels in the nucleus increase, however, asubstantial part is still present in the cytoplasm. In addition,very little speckle formation is observed. After 4 h thetranslocation is almost complete showing the presence of alarge number of small speckles, whilst after a further 2 h GFPfluorescence can only be observed in the nucleus in the formof fewer but larger speckles. This time course clearly unveiledthat the translocation event and the speckle formation areclosely linked, both being part of a highly dynamic process.

These observations were corroborated and further extendedby Nagy, Schäfer and colleagues (Kircher et al., 1999a; Gilet al., 2000) using a tobacco CaMV35S-phyB/GFP fusion intransgenic tobacco plants. As shown for Arabidopsis (Yamaguchiet al., 1999), the tobacco phyB/GFP fusion protein was ableto complement a phyB-deficient Nicotiana mutant (Kircheret al., 1999a). Kircher et al. (1999a) demonstrated, as Yamaguchiet al. (1999), that the accumulation of phyB/GFP in thenucleus is slow, taking approx. 2 h to reach a fluorescence levelabove the detection threshold. This could either represent areal physiological phenomenon or an artifact due to the pres-ence of the GFP fusion partner. Although the phyB/GFPfusion protein was shown to complement a phyB-deficientmutant, the amount of translocated nuclear phyB needed forfunctional signalling to occur may be very little in which caseit is hard to determine if the slow import kinetics are in factreal. Although fusion protein technology is a useful tool forsubcellular localization studies, kinetic measurements of fusionprotein translocation events do not necessarily reflect thein vivo situation. More detailed in vivo immunolocalizationstudies of the endogenous phyB would however, clarify this.

Kircher et al. (1999a) clearly showed that the phyB trans-location event is highly dependent on the quality of light inthat neither continuous far-red light nor repeated pulses result



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in nuclear phyB accumulation or speckle formation. Takentogether these results indicate that phyB regulates its ownnuclear translocation, which is mediated by the low-fluenceresponse (LFR) of phytochrome (Kircher et al., 1999a). Fur-ther characterization also showed that red-light-inducedspeckle formation is dependent on the fluence rate. Below afluence rate of 7 µmol m−2 s−1 the accumulation of nuclearspeckles follow a hyperbolic curve reaching saturation (c. 20speckles/nucleus after 3 h red light irradiation) at higherfluence rates (Gil et al., 2000). The authors also make theobservation that an equal number of photons at different timepoints result in different kinetic and saturation properties,indicating that nuclear import of phyB and speckle formationis dependent on both the fluence rate and time.

Subcellular localization of phyA As for phyB, it was of greatinterest to determine the dynamics of phyA translocation andsubcellular partitioning and it was partly expected that phyAwould behave differently from phyB due to their differentmodes of action. As for phyB, Kim et al. (2000) generated afull-length Arabidopsis PHYA cDNA/GFP fusion proteindriven by the endogenous PHYA promoter and showed thatthis fusion protein is able to complement and restore wild-type characteriztics in a phyA-deficient Arabidopsis mutant(phyA-201). Using these complemented lines it was shownthat the phyA/GFP fusion protein localizes to the cytoplasmin darkness with no detectable nuclear fluorescence. Theintracellular localization of phyA is strongly affected byirradiation showing rapid nuclear import upon brief exposureto pulses of far-red, red, and blue light. It was also noted bythe authors that after the far-red pulse, but before thetranslocation event, speckle formation was observed in thecytoplasm reminiscent of phyA sequestration in monocots(Speth et al., 1986; Speth et al., 1987) and rice phyA/GFPspeckle formation in tobacco plants (Kircher et al., 1999a).Continuous red light irradiation for 5 h failed to inducenuclear import and cytosolic speckle formation of phyA/GFP.Conversely, continuous far-red and blue light irradiation for5 h induced both nuclear import of phyA and speckleformation whilst very little cytoplasmic speckling wasobserved (Kim et al., 2000). Taken together these findingssuggest that the nuclear import of Arabidopsis phyA/GFP isnot only mediated by the very low fluence response (VLFR)but also by the far-red high irradiance response (HIR). Indicots the far-red HIR is diminished by red light pretreatmentand transgenic seedlings subjected to a red light pretreatment,followed by far-red light irradiation, showed lack of phyAnuclear import (Kim et al., 2000).

In tobacco, the rice phyA/GFP fusion protein translocatesto the nucleus in response to short pulses of red and far-redlight suggesting VLFR-mediated nuclear import (Kircheret al., 1999a). This analysis was extended by generating trans-genic tobacco harbouring the Arabidopsis phyA/GFP fusion.Kim et al. (2000) found that in contrast to Arabidopsis phyA

in Arabidopsis and rice phyA in tobacco, Arabidopsis phyAin tobacco does not show VLFR-mediated nuclear transloca-tion. These results clearly demonstrate another intriguingproperty of phytochrome; the differences in the spectralsensitivity of Arabidopsis phyA are indicative of differentphysiological roles depending on the cellular background.The authors speculate that this is probably exerted at the levelof light-dependent degradation or by the mechanism mediat-ing phyA retention or import into the nucleus (Kim et al.,2000). A cautionary note from this is that localization datafrom heterologous systems should be analysed carefully.

Subcellular localization of phyC, phyD and phyE It is be-coming increasingly clear that the two major phytochromes,phyA and phyB, exhibit very different subcellular localizationand translocation dynamics dependent on the quality andquantity of light. To extend this analysis Nagy, Schäfer andcolleagues generated transgenic Arabidopsis and tobaccoplants harbouring phyC-E/GFP fusions proteins (Nagy et al.,2001). Analyses of the transgenic plants showed that, phyC-phyE are constitutively localized to the nucleus, that nuclearstaining in the dark is always diffuse and that speckleformation can be induced by red light and reversed by far-red.These findings indicate that phyC-phyE can be imported intothe nucleus in an inactive Pr form but yet speckle formationis dependent on the active Pfr form. Interestingly, the phyC-phyE speckle formation is much more heterogeneous thanthat of phyA and phyB and can vary spatially within tissues(S. Kircher, pers. comm.).

There are a number of obvious questions arising from theaccumulation of recent data. Why is the regulation andtranslocation of the different phytochromes so very different?Why, in contrast to phyA and phyB, are phyC-phyE localizedconstitutively to the nucleus and do they have any biologicalrole in darkness? If phyC-phyE have a role in the nucleusin darkness then why is speckle formation light-dependentIndeed what is the significance of light-induced nuclear, andoccasional cytoplasmic, speckle formation?

3. All phytochromes localize to nuclear speckles

Nuclear speckling has been documented in animal cells(Lamond & Earnshaw, 1998), however, the precise physio-logical role of speckle formation remains somewhat obscure.In plants, several proteins have been shown to localize tospeckles including COP1 (constitutively photomorphogenic1), all phytochromes, CRY2 (a blue light photoreceptor),RPN6 (a component of the 26S proteasome) and LAF1 (longafter far-red 1) (von Arnim et al., 1997; Mas et al., 2000;Ballesteros et al., 2001; Nagy et al., 2001; Peng et al., 2001).Initially there was some concern whether speckle formationwas merely an artifact of phytochrome overexpression(Neff et al., 2000). However, Kim et al. (2000) has clearlyshown that phyA forms speckles when expressed as a fusion



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protein from its own endogenous promoter. It is tempting tohypothesise that speckle formation may represent general sitesof physical interaction between phytochromes, other lightreceptors and nuclear signalling components. Indeed, recentstudies by Mas et al. (2000) clearly show, using fluorescenceresonance energy transfer (FRET) that phyB and cry2physically interact in light-induced nuclear speckles. Similarly,Quail and colleagues have convincingly shown that bothphyA and phyB can interact with nuclear proteins. The bestcharacterized of these is PIF3, a HLH-type protein, whichinteracts with both phyA and phyB, albeit with differentaffinities, in a photoreversible fashion (Ni et al., 1998; Niet al., 1999; Zhu et al., 2000). Moreover, it has been shownthat PIF3 together with phyB can bind, photoreversibly topromoter elements of several light-regulated genes (Martinez-Garcia et al., 2000). These results clearly indicate that for atleast some phyB-mediated responses the chain of signallingevents is very short (Section III/1). An attractive possibilitythat may explain the variation in speckle formation betweenthe different phytochromes is that these may representdistinct functional nuclear complexes. It is curious to notethat neither PIF3 nor HFR1 by themselves form speckles. Tothis end it will be interesting to examine the phytochrometranslocation kinetics in various phytochrome signallingmutant backgrounds.

Despite the compelling available evidence, nuclear speckleformation may also serve a separate discrete role. Recently, itwas shown in onion epidermal cells, that COP1 and LAF1speckles are different from phyA speckles indicating that nei-ther COP1 nor LAF1 are in close physical proximity to phyA(Ballesteros et al., 2001). Indeed, LAF1 does not interact withphyA in yeast two-hybrid assays (Ballesteros et al., 2001). Inaddition, the diffusion and complete loss of nuclear specklesafter light-to-dark transition may imply that speckle forma-tion is involved in the controlled degradation of phyto-chrome. Speckle formation has also been implicated inplaying a role in the process of SUMO (small ubiquitin-likemodifier)-mediated protein protection and protein–proteininteractions via altered subcellular localization patterns(Melchior, 2000; Muller et al., 2001). To this end Chua andcolleagues demonstrated recently that LAF1, a positive com-ponent of phyA signalling, contains a putative sumolation site(KKQE) which if mutated (KRQE) abolishes in vivo nuclearspeckle formation (Ballesteros et al., 2001). This suggests thatthe recruitment of LAF1 to subnuclear foci require this putat-ive sumolation site. It will indeed be interesting to learnwhether SUMO interacts with LAF1 in yeast two-hybridassays. Likewise, colocalization studies in transgenic plants,using both wild-type and mutant LAF1 fusion proteins, mayclarify more firmly the possible in vivo physical interactionbetween LAF1 and SUMO. The potential role of proteindegradation and protection as part of controlling lightsignal transduction will be covered in more detail in SectionIII/4.

4. Possible partitioning mechanisms

It is now evident that the various phytochromes make up acomplex network of partitioning events involving both light-dependent and light-independent nuclear translocations.However, questions such as ‘How are phyA and phyB retainedin the cytoplasm in darkness and how is this controlled?’beg an answer. The Pr to Pfr conformation change is clearlyrequired for the translocation event in that a phyB mutant,unable to bind its chromophore, is constitutively localizedto the cytoplasm (Kircher et al., 1999a). The simplestexplanation to this would be that either the Pr form ‘masks’the nuclear import region or uncovers a region that is involvedin binding to cytoplasmic proteins. The long awaited three-dimensional structure of phytochrome would clearly unveilsuch possibilities. However, Nagy, Schäfer and colleagues havenoted that strong overexpression of both phyA and phyB canresult in weak nuclear staining in darkness indicating that thePfr conversion is not strictly necessary for the translocationevent to take place. This suggests that a separate, but notmutually exclusive, mechanism exists. It is possible that thePr form of phyA and phyB are retained in the cytoplasmin darkness by cytosolic ‘retention proteins’ and that uponstrong overexpression some of the phytochrome escapes theretention mechanism due to ‘retention protein’ saturation.Assuming that different phytochromes team up with different‘retention proteins’ it is conceivable that these exist at distinctendogenous levels reflecting the in vivo physiological amountsof the different phytochromes. Since phyC, phyD, and phyEare only present at low endogenous concentrations it is possiblethat by overexpressing these they largely escape the retentionmechanism and therefore appear to be constitutively nuclearlocalized. One way of addressing this would be to use theendogenous phyC-phyE promoters for the localization studiesalthough this may present a problem due to low expressionlevels. Equally conceivable is the possibility that the differentphytochromes have different affinities for the same ‘retentionprotein’. It will be interesting to learn whether any cytosolicphytochrome-interacting ‘retention proteins’ will be isolatedfrom yeast two-hybrid screens and if these prove to bephotoreversible in terms of their binding capacities?

Cytoplasmic retention of proteins as a control point forcellular activity is a common process. For instance, the zinc-finger protein BRAP2 binds to the NLS region of the tumoursuppressor protein BRCA1 with similar affinity as importinα/karyopherin α thereby overriding the nuclear translocationevent and acting as a cytoplasmic retention protein (Li et al.,1998). Similarly, the transduction of type 1 interferon is con-trolled by STAT2/p48 cytoplasmic complexes, which uponstimulation dissociate leading to rapid nuclear translocationof p48 (Lau et al., 2000). By substituting p48 with phyA inthe above scenario it is tempting to speculate that phyto-chrome is part of a cytoplasmic protein complex in darkness,which rapidly dissociates when irradiated leading to rapid



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nuclear translocation. More recently, evidence has alsodemonstrated the involvement of heterotrimeic G-proteinsin cytoplasmic retention and release. TUBBY, a transcriptionregulator involved in maturity-onset of obesity, is releasedfrom the plasma membrane by receptor-mediated activa-tion of G-proteins via phospholipase C action resulting inrelease and nuclear translocation of TUBBY to the nucleus(Santagata et al., 2001).

Various studies have demonstrated that phyB has serine/threonine kinase activity capable of both autophosphoryla-tion and phosphorylation of other proteins (Section IV/2).Limited evidence exists, but Park et al. (2000) has speculatedthat phosphorylation of phytochrome may contribute to thePr to Pfr conformation change which may imply that phos-phorylation patterns may be involved in the retention andrelease of phytochromes. It is possible that PKS1 (phyto-chrome kinase substrate 1; Fankhauser et al., 1999a) may playa role in this retention mechanism. Conversely, the phospho-rylation patterns of retention proteins may be important. Inmammalian systems it has been shown that the phosphor-ylation of cytoplasmic proteins can either change bindingaffinities or alternatively mask binding regions involved inprotein complex formation (Stanley, 1996). This may be thecase for phytochrome.

Signal transduction pathways cannot be viewed as linearchains of events but should be viewed as an interconnectednetwork comprising a multitude of different pathways. Thereare numerous interactions and intersections between lightsignal transduction and other signalling pathways (Section V)and these probably take place both in the cytoplasm and in thenucleus (Møller & Chua, 1999). It is feasible therefore thatinteraction among signalling cascades in the cytosol mayaffect phytochrome translocation ultimately influencingthe regulation and transcription of light-regulated genes inthe nucleus. Indeed it was demonstrated very recently that thepotato photoperiod-responsive 1 (PHO1) locus, representinga general component of the gibberellic acid (GA) pathway,translocates rapidly to the nucleus in response to GAapplication (Amador et al., 2001). It will be of great value toexamine the phytochrome translocation dynamics in mutantsaffected in other signal transduction pathways.

III. The nucleus

The fairly recently discovered light-triggered nucleartranslocation of phyA and phyB, probably representing one ofthe earliest check points in phytochrome signal transduction,has created a lot of excitement and has opened up new waysof viewing the intracellular light signalling pathways. Thenucleus undoubtedly plays a key role not only in recruitingbiologically active phytochrome but also in the subsequentsignal relay mechanisms from the photoreceptors to changesin gene expression. Although the overwhelming importanceof the nucleus in light signalling is only now becoming

apparent, several approaches have been taken during the last5–10 yr in efforts to identify nuclear localized phytochromesignal transduction components. Although not selective fornuclear proteins, various genetic screens have been employed(Neff et al., 2000) resulting in the isolation of mutants withgenetic lesions in nuclear-localized phytochrome signallingcomponents. A more selective approach, using phytochromeas bait in yeast two-hybrid screens, has also been employed.From these two main approaches it is possible to divide theidentified nuclear proteins into phytochrome-interacting and‘phytochrome-free’ components.

1. Phytochrome-interacting nuclear components

PIF3 The first bona fide phytochrome interacting partneridentified was PIF3 (phytochrome interacting factor 3) (Niet al., 1998). PIF3, a helix-loop-helix protein, was isolatedin a yeast two-hybrid screen using the nonphotoactiveC-terminal domain of the Arabidopsis phyB as bait (Ni et al.,1998). It was subsequently shown that PIF3 could also bindto the C-terminal domain of phyA suggesting that PIF3 mayact as a common interaction partner for both phyA and phyB(Ni et al., 1999). The analysis was extended further and itwas shown that mutant versions of phyB (A776V, G793R,E838K), that disrupt signal transfer, show dramaticallyweaker binding to PIF3. To show that PIF3 is part of thephytochrome signal transduction pathway, Quail andcolleagues generated Arabidopsis plants containing a PIF3antisense transgene and demonstrated that reduced PIF3levels result in reduced photoresponsivenss towards red lightirradiation but only minimally towards far-red light (Ni et al.,1998). Although the effects in terms of hypocotyl length arenot striking these results show that PIF3 is a component ofboth phyA and phyB signalling. Moreover, the weak PIF3-deficient phenotype could indicate functional redundancy,which is often observed due to the promiscuous behaviour oftranscriptional regulators. Is the binding of PIF3 to phyBdependent on an active Pfr form? Quail and colleagues wenton to show, using in vitro pull-down assays, that PIF3 bindstightly to the red-light activated full-length phyB in darknessbut dissociates rapidly upon conversion back to its Pr formupon far-red light irradiation (Ni et al., 1999). More recentlyit has also been shown that although phyA interacts with PIF3in a photoreversible manner the apparent affinity for phyA isapprox. 10-fold lower than that for phyB (Zhu et al., 2000).This could of course explain the weaker phenotype observedin PIF3 antisense plants in response to far-red light irradiation(Ni et al., 1998). In addition, in vitro pull-down assays anddeletion mapping has shown that a 37 amino acid stretchpresent at the N-terminal region of phyB, but absent in phyA,contributes to the stronger binding of phyB to PIF3 (Zhuet al., 2000). It follows from this that PIF3 probably has amore predominant role in phyB signalling with only a minorrole in phyA signalling.



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Martinez-Garcia et al. (2000) investigated whether, asmember of the helix-loop-helix transcriptional regulator fam-ily of proteins PIF3, can bind to promoter elements. Using arandom binding site selection procedure it was shown thatPIF3 can bind DNA in a sequence-specific fashion with thecore binding sequence being the palindromic hexanucleotideG-box motif CACGTG found in many light-regulated genes(Martinez-Garcia et al., 2000). It was subsequently shownthat in PIF3-deficient seedlings the expression levels of thephytochrome-regulated genes CCA1 and LHY were reduced.Interestingly, it has also been shown that the bZIP proteinHY5 binds to G-box elements in Arabidopsis (Chattopadhyayet al., 1998) however, in the hy5 mutant CCA1 and LHYshow normal phyB-mediated expression dynamics. Theseresults suggest that G-box elements can discriminate betweendifferent DNA-binding proteins. It is possible that PIF3,through phyB, regulates genes such as CCA1 and LHY whichthemselves encode MYB transcription factors suggestingthat PIF3 may act as a control point for different branches ofphotomorphogenesis.

Another intriguing aspect of PIF3 biology is that the Pfrform of phyB binds G-box-bound PIF3 forming a phyB/PIF3/DNA complex as observed in gel retardation assays(Martinez-Garcia et al., 2000). As for the PIF3/phyB bindingcharacteriztics, the Pr form of phyB does not interact with thePIF3/DNA complex and moreover bound Pfr phyB rapidlydissociates following far-red light irradiation. Taken togetherthis infers that PIF3 may acts as a recruitment agent, directingincoming photoconversion-induced phytochrome to targetpromoter sites and by doing so controlling the expression oflight-regulated genes.

The Arabidopsis genome contains a large number ofbHLH proteins. It has been well documented that bHLHfamily members form both homodimers and heterodimercombinations thereby modifying their DNA binding andprotein–protein interaction properties (Massari & Murre,2000). If this is the case for phytochrome signal transduction,different combinations of bHLH proteins may form,depending on the light conditions, thereby generating avast array of different phytochrome- and DNA-bindingaffinities.

Hfr1 The possibility of having a combinatorial network,consisting of different bHLH proteins directing differentphytochromes to their target promoter sequences, has beenstrengthened by the isolation of HFR1 (Fairchild et al.,2000). HFR1 is an atypical bHLH protein, specific for phyAsignal transduction in Arabidopsis, which does not interactwith phyA or phyB directly but forms photoreversibleheterodimers with PIF3/phyA complexes. Since the mutantphenotype of hfr1 is more prominent in response to far-redlight than that of PIF3-deficient seedlings it is clear thatdifferent bHLH combinations have different effects underdifferent light regimes.

Ndpk2 Another protein found to interact with the C-terminal domain of phyA is nucleoside diphosphate kinase 2(NDPK2) (Choi et al., 1999). Using a quantitative yeast two-hybrid assay Song and colleagues showed that NDPK2 hasreduced binding affinity towards phyA missense mutants asseen for PIF3. Moreover, biochemical cross-linking/SDS-PAGE studies showed that NDPK2 interacts preferentiallywith the Pfr form of purified oat phyA with approximately1.8-fold higher binding affinity than for the inactive Pr form.The photoreversible binding characteriztics of NDPK2 is notas clear-cut as for PIF3. To this end Song and colleagues testedwhether the enzymatic activity of NDPK2 is influenced bythe presence of photoactive phyA. It was shown that theintrinsic γ-phosphate-exchange activity increased approx. 1.7-fold upon incubation with the Pfr form of oat phyA whilst thePr form had no effect. From these data it is evident thatNDPK2 interacts directly with phyA and that this interactionis stronger upon phyA activation.

Analysis of an ndpk2 loss-of-function mutant has shownthat NDPK2 is involved in the deetiolation process in Arabi-dopsis, displaying reduced responsiveness in both red- andfar-red-induced hook opening and cotyledon expansion (Choiet al., 1999). Hypocotyl growth inhibition was however, un-affected. This does indicate that NDPK2 is involved in bothphyA and phyB signalling events. It would be interesting tolearn whether NDPK2 interacts with phyA or other phyto-chromes and whether these interactions are photoreversible.

NDPK2 localizes to both the nucleus and the cytoplasm(Choi et al., 1999; Zimmermann et al., 1999). Mechanisticdata is still lacking but it is possible that NDPK2 acts as a tran-scriptional regulator. Zimmermann et al. (1999) has reportedthat NDPK1a, which is in fact NDPK2, is capable of bindingto the HIS4 promoter in yeast, which is the target site for theGCN4 transcription factor. Moreover, NDPK1a can fullycomplement the gcn4 mutant (Zimmermann et al., 1999).Although this may indicate that NDPK2 is involved innuclear transcriptional control, NDPK2 also localizes to thecytoplasm. Whether the cytoplasmic localization data ismerely an artifact due to saturation of the nuclear importmechanism or whether it proves to be real, it is conceivablethat NDPK2 has some functional overlap with PKS1 (phyto-chrome kinase substrate 1) (Fankhauser et al., 1999). NM23,a mammalian NDPK, is postulated to be involved in aphosphorelay/phosphotransferase mechanism (Engel et al.,1995; Lu et al., 1996) and PKS1 has been postulated to be asubstrate for a phyA-mediated phosphotransfer reaction(Fankhauser et al., 1999). PKS1 will be discussed in moredetail in Section IV/1.

Elf3 The elf3 mutant was initially characterized as beingphotoperiod-insensitive early flowering, impaired in thetransduction of light signals to the circadian clock (Hickset al., 1996; Zagotta et al., 1996). Recently, Millar andcolleagues showed that ELF3 affects light input to the



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circadian oscillator (McWatters et al., 2000) and ELF3 hasreceived a lot of attention (Covington et al., 2001; Hickset al., 2001; Liu et al., 2001). The EL3 transcript is regulatedin a circadian fashion and the gene has now been cloned andit encodes a novel 695 amino acid protein that may act as atranscriptional regulator (Hicks et al., 2001). ELF3 is asexpected a nuclear protein and accumulates in a periodicmanner showing the highest levels just before the onset ofdarkness during 24-h light/dark cycles (Liu et al., 2001). Thepattern of nuclear protein oscillation is almost identical to theELF3 transcript dynamics suggesting that ELF3 function istranscriptionally regulated. Further, in a yeast-two hybridscreen using ELF3 as bait, Liu et al. (2001) isolated ELF3 andphyB, suggesting that ELF3 may act as a dimer interactingwith phyB. It was shown that ELF3 interacts with the C-terminal domain of phyB but not with phyA. Moreover, theelf3 phenotype includes partial insensitivity towards red-lightinduced hypocotyl growth inhibition implying that ELF3may form a light-induced nuclear complex with phyB in vivo.Conversely, genetic analysis shows that ELF3 controlsflowering independent of phyB. It remains to be shownwhether ELF3 regulates flowering by interaction with otherphytochromes or by regulating the circadian clock.

CRY1 and CRY2 Both genetic and physiological evidenceshow that there is cross-talk between the phytochrome andcryptochrome signalling pathways. Indeed it has been shownin yeast two-hybrid assays that the C-terminal part of phyAcan interact with the C-terminal part of CRY1 (Ahmad et al.,1998). To this end Cashmore and colleagues have shown thatoat phyA can in vitro phosphorylate recombinant CRY1 andCRY2 specifically on serine residues. However, no differencein phosphorylation patterns is observed between the Pr andPfr form of phyA. Conversely, using dark-adapted Arabidopsisseedlings, phosphorylated CRY1 can be affinity purifiedafter a red light pulse whilst after a red/far-red pulse cycleno phosphorylation occurs. These data suggest that thephotoactive form of phyA mediates in vivo phosphorylationof CRY1 resulting in enhanced blue light activation (Ahmadet al., 1998). More recently, Cashmore and colleagues ( Jarilloet al., 2001) showed that the Arabidopsis circadian clock PASdomain protein ADAGIO1, described originally as ZTL(Somers et al., 2000), interacts with both CRY1 and phyB inyeast two-hybrid and in vitro interaction studies. Knowingthat both phytochrome and cryptochrome are nuclearlocalized it is feasible that there is functional, directinteraction between these two photoreceptors resulting in acoordinated nuclear network of red and blue light responses.

2. ‘Phytochrome-free’ nuclear components

Yeast two-hybrid screening, using phytochrome as bait,has undoubtedly contributed enormously towards ourunderstanding of early phytochrome signalling events. How-

ever, by employing light-specific genetic screens a number ofnuclear-localized phytochrome signalling components havebeen identified whose deficiencies result in either insensitivityor hypersensitivity in terms of hypocotyl elongation.Although these components do not physically interact withphytochrome, their photomorphogenic phenotypes areoften more dramatic than those observed for the null-mutants of phytochrome-interacting components, clearlyimplying an important role in phytochrome signalling. The‘phytochrome-free’ nuclear components can be furtherdivided into three main classes: phyA-specific, phyB-specificand phyA/phyB components, with phyA-specific mutantsbeing most abundant.

PhyA-specific The first bona fide phyA-specific signallingmutants identified were fhy1 and fhy3 (Whitelam et al.,1993). Apart from the photoreceptor mutants, they showthe strongest far-red insensitive phenotype amongst isolatedmutants to date, and maybe this is why fhy1 and fhy3 were thefirst ones to be identified. The disrupted gene in fhy1 has veryrecently been cloned and encodes a small (202 amino acids,23 kDa), novel protein that is nuclear localized in dark-grownseedlings (Desnos et al., 2001). Expression of the FHY1 geneis down-regulated by light and phyA is involved in thisprocess, suggesting negative-feedback regulation of far-redlight signalling via phyA. However, additional photoreceptorsare also involved and the regulation of FHY1 mRNA levels byphotoreceptors other than phyA may reflect a cross-talk pointbetween the signalling pathways associated with differentphotoreceptors.

The far1 mutant was isolated based on its partial insens-itivity towards far-red light irradiation showing partial lackof hypocotyl growth inhibition (Hudson et al., 1999). TheFAR1 gene was cloned by positional cloning and encodes anovel nuclear localized protein with no known function. Thepartial insensitivity towards far-red light irradiation may be aresult of functional redundancy amongst FAR1 family mem-bers since FAR1 is part of a multigene family in Arabidopsis.

Recently, Chua and colleagues isolated a phyA-specificmutant, laf1, which is partially insensitive towards far-redlight irradiation in terms of hypocotyl growth (Ballesteroset al., 2001). Moreover, a careful detailed physiological char-acterization of laf1 showed that far-red responses such asapical hook opening, cotyledon expansion and gravitropismwere unaffected. LAF1 was cloned and encodes a 283 aminoacid MYB protein previously identified as atMYB18 (Kranzet al., 1998) belonging to the two-repeat (R2R3-type) MYBproteins of which there are approximately 130 members inArabidopsis. As for FAR1, the presence of this large R2R3-likeMYB family could explain the partial insensitivity towardsfar-red light. Ballesteros et al. (2001) demonstrated usingyeast transactivation experiments that LAF1 can act as a tran-scriptional activator suggesting direct involvement in light-dependent gene regulation. The nuclear localization of LAF1



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was determined using a LAF1/GFP fusion protein in onionepidermal cells. By contrast to the uniform nuclear stainingobserved with other phytochrome signalling components,LAF1 localizes to subnuclear foci or speckles in a time-dependent manner. After the transfection (4–6 h) LAF1/GFPfluorescence is observed diffusely throughout the nucleus withspeckle formation occurring only after 8–10 h. Followingthis the GFP signal fades until it eventually disappears 4–8 hlater suggesting that speckle formation precedes degradationof the protein. Domain mapping experiments demonstratedfurther that the first 70 amino acids of LAF1 is sufficient fornuclear translocation but not for speckle formation whilst an84 amino acid region (amino acids 176–260) is responsiblefor the speckle formation. Upon closer examination a putativesumolation site (KK QE) was found in this region (aminoacids 257–260) of the protein and studies have shown thatproteins can localize to speckles when conjugated to SUMO(Muller et al., 1998). To test whether sumolation could beinvolved in LAF1 speckling, Ballesteros et al. (2001) mutatedthe putative sumolation site (KK QE to KR QE), whichabolishes in vivo nuclear speckle formation. This suggests thatthe recruitment of LAF1 to subnuclear foci requires SUMOconjugation. Whether SUMO physically interacts with LAF1remains to be determined. LAF1 does not interact with phyAand phyA speckles do not colocalize with LAF1 speckles(Ballesteros et al., 2001). It is of course tempting to speculatethat LAF1 may interact with PIF3 or some other phyA-interacting proteins or LAF1 may actually activate transcrip-tion of phyA-interacting components. Regardless of themechanism, these findings clearly indicate that transcriptionalactivators that do not interact with photoreceptors are alsoimportant for correct phytochrome signal transduction.

The hypersensitive mutants spa1 was identified from a sup-pressor screen using a weak phyA allele (Hoecker et al., 1998).In wild-type plants the spa1 mutation causes hypersensitivity(short hypocotyl) towards red and far-red light however,genetic studies have shown that this hypersensitivity is lost ina phyA null background. This implies a role for SPA1 specificfor phyA signalling and SPA1 must therefore encode a neg-ative component of the phyA signal transduction pathways.The spa1 locus has been cloned and SPA1 encodes a constitu-tively nuclear localized novel protein kinase containing aWD-repeat motif (Hoecker et al., 1999). Interestingly, it hasbeen suggested that SPA1 may counteracts phy-mediatedgrowth inhibition during de-etiolation: phytochrome inhibitselongation whilst SPA1 promotes elongation (Parks et al.,2001). Recently, SPA1 was shown to interact with the coiled-coil domain of COP1 in a yeast two-hybrid screen and byin vitro interaction studies (Hoecker & Quail, 2001). Theimplications of this are that SPA1 may link phyA signalling tothe light signalling pathway of COP1.

Another phyA-specific hypersensitive mutant recentlyisolated is eid1 (Büche et al., 2000). The eid1 mutant showshypersensitivity towards red and far-red light, but as for spa1

this hypersensitivity is abolished in a phyA null mutant. Anintriguing feature of the eid1 mutant is that it shifts theresponsiveness of the phyA-signalling pathway from far-red tored wavelengths. EID1 has been cloned and encodes a novelF-box protein (Dieterle et al., 2001). The N-terminal domainof EID1 shows homology to F-box proteins that form partof the SCF (Skp1, Cdc53 and F-box) complex. The SCFcomplex functions as an ubiquitin-ligase involved in theproteasome-dependent degradation of proteins (Craig &Tyers, 1999) (Section III/4). The EID1 protein also containsa leucine zipper domain, important for its function, whichmay imply that EID1 acts as a homo- or heterodimer in vivo.The presence of an F–box domain suggests interactions withother members of the SCF complex. To this end Dieterle et al.(2001) used EID1 as bait in a yeast two-hybrid screen andisolated ASK1 and ASK2, two Arabidopsis homologs of theyeast Skp1 protein (Gray et al., 1999). These interactionswere verified using in vitro pull-down assays. Moreover,deleting part of the F-box domain and changing a conservedN-terminal proline residue in EID1 abolished the ASK1 andASK2 interactions. EID1 is a constitutively nuclear localizedprotein as shown by EID1/GFP fusion studies in protoplasts.It is tempting therefore to speculate that EID1 may beinvolved in the proteasome-mediated degradation ofphytochrome. However, a role for EID1 in phyA degradationcan be excluded because phyA levels are not altered in theeid1 mutant. Therefore, EID1 is probably involved in thedegradation of positively acting phyA signalling components(Section III/4) but this remains to be demonstrated.

PhyB-specific Mutants specific for phyB signalling are morerare. This could partly be explained by the fact that phyB-phyE show redundancy in terms of red light perception(Devlin et al., 1998; Devlin et al., 1999). In addition, redlight plays multiple roles during development, separatefrom phytochrome signalling, and it has therefore provenproblematic to isolate bona fide phyB-specific signallingmutants.

Putative phyB-specific mutants have been isolated andinclude red1, pef2, pef3, and srl1 (Ahmad & Cashmore, 1996;Wagner et al., 1997; Huq et al., 2000a). However, the dis-rupted genes in these mutants have not yet been cloned so itis difficult to assign any specificity as yet.

As far as we are aware there are only two mutants that havebeen shown to be specifically disrupted in phyB signalling.One of these is poc1 (photocurrent 1), which exhibitsenhanced responsiveness towards red light (Halliday et al.,1999). Moreover, the poc1 mutant phenotype is abolished inphyB-deficient seedlings demonstrating the phyB specificityand suggesting a role of POC1 in enhancing phyB signalling.Interestingly, the T-DNA insertion in poc1 is located in thepromoter region of PIF3 (Section III/1), which causes PIF3overexpression in response to red light irradiation. Themechanism by which the promoter insertion results in red



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light-induced overexpression is unknown but it is possible thatthe T-DNA insertion disrupts a negatively acting red light-specific PIF3 promoter region. It is interesting to note thedifferences in hypersensitivity between wild-type Arabidopsisseedlings overexpressing PIF3 (Ni et al., 1998) and poc1(Halliday et al., 1999). Under identical red light fluence rates(20 µmol m-2 s-1) the hypersensitivity of PIF3 overexpressionin wild-type seedlings is marginal whilst PIF3 overexpressionin poc1 results in a marked red-light induced hypersensitivity.However, the reported differences may simply reflect differencesbetween endogenous promoter insertions and CaMV35S-driven PIF3 overexpression or simply differences in ecotypes.

Recently, Quail and colleagues isolated a mutant, gi-100,that is partially insensitive specifically towards red light irradi-ation (Huq et al., 2000b). Cloning of the mutant locusrevealed that the disrupted gene was the previously identifiedGIGANTEA gene (Fowler et al., 1999). Since the mutant wasidentified from an activation-tagged pool of mutants, Huqet al. (2000b) tested whether the expression profiles ofGIGANTEA or any other neighbouring genes was affected ingi-100. It was shown that the insertion results in a truncationof the GIGANTEA transcript, with only marginal effects ontranscriptional activities of neighbouring ORFs. By contrastto previous data indicating that GIGANTEA is a membraneprotein (Fowler et al., 1999), Huq et al. (2000b) showedconclusively, using GIGANTEA/GUS fusions, thatGIGANTEA is constitutively nuclear localized. Takentogether these data indicate that GIGANTEA is involved inthe nuclear localized phyB-signalling pathway.

PhyA, phyB and cryptochrome components Due to theinteraction between the phyA and phyB signalling pathwaysit is not surprising that there are mutants that show aphenotype in both red and far-red light. To date there are fourdescribed mutants that show red and far-red light inducedphotomorphogenic phenotypes, pef1, psi2, dfl1, and shl(Ahmad & Cashmore, 1996; Genoud et al., 1998; Nakazawaet al., 2001; Pepper et al., 2001).

pef1 shows partial insensitivity towards red and far-red lightsuggesting that PEF1 may represent a lesion in an early stepof the phytochrome signal transduction pathway which mayalso be indicative of nuclear localization. This however,remains to be shown.

The second dual phyA/phyB phytochrome signallingmutant isolated is psi2. psi2 was identified based on elevatedactivity of a chlorophyll a/b binding protein-luciferase(CAB2-LUC ) transgene in Arabidopsis (Genoud et al., 1998).This mutant shows hypersensitive induction of light-regulated genes in the VLF range of red light and a hypersen-sitive hypocotyl growth response towards high fluence red lightirradiation. Double mutant analysis further demonstratedthat the psi2 phenotype is dependent on both phyA and phyB.Surprisingly, at high fluence rates, psi2 shows light-dependentdevelopment of spontaneous necrotic lesions. Recently, it

has also been shown that the putatively disrupted gene inpsi2 is constitutively and uniformly distributed in the nucleus(S. G. Møller and N. H. Chua, unpublished). This suggeststhat PSI2 may act as a nuclear negative component of phyAand phyB signalling.

dfl1 exhibits hypersensitivity in terms of hypocotyl growthunder blue, red and far-red light conditions but also showsauxin related phenotypes such as altered lateral root number(Nakazawa et al., 2001). DFL1 encodes a GH3 homologproviding a link between light signalling and auxin responses(Section V/I).

Arabidopsis seedlings containing mutant shl alleles showenhanced hypocotyl growth inhibition in red, far-red, blue,and green light over a range of fluences indicating that theSHL proteins act as negative regulators of photomorphogenicresponses in a downstream signalling cascade shared byCRY1, PHYA, and PHYB and possibly CRY2, PHYC,PHYD, and PHYE (Pepper et al., 2001). However, themolecular evidence for this remains to be shown (Fig. 2).

3. cop/det/fus

The 11 recessive cop/det/fus (constitutive photomorphogenesis/de-etiolated/fusca) mutants of Arabidopsis resemble light-grown seedlings when grown in darkness, are pleiotropic innature and were identified from a number of genetic screens(Chory et al., 1989; Deng et al., 1991; Wei & Deng, 1992;Misera et al., 1994). As for light-grown wild type seedlings,cop/det/fus mutants exhibit hypocotyl growth inhibition, opencotyledons, and express light-regulated genes in darknessimplying that the disrupted proteins act as negativecomponents of light signal transduction. To date 5 cop/det/fusmutant loci have been cloned and include COP1, FUS2/DET1, COP8, COP9, COP11, and FUS5 (Schwechheimer &Deng, 2000).

Cop1 The cop1 mutant was isolated back in 1991 (Denget al., 1991) and COP1 was the first cop/det/fus mutant locusto be cloned and fully characterized (Deng et al., 1992).COP1 encodes a soluble protein of 76 kDa and can bedivided into three structural domains: an N-terminalzinc-finger domain, a putative coiled-coil domain and aC-terminal WD-40 repeat domain with homology to the β-subunit of trimeric G-proteins. Several recessive cop1 mutantshave been identified which have highlighted the importanceof the different protein domains and also enabled furtheranalysis into COP1 function beyond the seedling stage. Forinstance, in weak cop1 alleles both the phyA-mediated end-of-day far-red response and the shade avoidance responseare impaired (Deng et al., 1991; McNellis et al., 1994). Inaddition, weak cop1 mutants flower early under short dayconditions and also flower in the dark (McNellis et al., 1994).

COP1 is not regulated at the transcript or protein level(Deng et al., 1992) but at the level of subcellular localization.



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In darkness COP1 accumulates in the nucleus but is excludedwhen transferred to light. The cytoplasmic localization of COP1is probably mediated through a cytoplasmic localization orretention signal, which neutralizes the COP1 NLS in a light-dependent manner (von Arnim & Deng, 1996; Stacey & vonArnim, 1999). Moreover, using various photoreceptormutants it has been shown that the subcellular localizationpatterns are mediated by phyA, phyB and CRY1 implyingthat COP1 acts downstream in at least three different lightsignalling pathways (Osterlund & Deng, 1998). As for thephytochromes and LAF1, COP1 localizes to subnuclear fociboth in onion epidermal cells and in transgenic Arabidopsisplants (Ang et al., 1998; von Arnim et al., 1998) and moredetailed analysis has shown that a 58 amino acid stretchbetween residues 120–177 confer speckle formation. Interest-ingly, mutations in the C-terminal WD-40 repeat domain arephenotypically lethal and result in loss of speckle formationimplying that COP1 accumulation at subnuclear sites has afunctional role (Stacey & von Arnim, 1999).

Another regulatory function of COP1 is mediated byinteractions to other nuclear proteins and several have beenisolated including CIP1, CIP4 and CIP7 (Matsui et al., 1995;Yamamoto et al., 1998; Yamamoto et al., 2001). CIP1 wasisolated using biotinylated COP1 as a probe to screen anArabidopsis expression library. CIP1 encodes a cytoplasmicprotein probably associated with cytoskeletal structures in thehypocotyl and cotyledons and it is possible that CIP1 regu-lates the nucleo-cytoplasmic partitioning of COP1 (Matsuiet al., 1995). Conversely, CIP4 is a nuclear protein and can actas a transcriptional activator required for the promotion ofphotomorphogenic responses (Yamamoto et al., 2001). CIP4transcript levels are induced by light, regulated by COP1 andCIP4 deficient plants show elongated hypocotyls in responseto light treatment. CIP7 is also a nuclear protein shown to actas a transcriptional activator (Yamamoto et al., 1998) and asfor CIP4, CIP7 expression is induced by light and repressedby COP1 in darkness. Furthermore, CIP7 antisense plantsshow reduced expression of light-regulated genes but in

Fig. 2 Phytochrome signalling in the nucleus. The figure illustrates the multitude of known phytochrome pathways and interactions that take place in the nucleus. PhyA and phyB are translocated to the nucleus in response to light where they interact with various signalling components. Note that phyA also interacts with CRY1 and that not all signalling components interact with the photoreceptors. Solid arrows represent demonstrated steps whilst dashed arrows represent speculative steps.



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contrast to CIP4 antisense plants exhibit no hypocotyl growthinhibition defects. This clearly implies that COP1 can inter-act with a spectrum of proteins, which in turn regulate differ-ent light-dependent physiological responses.

Interestingly, it has been shown that the signallingmechanism of Arabidopsis CRY1 and CRY2 are mediatedby the C-terminus and that plants overexpressing thesedomains exhibit a COP1 phenotype (Yang et al., 2000).This prompted Deng and colleagues (Wang et al., 2001) toexamine whether COP1 physically interacts with CRY1 andCRY2 and indeed it has now been shown that CRY1 andCRY2 repress COP1 activity via direct physical interactions.

The best studied COP1 interacting partner is thebZIP transcription factor HY5 (Oyama et al., 1997). By con-trast to COP1, HY5 localizes constitutively to the nucleus(Chattopadhyay et al., 1998) but is more abundant in thelight than in darkness. Using GFP/HY5 fusion proteins it hasalso been shown that COP1 can recruit HY5 to subnuclearfoci demonstrating in vivo physical COP1/HY5 interactions(Ang et al., 1998). Interestingly, HY5 is subject to dark-specificdegradation by the 26S proteasome and physical interactionbetween HY5 and COP1 is necessary for degradation to occur(Osterlund et al., 2000). These findings imply that HY5abundance is regulated by nuclear COP1. Moreover, COP1 isa RING finger protein and it is possible that COP1 may assistin the ubiquitin-mediated degradation of HY5 by the 26Sproteasome (Lorick et al., 1999) and this will be discussed inmore detail in Section III/4.

Cop9 COP9 is part of a 450–600 kDa protein complexconsisting of a number of subunits which are not present incop9 mutants nor in several other cop/det/fus mutants (Weiet al., 1994; Chamovitz et al., 1996). Some of the genesencoding these subunits (CSNs) have been cloned includingCOP9/CSN8, COP8/CSN4, COP11/CSN1, and FUS5/CSN7 whilst some still remain to be isolated (FUS8, FUS11,FUS12). The finding that the loss of one subunit results in theloss of the entire protein complex has enforced the notion thatthe CSNs are involved in the COP9 complex assembly (Kwoket al., 1998). The protein complex has now been named theCOP9 signalosome (Deng et al., 2000), which is thoughtto act as the ‘lid’ subcomplex (19S regulatory particle) ofthe 26S proteasome (Chamovitz et al., 1996). The COP9signalosome subunits have orthologues in other eukaryotesand both the mammalian and Drosophila COP9 signalosomeshave been copurified with the 26S proteasome based on theirhomologies to the plant COP9 complex (Wei & Deng, 1998;Wei et al., 1998).

The COP9 signalosome subunits have been shown to con-tain protein domains (PCI and MPN) present in the subunitsof the 26S proteasome and the translation initiation factoreIF3 (Aravind & Ponting, 1998; Hofmann & Bucher, 1998).Although the functions of these domains remain unknownthe similarity between the subunits of the COP9 signalosome

and the 26S proteasome is astonishing with all the COP9CSNs showing extensive homology to the subunits of the 19Sregulatory particle (Glickman et al., 1998; Henke et al., 1999,Wei & Deng, 1999). The 26S proteasome is probably themain protein degradation apparatus in the cell (Fig. 3) andconsists of a central 20S proteasome core complex togetherwith a 19S regulatory particle, which act as lids (Hershko &Ciechanover, 1998). Due to the extensive homologies itpossible that the COP9 signalosome can interact with the 20Sproteasome core complex forming an alternative type ofproteasome involved in the regulation of photomorphogenicresponses (Section III/4). Although this remains to bedemonstrated, Deng and colleagues have shown that ArabidopsisCSN1 can interact with the 19S regulatory AtS9 subunit(Kwok et al., 1999) suggesting that the COP9 signalosomeforms a supercomplex with the 26S proteasome (Fig. 3). The26S proteasome and its implication in regulating photo-morphogenic responses will be discussed in more detail below.

4. Degradation

It is becoming increasingly evident that the regulation ofcellular events is not only controlled by gene expression andprotein synthesis but also by targeted protein degradation. Inplants, protein degradation has been shown to be involved innumerous processes including cell death, circadian rhythms,defence responses, photosynthesis and photomorphogenesis.Here we will describe recent findings demonstrating the roleof proteolysis as part of photomorphogenesis, with particularemphasis on ubiquitination and SUMOlation.

Ubiquitin and the 26S proteasome Ubiquitin is a conserved76 amino acid polypeptide that marks proteins fordegradation by the multisubunit 26S proteasome (Hershko& Ciechanover, 1998; Pickart, 2001). The process ofubiquitination, i.e. the marking of proteins destined fordegradation, consists of several critical steps (Hershko, 1996).Briefly, the first step involves the activation of the C-terminalglycine residue of ubiquitin by an ATP-dependent reactioninvolving a specific ubiquitin-activating enzyme (E1). Onceactivated, the ubiquitin is transferred to an active site cysteineresidue of an ubiquitin-carrier protein (E2). Following this,a ubiquitin protein ligase (E3) catalyses the formation of anamide isopeptide linkage between the ubiquitin C-terminusand lysine residues on the protein to be degraded. Subsequ-ently, activated ubiquitin is added to the previously conjugatedubiquitin leading to the formation of a polyubiquitin chain.Once containing polyubiquitin chains, the 26S proteasomecomplex usually degrades the proteins by an ATP-dependentreaction (Coux et al., 1996).

The 26S proteasome is a remarkable protein complex con-sisting of the 20S core particle, which harbours the proteolyticsites, and the 19S regulatory particle, which make up the ‘lids’(Fig. 3). The 19S regulatory particle contains a multitude of



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ATPases and other subunits probably involved in the specificaction of the 26S proteasome. The precise mode of action ofthe 26S proteasome is not well understood, however, recentevidence has started to unravel the functional role of theCOP9 signalosome/19S regulatory particle in plant signallingevents as discussed in sections below.

The E1-E2-E3 cascade is somewhat hierarchical. In mostorganisms there are only one or very few E1 enzymes that areresponsible for all ubiquitin activation and because of this thespecificity of E1s is very low (McGrath et al., 1991). In con-trast, there are several E2s, 36 isoforms already identified inArabidopsis (Callis & Vierstra, 2000), which have more spe-cialised functions depending on the cellular localization andtheir interaction with various E3s. Similarly, the E3 proteinsinteract directly with the proteins to be degraded and aretherefore very diverse with varying specificities. E3s can befurther divided into HECT-domain proteins, Ubr-1-like E3s,APC (anaphase-promoting complex), monomeric RING-H2

E3s and SCF (Skp1/cullin/F-box-type) E3s. Although allclasses have been found in plants (Callis & Vierstra, 2000),the involvement of SCF E3s in photomorphogenesis and inthe interaction with auxin signalling has recently beendemonstrated.

The SCF complex was originally identified in yeast andconsists of the subunits Skp1, Cdc53 or cullin and an F-boxprotein, all making up the ubiquitin ligase protein complex(Patton et al., 1998). The F-box protein gives the SCF-E3complex its specificity, acting as the receptor, bringing E2s inclose proximity with the ubiquitinated proteins. Indeed sev-eral F-box proteins can interact with identical Skp1 and cullinsubunits (Patton et al., 1998). The first SCF complex identi-fied in plants was SCFTIR1 (Transport Inhibitor Response 1),which is most likely involved in the degradation of inhibitorsof the auxin response (Gray et al., 1999). It is possible, thatthe substrates for SCFTIR1 are the very unstable nuclearlocalized Aux/IAA proteins (Section V/1), which themselves

Fig. 3 Specific degradation of light signalling components by ubiquitination and the 26S proteasome. COP1 translocates to the nucleus in darkness where it recruits HY5 for ubiquitination by the SCF complex and subsequent degradation by the 26S proteasome. EID1 may function as an F-box protein in the SCF complex involved in the degradation of phytochrome signalling components (SP). Also, phyA itself may be degraded by ubiquitination and the 26S proteasome. Note that the 19S regulatory complex of the 26S proteasome is in fact the COP9 complex. Solid arrows represent demonstrated steps whilst dashed arrows represent speculative steps.



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can act as repressors of auxin-regulated genes (Ulmasov et al.,1997). Indeed mutational analysis have identified IAAdomains that disrupt the auxin response by increasing proteinstability suggesting that the specific degradation of Aux/IAAproteins are important for a proper auxin response (Leyser,1998; Gray & Estelle, 2000). More recently, two SCF-typeE3s were isolated involved in circadian responses. Deficiencyof the Arabidopsis proteins ZTL (Somers et al., 2000) andFKF1 (Nelson et al., 2000) result in impaired circadianrhythm responses and both proteins have been shown tocontain an F-box implying that they are components of anSCF complex. This further suggests that ubiquitin-dependentdegradation is important for correct circadian responses.

In terms of phytochrome signalling it has for a long timebeen speculated, and almost assumed, that phyA is subjectedto ubiquitin-mediated degradation in its Pfr form ( Jabbenet al., 1989; Fig. 3). This has now been shown to be the casedemonstrating that both the N-terminal and C-terminal partsof phyA are important for Pfr ubiquitination and breakdown(Clough et al., 1999). However, only very recently was itshown that phytochrome signalling intermediates are indeedinvolved in ubiquitin-mediated proteolysis. The phyA-specific hypersensitive mutant eid1 has been isolated(Büche et al., 2000) and as discussed in Section III/2, it hasbeen shown that EID1 contains homology to F-box proteins(Dieterle et al., 2001). This does suggest that EID1 may interactwith other members of the SCF complex and indeed Dieterleet al. (2001) demonstrated that EID1 can interact with ASK1and ASK2, two Arabidopsis homologs of the yeast Skp1 pro-tein (Gray et al., 1999). This interaction does seem specificin that a mutant variant of EID1, lacking the F-box, can notbind ASK1 and ASK2 (Dieterle et al., 2001). It is tempting tospeculate that EID1 may be involved in ubiquitin-mediatedprocesses in response to light. Since phyA levels are not alteredin the eid1 mutant, EID1 could be involved in the degrada-tion of positively acting phyA signalling components (Fig. 3)where the F-box moiety of EID1 confers light-specific SCFligase activity.

Apart from the HECT-domain E3s (Bates & Vierstra,1999), the members of the different E3 classes harbour a sub-unit with a RING-finger motif. This motif is thought to inter-act with the E2 enzyme and by doing so mediating thetransfer of ubiquitin from E2 to the protein substrate. This isof course not the case for SCF as discussed above. COP1 con-tains a RING-finger motif suggestive of E3 activity and it hasbeen shown by Deng and colleagues that COP1 most prob-ably targets HY5 for degradation in darkness (Fig. 3; vonArnim & Deng, 1994; Osterlund et al., 2000). In darkness,COP1 translocates to the nucleus and interacts with HY5, abZIP protein that activates transcription of light-regulatedgenes in the light, and this interaction results in HY5 degra-dation (Fig. 3). Conversely, in the light COP1 is excludedfrom the nucleus resulting in HY5 stabilisation ultimatelyleading to transcription of light-regulated genes. This point is

further underlined by the fact that HY5 is not degraded inCOP9 signalosome mutants (see below) and it has been dem-onstrated that COP1 does not translocate to the nucleus inthese seedlings (Osterlund et al., 2000). An additional HY5control point involves a kinase, possibly a caseine kinase,which phosphorylates HY5 in darkness resulting in a lessactive form, that also binds COP1 less tightly (Hardtke et al.,2000). HY5 activity is therefore regulated by two intertwinedmechanisms strongly dependent on nuclear COP1 activity.

As discussed previously, COP9 is part of a large proteincomplex (COP9 signalosome), consisting of several subunits,which are lacking in cop9 and in many of the constitutivelyphotomorphogenic cop/det/fus mutants. The eight subunits ofthe COP9 signalosome are all paralogous to the subunits ofthe 19S regulatory particle and therefore the COP9 signa-losome probably interacts with the 20S core particle to formalternative 26S proteasome complexes. Here there are twopossibilities: the COP9 signalosome interacts with the 20Score particle directly forming a COP9 proteasome; or theCOP9 signalosome interacts with the 19S regulatory particleforming a 26S proteasome/COP9 signalosome supercom-plex. Both are possible and certainly not mutually exclusive.The characteriztics of the various COP9 signalosome sub-units (CSNs) are indeed interesting (cf. Wei & Deng, 1999)and one subunit, which is different from the rest, CSN5,not only acts as a COP9 signalosome subunit but also as afunctional monomer (Kwok et al., 1998). For instance, inArabidopsis COP9 signalosome mutants there is no proteincomplex but CSN5 monomers can be detected. CSN5 hasbeen shown to affect the nucleo/cytoplasmic distribution ofseveral interacting proteins ultimately mediating proteolysisimplying that CSN5 does not only act as part of the COP9signalosome. However, it still remains to be shown whetherthe monomeric activity of CSN5 is related to the COP9signalosome.

Although it is clear that the COP9 signalosome is part ofthe 26S proteasome proteolytic pathway involved in the reg-ulation of photomorphogenic responses, cop/det/fus mutantsare very pleiotropic suggesting that the affected proteins areinvolved in other developmental processes. Using transgenicplants with reduced CSN5 levels, Deng and colleagues(Schwechheimer et al., 2001) found that COP9 signalosomedeficiency results in auxin related phenotypes typical of auxinresponse mutants such as axr1–3 and tir1–1 (Lincoln et al.,1990; Gray et al., 1999). As described earlier, auxin responsesare controlled by the SCFTIR E3 ubiquitin ligase, whichdegrades Aux/IAA proteins (Gray et al., 1999; Gray & Estelle,2000) and indeed decreased degradation of the pea IAAprotein PSIAA6 is seen in CSN5 transgenic plants comparedto wild type (Schwechheimer et al., 2001). In addition, thecullin or Cdc53 subunit of various SCF complexes has beenshown to interact with the COP9 signalosome (Lyapina et al.,2001) and it was therefore tempting to suggest that maybeSCFTIR interacts with the COP9 signalosome. This is indeed



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the case (Schwechheimer et al., 2001) suggesting that theCOP9 signalosome may be important in mediating E3 ubiq-uitin ligase responses. It is of course tempting to speculate thatthe pleiotropic nature of cop/det/fus mutants is a result ofimpairment of several E3 ubiquitin ligase functions.

As part of a normal auxin response, the ubiquitin-relatedprotein RUB1, becomes conjugated to the atCUL1 subunitof SCFTIR (del Pozo & Estelle, 1999) and it has been shownthat the COP9 signalosome promotes RUB1 deconjugation(Lyapina et al., 2001). It is possible that RUB1 modificationsmay affect COP9/SCFTIR interactions thereby regulatingthe nucleo/cytoplasmic partitioning of SCFTIR. This wouldbe analogous to the hypothesis that COP9 signalosome isrequired for the light-dependent subcellular distribution ofCOP1 (von Arnim & Deng, 1994). Once again the import-ance of controlled subcellular distribution patterns on majorcellular processes is clear and it remains an exciting challengeto dissect the network as part of developmentally controlledproteolytic events.

SUMO There is a growing family of ubiquitin-relatedproteins and one of these is the small-ubiquitin-related-modifier, or SUMO. SUMO resembles ubiquitin in manyways in terms of structure, in its ability to ligate to proteinsand also in the mechanism of conjugation (Melchior, 2000).However, in contrast to ubiquitination, SUMOlation doesnot lead to protein degradation but rather protection. It hasindeed been speculated that SUMO acts as an antagonist toubiquitin and by doing so adding another level of control andcomplexity in the degradation of specific proteins. HowSUMO exerts its role is largely unknown but there is evidencethat SUMOlation regulates both protein–protein interactionsas well as subcellular localization patterns. As described inSection III/2, the phyA-specific signalling component LAF1encodes a nuclear two-repeat MYB protein containing aputative SUMOlation site (Ballesteros et al., 2001). AlthoughSUMOlation consensus sites are minimal (KKQE),Ballesteros et al. (2001) demonstrated that by mutating theconserved second lysine residue to an arginine the recruitmentof LAF1 to subnuclear foci was abolished suggesting a role forSUMO in this process. If this is indeed the case it is possiblethat SUMO conjugation recruits LAF1 to subnuclear foci, asseen for HIPK2 (Kim et al., 1999), and by doing so bringsLAF1 in close proximity to other MYB factors or directly topromoter elements of light-regulated genes. Transcriptionfactors/activators are often controlled by degradation and it ispossible that SUMOlation and speckle formation protectsLAF1 from the 26S proteasome. It would indeed beinteresting to learn whether LAF1/SUMO subnuclear speckleformation and LAF1 stability is affected in COP9signalosome mutants.

The identification of specific degradation of proteins inresponse to both developmental and environmental cues hasadded to the complexity of cellular regulation. Although

recent research has provided exciting findings linking phyto-chrome signalling to protein degradation there still remains alot of uncharted territory. The recent identification of 37multiubiquitin chain assembly factor (E4) proteins contain-ing a U-box motif (Azevedo et al., 2001) and the finding thatArabidopsis has 337 F-box containing and 358 RING fingermotif containing proteins (Estelle, 2001) does suggest that wehave a long way to go before we understand how phyto-chrome signalling and protein degradation are functionallyconnected.

Clearly, there has been substantial recent activity centredon the nucleus. The isolation of phytochrome interactingproteins and the elucidation of the light-induced nucleartranslocation of phyA and phyB has certainly created a lotof excitement. These data suggest that at least some of thephytochrome signalling pathways are short and exclusivelynuclear localized. In addition, the characterization of EID1and LAF1 has indicated the involvement of protein degrada-tion and protection as being part of the nuclear phytochrome-signalling network. Also, recent studies into the structure ofthe COP9 signalosome have unveiled new insight into therole of protein degradation as part of light signalling. To date,the available data has probably only touched the tip of the ice-berg regarding phytochrome signalling and regulation in thenucleus. One would anticipate the future isolation of phyto-chrome interaction proteins involved in cytoplasmic reten-tion, in the import/export process itself as well as componentsof the ubiquitin and SUMO pathways. Regardless, it will nowbe important to assemble all the ‘nuclear’ data and to expandthe findings and interpretations to the whole cell level.

IV. The cytoplasm

Traditionally, signal transduction pathways were generallyviewed as signal perception via plasma membrane-boundreceptors followed by signal relay through the cytoplasmultimately resulting in micro or macro morphological changesunderpinned by alterations in nuclear gene expression. Thevital role of the nucleus in phytochrome signal transductionis undisputable, however, the cytosol and the organellessuspended in it clearly form integrated compartments ofthis network. For example, the cytosolic retention of phyAand phyB in darkness, the isolation of cytosolic phyto-chrome interacting proteins, the possible involvement ofheterotrimeric G-proteins during early steps of phytochromesignalling and the involvement of cytoplasmic organellesin photomorphogenic responses all demonstrate that thenucleus does not act alone in light signal transduction.

1. The cytosol

Dissecting secondary messengers: Ca2+/CaM, cGMP and heterotrimeric G-proteins The activation of heterotrimericG-proteins and subsequent signal relay via secondary



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messengers have been implicated in signal transductioncascades for a very long time. To this end Chua and colleagues(Neuhaus et al., 1993; Bowler et al., 1994) investigatedwhether this is the case for phytochrome signal transduction.Using a microinjection approach it was demonstrated thatputative heterotrimeric G proteins might act as an upstreamcomponent of the phyA signal transduction pathway (Bowleret al., 1994; Neuhaus et al., 1993). Moreover, it was shownthat at least three different pathways, involving calciumand/or cGMP, controlled phyA responses downstream of theG protein complex. The data indicate that: cGMP stimulateschalcone synthase (CHS ) expression and anthocyaninbiosynthesis; that Ca2+/calmodulin (CaM) activateschlorophyll a/b binding protein (CAB ) expression and partialchloroplast development; and that both cGMP and Ca2+

activates photosystem I (PSI) genes such as ferredoxinNADP+ oxidoreductase (PETH ) expression to produce fullyactive chloroplasts. Using agonists and antagonists to disruptsignal flow, cGMP and Ca2+/CaM were shown to exhibitinterpathway regulation where downstream regulators of onepathway can negatively regulate another and vice versa, acontrol mechanism termed reciprocal control (Bowler et al.,1994). Although this approach yielded insights into phyAsignal transduction, in terms of cytoplasmic signal relaymediated by G-proteins and secondary messengers, there is todate very little genetic evidence supporting these findings.Despite this and as discussed in Section II/4, there is newevidence suggesting the involvement of heterotrimeric G-proteins in cytoplasmic retention and release mechanisms(Santagata et al., 2001) implying that cytoplasmic G-proteinsmay be involved in the cytoplasmic retention of photo-chemically inactive phytochrome. Direct genetic evidence ofheterotrimeric G-protein involvement in phytochrome signaltransduction came however, with the very recent finding thatoverexpressed heterotrimeric G-protein α-subunit (Gα)results in decreased hypocotyl cell elongation in response tolight (Okamoto et al., 2001). Deng and colleagues (Okamotoet al., 2001) have shown that this hypersensitivity occurs inresponse to far-red, red, and blue light irradiation implying ageneral spectrum-wide role for Gα. However, more detailedgenetic studies demonstrate that the Gα-induced phenotyperequires functional phyA and FHY1 (Whitelam et al., 1993)but not FHY3 (Whitelam et al., 1993) or FIN219 (Hsiehet al., 2000). Since FHY1 has been shown to represent abranch-point in the phyA signalling pathway (Barnes et al.,1996), these results suggests that Gα is only involved incertain branches of phyA signalling. The possible mechanismof heterotrimeric G-protein activation via phytochrome stillremains to be unravelled. In Dictyostelium, G-proteins areactivated by NDPK (Bominaar et al., 1993) and it is temptingto speculate that either NDPK2 activates Gα after it has beenactivated by phyA or that Gα interacts with phyA directly.In addition, the latest finding that PRA2, a small G-proteinin pea, may act as a cross-talk mediator between light and

brassinosteroid signalling in plants is exciting (Kang et al.,2001).

To date, the involvement of the secondary messengers Ca2+

and cGMP in phytochrome signalling has little supportgenetically. This may partially be due to the presence of sucha large cellular Ca2+ signalling network making isolationof ‘Ca2+’-proteins specifically involved in phytochromeresponses difficult. However, it is assumed that protein kinasesand phosphatases, being interpreters of Ca2+ signals, will beisolated from either forward or reverse genetic screens. To thisend Lin and colleagues (Guo et al., 2001) isolated an Arabi-dopsis photomorphogenic mutant, sub1, which is hypersensi-tive towards both blue and far-red light irradiation. Geneticstudies further showed that SUB1 functions as a signallingcomponent downstream of CRY1 and CRY2. Conversely, theactivity of phyA is not dependent on SUB1 indicating thatSUB1 may act as a modulator of phyA signalling. The cloningof the mutant lesion in sub1 revealed that the disrupted geneencodes a novel protein containing EF-hand-like motifssuggestive of Ca2+ binding and indeed heterologouslyexpressed SUB1 can bind Ca2+. Moreover, SUB1 localizes tothe cytoplasm apparently enriched in the nuclear periphery.Together with the proposed role of SUB1 being a crypto-chrome signalling component that modulates phyA signal-ling, SUB1 probably defines a point of cross-talk betweencryptochrome and phyA and may play a role in monitoringlight-induced cytoplasmic changes in ion homostasis.

To try to dissect the involvement of Ca2+ in photomorpho-genic responses it may be of value to use intracellular-directedCa2+ probes such as aequorin (Rizzuto, 2001) or CaMeleon(Miyawaki et al., 1997) to monitor cytosolic subregions andmicrodomains for changes in Ca2+ concentrations in responseto different light treatments.

Pat1 The first cytosolic phytochrome signalling componentto be isolated was PAT1 (Bolle et al., 2000). pat1 was isolatedin a genetic screen showing a far-red insensitive phenotypealmost as strong as that observed for fhy1 and fhy3. PAT1 is amember of the GRAS protein family, which include memberssuch as SCR (Di Laurenzio et al., 1996), GAI (Peng et al.,1997a) and RGA (Silverstone et al., 1998), all shown to beinvolved in plant signal transduction. The T-DNA insertionin pat1 leads to the production of a truncated protein andthe pat1 mutant phenotype can be recapitulated byoverexpression of the truncated PAT1 protein in wild-typeseedlings. These data indicate that the truncated version ofPAT1 acts as a dominant negative protein and since PAT1does not interact with the C-terminal region of phyA or phyB(S. G. Møller and N. H. Chua, unpublished), it suggests thatPAT1 may interact with other phyA signalling components.In both onion epidermal cells and in transgenic plants, PAT1/GFP fusions localize constitutively to the cytoplasm,suggesting that PAT1 is involved in very early signallingsteps even before phyA is imported into the nucleus.



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Fin219 In a suppressor screen developed to identify genesinvolved in the light inactivation of COP1, a repressor ofphotomorphogenic development, Deng and colleagues(Hsieh et al., 2000) identified a suppressor mutation thatshowed loss of hypocotyl growth inhibition in response tofar-red light irradiation. The mutant locus, fin219, wascloned and the disrupted gene encodes a 575 amino acidconstitutively cytoplasmic protein with very high similarity tothe GH3 family of proteins. The GH3 protein first identifiedin soybean (Hagen et al., 1984) as being auxin inducible andFIN219 shows the same auxin inducibility. To verify theinvolvement of FIN219 in far-red light signalling, it wasfurther shown that FIN219 overexpression results in far-redinduced hypersensitivity. In addition, FIN219 interactsgenetically with FHY1, which has recently been shown to bea nuclear protein, underlining the interaction between thenucleus and cytoplasm as part of light signal transduction.FIN219 may be involved in the far-red light mediatedinactivation of COP1 and although fin219 has no obviousauxin phenotype it may represent an intersection betweenlight and auxin signal transduction.

Pks1 The first phytochrome kinase substrate identifiedwas phytochrome kinase substrate 1 (PKS1; Fankhauser et al.1999). PKS1 was isolated in a yeast two-hybrid screen usingthe C-terminal domain of phyA as bait and it wassubsequently shown that PKS1 binds to both the phyA andphyB C-terminal regions. Unlike PIF3 and NDPK2, PKS1binds with equal apparent affinity to both the Pr and Pfrforms of phyA and phyB indicating no photo-selectivity ofphytochrome binding. PKS1 encodes a 439 amino acid novelprotein with no identifiable functional motifs. However,purified oat phyA was able to phosphorylate a GST-PKS1fusion protein and experiments showed that PKS1 was abetter substrate for the Pfr kinase form than for the Pr kinaseform of phyA. Also, immunoprecipitation experiments,using phosphatase treatments, showed that PKS1 is aphosphoprotein in vivo. In addition, both phytochromeautophosphorylation and PKS1 phosphorylation isstimulated approx. 2.5-fold upon red light irradiationdemonstrating that phyA shows light-regulated autophos-phorylation and PKS1 phosphotransferase/phosphorelayactivity. Since PKS1 is shown to localize to the cytoplasm itis possible, as discussed in Section II/4, that PKS1 is involvedin the cytoplasmic retention of phyA or phyB in the cyto-plasm (Fig. 2). PKS1 may retain phyA and phyB in aninactive state in the cytoplasm, however, this is difficult toreconcile given the lack of photoreversible PKS1/phytochromebinding characteriztics. To determine the role of PKS1in photomorphogenesis, Chory and colleagues (Fankhauseret al., 1999) generated transgenic seedlings overexpressingPKS1 and showed that in red light PKS1 overexpressionresults in partial insensitivity whilst far-red or blue light has noeffect. This suggests that PKS1 is a negative component of

phyB signalling probably influencing phyB kinase activity ormaybe its subcellular partitioning pattern acting as a retentionprotein. It will indeed be interesting to learn whether a PKS1loss-of-function mutant has altered phyA or phyB nucleartranslocation patterns.

2. Is phytochrome a kinase?

At this point we should address the old issue of whetherphytochrome actually is a kinase. Historically, it was proposedby Borthwick & Hendricks (1960), that phytochromes areindeed light-regulated enzymes. Quail and colleagues showedthat phytochrome was a phosphoprotein by in vivo 32Plabelling experiments in 1978, but it was almost a decade laterthat purified fractions of phytochrome were shown to haveprotein kinase activity (Wong et al., 1986, 1989; McMichael& Lagarias, 1990; Hamada et al., 1996). However, it wasunclear whether the kinase activity was due to phytochromeitself or a copurified protein with kinase activity. This concernwas based on studies describing loss of kinase activityfollowing more rigorous downstream purification methods(Grimm et al., 1989; Kim et al., 1989). Whether phytochromehas bona fide kinase activity and if so what type of kinase isstill somewhat unclear (Elich & Chory, 1997; Cashmore,1998; Fankhauser & Chory, 2000).

Bacterial ‘two component’ phosphorelay systems Work onbacteriophytochromes, phytochrome-like photoreceptorsfound in several bacteria, may provide relevant clues aboutphytochrome action. The first bacteriophytochromeidentified, RcaE (response to chromatic adaptation E)from the cyanobacterium Fremyella diplosiphon (Kehoe &Grossman, 1996), shows homology at the N-terminus tothe chromophore-binding pocket of phytochromes and iscapable of binding a variety of bilins generating red/far-redphotoreversible chromoproteins. Significantly, RcaE containsa region of homology to two component histidine kinasesystems, suggesting that bacterial photoreceptors cantransduce a light signal into a phosphorelay cascade.

Sequence searches with other bacteria, both photosyn-thetic and nonphotosynthetic, have revealed additionalphytochrome-like sequences showing similarities to plantphytochromes at the amino terminus and a histidine kinasedomain (Vierstra & Davis, 2000).

The Synechocystis phytochrome Cph1 has also been shownto be the photoreceptor of the two-component light sensorysystem (Hughes et al., 1997; Yeh et al., 1997; Park et al.,2000), exhibiting histidine kinase activity in vitro. The secondstep in the phosphorelay reaction is the transfer of phosphateto an aspartic acid residue in the Rcp1 response regulator.Cph1 also shows red/far-red photoreversible absorbanceproperties indicative of phytochrome activity. Interestingly,the degree of histidine autophosphorylation observed in vitrois greater in the Pr form compared to the Pfr form. It has been



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suggested that the Pr form has kinase activity whilst the Pfrform may act as a protein phosphatase (Hughes & Lamparter,1999).

Similarly, in a four-step phosphorelay model proposed byKehoe and Grossman (1997), RcaE is proposed to act as akinase in red light, phosphorylating RcaF, which then trans-fers the phosphate group to RcaC, whilst in green light RcaEappears to act as an RcaF phosphatase.

This dual activity of bacteriophytochromes is in sharp con-trast to plant phytochromes, where the Pr form is generallyconsidered to be inactive.

Sequence analysis shows that phytochrome seem to containtwo diverged histidine kinase related domains (HRKD) in atandem arrangement (Yeh & Lagarias, 1998). The firstHRKD domain also contains two motifs with homology toPAS (PER-ARNT-SIM) domains (Lagarias et al., 1995; Kay,1997). PAS domains are present in various signal transductionmolecules that sense environmental signals such us light, oxy-gen levels, and redox potential (Taylor & Zhulin, 1999). Theymay also mediate protein–protein interactions (See Fig. 1 fordetails of domains). Despite these sequence similarities, theproposal that higher plant phytochromes are light-activatedkinases remains controversial because the histidine residuescritical for kinase activity in most bacterial sensors are notconserved in all phytochromes (Shneider-PoetSchneider-Poetsch, 1992).

Autophosphorylation Purified oat phyA expressed inaccharomyces cerivisiae and Mesotaenium caldariorumphytochrome expressed in Pichia pastroris form activeholophytochrome and display autophosphorylation activityin a light-regulated fashion (Yeh & Lagarias, 1998). However,in contrast to earlier findings, this appears to be a serine/threonine kinase activity rather than histidine/aspartatekinase activity. Mass spectroscopy revealed that phyA hasthree phospho-acceptor sites, serine7, serine17 and serine598(McMichael & Lagarias, 1990; Lapko et al., 1997, 1999).Interestingly, serine7 phosphorylation is similar in both Prand Pfr, serine17 is phosphorylated in the Pr form by proteinkinase A in vitro whereas, serine598 is preferentiallyphosphorylated in the Pfr form. Additionally, when theamino acids shown to be important for histidine-kinaseactivity in prokaryotic systems are mutated there is no effecton phyA activity in transgenic plants (Boylan & Quail, 1996).This suggests that phytochrome A may be a serine/threoninekinase, that has diverged from a histidine kinase. Thissuggestion is not without precedent; other prokaryotic-likehistidine kinases found in eukaryotes have been shown tophosphorylate serine and threonine rather than histidine andaspartate (Popov et al., 1993; Harris et al., 1997). Fankhauserand Chory (1999), have also suggested that phytochromephosphorylation may proceed through less stable inter-mediates such as phospho-aspartate and phopho-histidine.

Taken together, it appears phytochrome is autophosphor-

ylated in both a light-dependent and a light-independentmanner, but what is the biological significance of this? Instudies where serine residues at the amino terminus of ricephyA were mutated to alanines an increase in biological activ-ity compared to wild type phyA can be observed (Stockhauset al., 1992; Jordan et al., 1997). From this observation it waspostulated that the light-independent phosphorylation ofserine7 down-regulates phytochrome activity.

Similarily, if serine598 is mutated to alanine, and themutated phytochrome is expressed in a phyA null back-ground, hypersensitivity to far-red light is observed; suggest-ing that serine598 phosphorylation can also negativelyregulate phytochrome activity (Park et al., 2000). Serine598 ispositioned in the hinge region between the chromophore-binding N-terminal domain and the C-terminal regulatorydomain (Fig. 1). In a model described in Park et al. (2000),serine/threonine phosphorylation is suggested to play a role inphotoinducible conformational changes of phytochrome andto modulate interdomain signalling.

In this model, serine598 phosphorylation would serve todesensitise Pfr, which could not then associate with phyto-chrome interacting factors (PIFs), whereas the unphosphor-ylated Pfr would be capable of binding PIFs. This is analogousto the desensitising mechanism for the rhodopsin photoreceptorin vision (Abdulaev & Ridge, 1998). It would be interestingto examine whether a serine598 to alanine substitution inphytochrome has an affect on nuclear transport vs. cytoplasmicretention. It should be noted that no phosphatase activity hasbeen associated with phytochrome and so a dephosphoryla-tion step would require an additional, as yet unidentified,factor. Although the available evidence does suggest thatphytochrome is capable of autophosphorylation, the questionof whether phytochrome shows specific kinase activity stillremains unresolved. Various phytochrome-specific signallingmutants and biochemical approaches do suggest that thismay be the case. However, most in vitro phytochrome kinaseassays described in the literature make use nonphysiologicalconditions in terms of incubation times, rendering the resultssomewhat difficult to interpret. If it is assumed that phyto-chromes do indeed have specific kinase activity it would beexpected that there are several proteins that are phosphor-ylated by phytochrome. Some of these have been discussedincluding PKS1, CRY1, CRY2, and NDPK2, however, therenow is recent evidence suggesting that the phytochrome–kinase boundary extends into other pathways such as auxinsignalling (Section V/1).

3. Chloroplasts

Chloroplasts are thought to have arisen from the engulfmentof free-living cyanobacteria-type organisms (Schwartz &Dayhoff, 1978). Although chloroplasts contain their ownautonomous genome the nuclear genome in a plant cellremains superior because it encodes most of the proteins



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(> 90%) necessary for chloroplast maintenance and function.An important light-dependent developmental pathway inplants involves the transition of nonphotosynthetically activeproplastids into photosynthetically active chloroplasts(Kendrick & Kronenberg, 1994), a process that requires thecoordinated expression of both plastidic and nuclear genes.This finely tuned developmental program requires precisecommunication between the nucleus and chloroplasts and itis now evident that the former senses the functional stateof the latter, via retrograde signalling, and orchestratessubsequent morphological and molecular manifestations(Oelmuller et al., 1986; Oelmuller, 1989; Susek et al., 1993;Lopez-Juez et al., 1998; Streatfield et al., 1999). Indeed ithas been shown that the herbicide Norflurazon, whichimpairs chloroplast functionality, results in reduced expressionof nuclear-encoded genes such as LHCB (chlorophyll a/b-binding protein) and RBCS (small subunit of ribulosebisphosphate carboxylase) (Oelmuller, 1989; Susek et al.,1993). The nature of the retrograde signalling pathway stillremains largely unknown. However, chlorophyll precursorshave been implicated and recent evidence has shown thatMg-protoporphyrin IX and protoporphyrin IX can act as asignal from the chloroplast to the nucleus.

Tetrapyrrole intermediates as retrograde signals Early studiesinto retrograde signalling suggested that chlorophyll precursors,such as protoporphyrin IX, could act as negative signallingmolecules in that they attenuate the expression of light-inducible nuclear genes in Chlamydomonas reinhardtii(Johanningmeier & Howell, 1984; Johanningmeier, 1988).Conversely, the Chlamydomonas brown mutants brs-1 andbrc-1, defective in Mg-protoporphyrin IX synthesis, areunable to induce nuclear heat shock protein (HSP70 ) geneexpression in response to light, suggesting that Mg-protoporphyrin IX may act as a positive retrograde signallingcomponent (Kropat et al., 1997; Kropat et al., 2000).Moreover, Mg-protoporphyrin IX-feeding experiments indarkness show that this tetrapyrrole can substitute for thelight signal in a dose-dependent manner as shown by thetransient increase in HSP70 expression (Kropat et al., 1997).Interestingly, protoporphyrin IX, protochlorophyllide andchlorophyllide could not act as signalling molecules, at leastfor HSP70 induction (Kropat et al., 1997; Kropat et al.,2000), suggesting that specific tetrapyrrole intermediates playdifferent roles as part of the retrograde signalling network.However, it is possible that nonphotosynthetic genes asmolecular probes may not represent the most accuratemeasure of retrograde signalling.

Using a combination of biochemical and molecular tech-niques Grimm and colleagues (Grimm, 1998) have unravellednumerous steps in the tetrapyrrole metabolic pathway (Grafeet al., 1999; Lermontova & Grimm, 2000; Papenbrock et al.,2000b, 2000a). For instance, it has been demonstrated that bydisrupting the Mg-chelatase protein complex, the branch-

point of tetrapyrrole biosynthesis, there is a reduction ofprotoporphyrin IX with a concomitant reduction in LHCBexpression (Papenbrock et al., 2000b; Papenbrock et al.,2000a).

gun mutants The genomes uncoupled (gun) mutants wereisolated (Susek et al., 1993) by exploiting the fact that in thepresence of Norfluorazon chloroplasts become photooxidisedresulting in the repression of nuclear genes encodingchloroplast proteins (Oelmüller, 1989). Chory and colleagues(Susek et al., 1993) screened a battery of transgenicArabidopsis plants containing the CAB promoter fused to ahygromycin-resistance gene in the presence of hygromycinand Norflurazon with the idea that if the retrograde signallingpathway is disrupted in some way, the damaged chloroplastswill not relay information back to the nucleus and CABexpression will remain high. Recently, Chory and colleagues(Mochizuki et al., 2001) cloned the disrupted gene in gun5and show that it encodes the Mg-chelatase H subunit locatedon the inner plastid envelope. In contrast to the studies ofHSP70 expression in Chlamydomonas (Kropat et al., 1997;Kropat et al., 2000), gun5 has reduced Mg-protoporphyrinIX and protoporphyrin IX levels. Taken together these datasuggests that the Mg-chelatase H subunit might monitor theflux through the chlorophyll biosynthetic pathway andsubsequently relay a positive signal to the nucleus or inhibit anegative signal.

The finding that gun2 and gun3 are alleles of hy1 and hy2(Vinti et al., 2000; Mochizuki et al., 2001) provide anotherinteresting twist to the retrograde signalling question. Bothhy1 and hy2, defective in photochromobilin synthesis, are dis-rupted in downstream components of the haem branch of thetetrapyrrole biosynthetic pathways and they were originallyisolated based on their loss of hypocotyl growth inhibition.The gun phenotype of hy1 and hy2 is most likely due to feed-back inhibition in that downstream disruptions in the haembranch can cause repression of early upstream steps ultimatelyleading to decreased flux through the chlorophyll branch(Terry & Kendrick, 1999). In fact, genetic studies have shownthat hy1 and gun5 affect the same retrograde signalling path-way (Vinti et al., 2000).

Clearly when dealing with complex biosynthetic path-ways, such as chlorophyll and haem biosynthesis, it can becomplicated to dissect the precise role of the various compo-nents due to extensive and different feedback mechanismsbetween the intermediates. However, the identification ofmutants, disrupted not only in components of the tetrapyr-role biosynthetic pathway itself, but also in components thatplay an indirect role in the chlorophyll biosynthetic pathwaywill aid in the understanding of the retrograde signallingnetwork.

Laf6 The laf6 mutant (Møller et al., 2001) was isolated in agenetic screen designed to identify components of the phyA



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signal transduction pathway. laf6 seedlings display a partialinsensitivity towards far-red light irradiation and the cloningof the disrupted gene revealed that it encodes an novel 557amino acid ATP-Binding-Cassette (ABC) protein (atABC1).Using atABC1/GFP fusion proteins in onion epidermal cellsand transgenic plants together with immunolocalizationstudies, it was shown that atABC1 localizes to the enveloperegion of chloroplasts (Møller et al., 2001). The disruption ofatABC1 in laf6 results in protoporphyrin IX accumulationand in repression of light-regulated genes suggesting that theaccumulation of protoporphyrin IX in this case results in anattenuation of nuclear gene expression. This again showsthat plastid-derived protoporphyrin IX is probably involvedin regulating nuclear gene expression. What is the role ofatABC1 or indeed protoporphyrin IX on hypocotylelongation? Clearly hy1 and hy2 show elongated hypocotyls inresponse to light, however, laf6 has normal phyA levels andcannot be rescued by chromophore feeding experiments(Møller et al., 2001). To further investigate this it has beenshown that the long hypocotyl phenotype of laf6 and theaccumulation of protoporphyrin IX in the mutant can berecapitulated by treating wild type seedlings with flumioxazin,a protoporphyrin IX oxidase (PPO) inhibitor. In addition,protoporphyrin IX accumulation and hypocotyl elongationin flumioxazin-treated wild-type (WT) seedlings can bereduced upon atABC1 overexpression. Knowing that ABCproteins are most often involved in transport processes, theseobservations suggest that atABC1 may be involved in thetransport and correct distribution of protoporphyrin IX. Thepreferential specificity of laf6 towards far-red light suggestfurther that elevated protoporphyrin IX levels may interactwith the cytosolic light-signalling pathway or trigger asecondary pathway ultimately coordinating nuclear geneexpression patterns. Since atABC1 does contain putativeporphyrin-binding motifs (S. G. Møller and N. H. Chua,unpublished) it will be interesting to learn whether atABC1can bind protoporphyrin IX directly. Moreover, the role ofatABC1 in coordinating the interplay between the nucleusand chloroplasts, ultimately modifying gene expressionaffecting hypocotyl elongation, remains to be solved (Fig. 4).

Nontetrapyrroles as retrograde signals Despite the availableevidence demonstrating the involvement of tetrapyrroles asretrograde signals, there are studies that suggest the presenceof nontetrapyrrole retrograde signalling pathways. Resultsfrom studies using the green alga Dunaliella tertiolecta hasshown that by changing the redox state of the plastoquinonepool in chloroplasts, nuclear CAB expression is affected(Escoubas et al., 1995). By reducing plastoquinone at highlight intensities CAB expression is enhanced whereas CABexpression is repressed upon inhibition of plastoquinoneoxidisation at low light intensities. This suggests that theredox state of the plastoquinone pool in chloroplasts mayact as a photon-sensing system regulating nuclear CAB

expression. Interestingly, the accumulation of chlorophyllat low-light intensities can be blocked by inhibition ofcytoplasmic phosphatases such as okadaic acid. This furtherimplies that CAB expression may be reversibly repressed by aphosphorylated component, which is in turn coupled to theredox state of plastoquinone, possibly via a chloroplast proteinkinase. Although an attractive model it still remains to beshown that the signal from the plastoquinone pool is relayedthrough a phosphorylation cascade.

The regulation of photosynthesis by carbon metabolitefeedback inhibition has been well established although thecellular mechanism still remains largely unknown. Forexample, CAB expression is increased upon sugar depletion.However, by blocking photosynthetic electron transport,sugar depletion does not lead to CAB induction (Oswaldet al., 2001). Taken together this suggests that the redox statein the chloroplasts can outweigh the carbohydrate-regulatedexpression of nuclear-encoded photosynthetic genes.

Recently, Lucas and colleagues (Provencher et al., 2001)cloned the disrupted gene in the maize mutant sucrose exportdefective 1 (sxd1), which encodes a novel chloroplast localizedprotein of 488 amino acids. sxd1 has been shown to be defect-ive in sugar transport, to accumulate anthocyanin and toform occlusions of plasmodesmata between bundle sheathcells. Many photosynthetic genes are suppressed by high sugarlevels through a hexokinase-mediated signalling cascade ( Janget al., 1997) however, in contrast to wild type, CAB expressionin sxd1 remains high despite the elevated sugar levels. It istempting to speculate that SXD1 may represent a chloroplastprotein required to integrate hexokinase-mediated signalswithin the cells.

The chloroplast to nucleus retrograde signalling network isunquestionably a more complex process than first anticipated.Although by a largely unknown mechanism, chlorophyllprecursors can clearly act as ‘plastid signals’. However, there iscompelling evidence that both the plastoquinone redox stateas well as sugars can have a dramatic effect on nuclear geneexpression of light-regulated genes. There are clearly multipleways in which these ‘chloroplast retrograde signals’ are trans-duced to the nucleus. Moreover the role of these ‘signals’ andindeed the role of the chloroplast in controlling photo-morphogenic responses, such as hypocotyl elongation andgene expression, remain unclear. The chloroplast undoubtedlyplays an important integrated role in photomorphogenicresponses along side the nucleus and the cytoplasm. It willindeed be interesting to learn, using molecular in vivo chloro-plast disruption techniques together with microarray analysis,which nuclear genes are affected and whether the majority ofthese encode photosynthetic genes.

V. Interactions with other signalling pathways

Signal transduction pathways have often been viewed as linearchains of events but it is becoming increasingly clear that there



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is extensive cross-talk between different pathways (Møller &Chua, 1999). The plethora of interactions that take place areprobably involved in modulating various pathway inputs sothat plants can develop in response to changing environ-mental and developmental cues. It has been conclusivelyshown that the phytochrome signalling cascade does indeedcommunicate and interact with various other signallingpathways. The isolation of hormone signalling mutants andthe recent characterization of key regulatory processes havegiven extensive insight into how light and hormone signallingare integrated.

1. Light and hormones

It is not surprising that hormones contribute to photo-morphogenic responses in that both light and hormonesgenerally act on similar cells and organs involving processessuch as cell elongation and expansion. For example cytokinin,ethylene and abscisic acid (ABA) inhibit cell elongation whilst

auxin, brassinosteroids, and gibberellic acid (GA) stimulatecell elongation.

Auxin The roles of auxin during plant development areseveral-fold. At the tissue/organ level, auxin is involved in rootelongation, lateral root development, meristem maintenanceand senescence whilst at a cellular level this hormone isinvolved in cell division, cell differentiation and cellelongation. Early studies into auxin signalling were mainlyfocused on the identification of auxin responsive DNAsequence elements (Guilfoyle et al., 1998). A number ofauxin-regulated genes have been isolated and the recent use ofArabidopsis mutants has started to unravel the auxin pathwayand its interaction with phytochrome signalling.

Several auxin-regulated genes have been found to encodeproteins that localize to the nucleus. For instance parA, anauxin-regulated gene in tobacco (Takahashi et al., 1995) andIAA1 and IAA2, early auxin-induced genes in Arabidopsis(Abel et al., 1994) are nuclear localized. Moreover, IAA1 and

Fig. 4 Retrograde signalling from the plastid to the nucleus. The figure illustrates possible retrograde signalling events that take place between plastids and the nucleus. Both LAF6 and GUN5 have been implicated in playing a role in this communication mechanism. It is still largely unknown how retrograde signals are transduced to the nucleus in response to both internal and environmental cues.



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IAA2 are very short lived proteins (t1/2 4–8 min) and showthe presence of DNA binding sequence motifs suggesting thatvery early primary responses in auxin signalling may involvetranscriptional control by IAA proteins (Abel et al., 1994).More recent data has indeed shown that a vast number of IAAgenes encode short lived nuclear proteins that can homo- andhetero-dimerise both in vivo and in vitro (Abel et al., 1994;Kim et al., 1997). Does this have implications for phyto-chrome signal transduction? The semidominant mutantshy2–1D (Kim et al., 1996) was isolated as a suppressor of thelong hypocotyl phenotype of hy2, a chromophore bio-synthetic mutant. SHY2 has been cloned and encodes IAA3(Tian & Reed, 1999), a member of the IAA auxin-induciblegene family. Loss-of-function shy2 mutants consistently havelonger hypocotyls than wild type (Tian & Reed, 1999) whilstgain-of-function mutations result in photomorphogenicresponses in darkness such as cotyledon expansion andhypocotyl growth inhibition (Kim et al., 1997). From thesedata it is clear that there is an interaction between phyto-chrome and IAA proteins. To this end Abel and colleagues(Colon-Carmona et al., 2000) have demonstrated, by in vitrointeraction studies, that SHY2/IAA3, AXR3/IAA17 (Ouelletet al., 2001), and Ps-IAA4 (Abel & Theologis, 1995) interactwith purified oat phyA. This suggests that one point of inter-action between auxin and light signalling is direct physicalcontact between early auxin inducible nuclear gene productsand phytochrome. Interestingly, it has now been shown thatphyA can phosphorylate IAA3, IAA17, IAA1, IAA9 and Ps-IAA4 in vitro independent of its Pr or Pfr form. In addition,it has been demonstrated, using metabolic labelling andimmunoprecipitation that SHY2-2 is phosphorylated in vivo.One potential problem with these data is that the in vitrokinase assay was performed for 25 min, which is outside thephysiological time scale for a primary phyA signalling event.Despite this, these data do suggest the possibility that a primaryinteraction point between phytochrome and auxin signallingresides in the nucleus and may involve phytochrome-mediatedphosphorylation of early auxin inducible proteins.

It is clear that auxin plays an important role duringhypocotyl elongation. For instance, when Arabidopsis seed-lings are grown in the light at 29°C they show a dramaticincrease in hypocotyl elongation compared to seedlingsgrown at 20°C (Gray et al., 1998). It was shown that seedlingsgrown at elevated temperatures have increased free auxinlevels suggesting that temperature regulates auxin synthesisand catabolism and by doing so mediates growth responses.In addition, Estelle and colleagues ( Jensen et al., 1998) haveshown, using polar auxin transport inhibitors, that auxintransport is required for hypocotyl elongation in the lightbut not in darkness. Similarly, it has been shown that thecalossin-like protein BIG is required for polar auxin transportand that BIG-deficiency results in altered expression oflight-regulated genes (Gil et al., 2001). Auxin transport hasalso been implicated in playing a role in the shade-avoidance

response (Morelli & Ruberti, 2000). Steindler et al. (1999)showed that by overexpressing ATHB-2, a homeodomainleucine zipper protein that is down-regulated by red andfar-red light, hypocotyl elongation increases whilst in moremature plants there are obvious auxin phenotypes. Thecross-talk between ATHB-2-mediated shade avoidanceresponse and auxin was further verified by IAA feedingexperiments which rescued the ATHB-2 phenotype. Simi-larly, HY5, a bZIP transcription factor acting downstream ofboth phytochrome and cryptochrome, was identified as apositive regulator of photomorphogenesis (Ang & Deng,1994). Although hy5 shows light insensitivity, the pleiotropicnature of this mutant suggests its involvement in otheraspects of plant development and auxin signalling hasbeen implicated as one of these (Oyama et al., 1997).Recent evidence has also emerged that Arabidopsis seedlingswith reduced COP9 signalosome, a negative regulator ofphotomorphogenesis, exhibits reduced auxin response similarto loss-of-function E3 ubiquitin ligase SCFTIR plants(Schwechheimer et al., 2001). In addition, it was shown thatCOP9 interacts with SCFTIR in vivo and that COP9 is in factneeded for the degradation of the pea IAA protein PSIAA6.These results suggest that COP9 may play an importantrole in mediating E3 ubiquitin-ligase responses involved thedegradation of IAA proteins (Section III/4).

Recently, the relationship between hypocotyl elongationand auxin was further strengthened by studies showing thatthe reduced auxin response mutant, axr1–12, and plants over-expressing iaaL, which conjugates free auxin to lysine, havereduced hypocotyl lengths in the light (Collett et al., 2000).Moreover, if auxin is added hypocotyl growth is promoted.Conversely, the enhanced auxin response mutant axr3–1 doesnot respond to exogenous auxin application. It has also beenshown that IAA7-deficient seedlings show longer hypocotylsthan wild-type in response to light (Nagpal et al., 2000).Once again it is clear that the nucleus plays an important role,not only as part of light signalling, but also as an interactionpoint between different pathways.

The nucleus is not the only subcellular compartment thatcontains auxin/light pathway integrators. Deng and col-leagues (Hsieh et al., 2000) have shown that fin219 exhibitsfar-red light induced hypocotyl elongation. fin219 was iso-lated as a suppressor of COP1 and the disrupted gene encodesan auxin-inducible cytoplasmic protein with good homologyto GH3-like proteins. Similarly, Nakazawa et al. (2001) haveshown that a second GH3 protein, DFL1, is involved inhypocotyl elongation. DFL1 overexpression results inreduced hypocotyl length in response to red, far-red and bluelight but not in darkness implying again interaction betweenauxin and photoreceptors.

Although it is still early days, it is clear that auxin plays amajor role in a variety of developmental processes, both at thephysiological and molecular level, and that a number of theseprocesses are modulated through phytochrome action.



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Brassinosteroids The possibility that brassinosteroids areinvolved in the interaction between light and hormonesignalling has created a lot of excitement. This enthusiasmwas largely prompted by the finding that a number of dwarfmutants such as det2, dwf1, dim1 and cpd, previouslyidentified as light signalling mutants, also exhibit defectivebrassinosteroid biosynthesis (Ecker, 1997). Together withthe recent identification of the brassinosteroid insensitivemutant bri1 (Li & Chory, 1997), the demonstration thatBRI1 is a brassinosteroid receptor (He et al., 2000), thecloning of BAS1 (Neff et al., 1999), and the discovery thatlight and brassinosteroids are integrated via a dark-inducedsmall G protein (Kang et al., 2001), has kept the enthusiasmalive.

The pleiotropic mutant det2 shows a number of lightresponsive traits in darkness including a short hypocotyl,anthocyanin accumulation, expanded cotyledons and pri-mary leaf buds (Li et al., 1996). Moreover, light responsivegenes show a 10–20-fold derepression. In contrast, det2 issmaller and greener than wild type in the light with reducedcell size, apical dominance and altered circadian rhythmresponses. The disrupted gene in det2 encodes a protein withhigh similarity to mammalian steroid 5α-reductases whichcatalyse the conversion of testosterone to dihydrotestosterone,a key step in steroid biosynthesis. In plants, one of the earlyreductive steps in brassinosteroid biosynthesis involves thereduction of campesterol to campestanol, similar to the reac-tion catalysed by the mammalian steroid 5α-reductases. Totest the role of DET2 in brassinolide biosynthesis Chory andcolleagues (Li et al., 1996) demonstrated that the shorthypocotyl phenotype of det2 in darkness can be rescued by10−6 M brassinolide. As for det2, the det3 mutant showsphotomorphogenic responses in darkness and exhibits reducedresponsiveness towards exogenous brassinosteroids. However,in contrast to det2, the disrupted gene in det3 encodes subunitC of a vacuolar H ± ATPase (Schumaker et al., 1999). Theseresults indicate that brassinosteroids are involved in severalprocesses, including modulation of light-regulated genes,induction of cell elongation, floral initiation, and in leaf deve-lopment, which in turn may be regulated by light or by otherhormones.

Another example illustrating the interaction between lightand brassinosteroid signalling is the cpd mutant, which dis-plays deetiolation and derepression of light-regulated genes indarkness. CPD has been cloned and encodes a cytochromeP450 with good sequence homology to steroid hydroxylases.As with det2, and the cpd mutant phenotype can be fullyreversed by feeding C23-hydroxylated brassinolide precursors(Szekeres et al., 1996). Similarly, the Arabidopsis dim mutantwas shown to be impaired in brassinosteroid biosynthesis(Klahre et al., 1998) resembling the det2 mutant phenotype(Takahashi et al., 1995). However, in contrast to det2, dim1mutant seedlings, grown in darkness, do not exhibit derepres-sion of light-regulated genes.

The Arabidopsis bri1 (brassinosteroid insensitive) mutantwas originally isolated based on its ability to elongate roots,compared to wild type seedlings, on 10−7 M 24-epibrassinolide(Clouse et al., 1996). The bri1 mutant also resembles thedet2 and cpd mutant phenotypes in light and darkness. BRI1has been cloned and encodes a receptor-like protein whichbelongs to a family of plant receptor-like transmembranekinases (RLK) known as leucine-rich repeat (LRR) kinases (Li& Chory, 1997). Chory and colleagues (He et al., 2000) havenow shown that BRI1 is most likely a brassinosteroid receptor.By fusing the extracellular leucine-rich repeat and transmem-brane domain of BRI1 to the kinase domain of the rice diseaseresistance receptor XA21, it was shown that transgenic plantselicit a defence response when treated with brassinosteroids.One interesting point to make is that, as far as we are aware,the only ‘new’ brassinosteroid insensitive mutants isolated todate, from numerous genetic screens, have proven to be newbri1 alleles.

In a suppresser screen using a phyB mutant, Chory and col-leagues (Neff et al., 1999) isolated what could be the closestpoint of interaction between light and brassinosteroid signal-ling identified to date. bas1 suppresses the long hypocotyl ofa phyB mutant and has reduced levels of brassinosteroidsshowing an accumulation of 26-hydroxybrassinolide duringfeeding experiments. The bas1 mutant phenotype is due tothe overexpression of a cytochrome P450. The phenotype isred-light specific, however, a partial suppression of a crypto-chrome mutant is also observed. Conversely, reduced levels ofBAS1 renders seedlings hypersensitive towards brassinoster-oids in a light-dependent fashion indicating that this P450may represent a light/brassinosteroid control point.

Very recently another cytochrome P450 was shown toplay a role in coordinating light and brassinosteroid signal-ling. The small G protein PRA2 from pea is induced indarkness and it has now been shown that PRA2 regulatesa cytochrome P450 (DDWF1) that in turn catalyses C-2hydroxylation in brassinosteroid biosynthesis (Kang et al.,2001). In fact PRA2 interacts with DDWF1 in vitro and thisinteraction is strictly GTP-dependent. As for other brassinos-teroid mutants, seedlings with reduced PRA2 levels showdwarfism, which can be rescued by feeding exogenousbrassinosteroids. By contrast, overexpression of DDWF1results in elongated hypocotyls in the light pointing towardsPRA2 acting as a mediator between light and brassinosteroidssignalling during etiolation.

The point of interaction between the light and brassinos-teroid pathways lies probably within the ability of light tomodulate brassinosteroid signal transduction in target cells orperhaps even by regulating their biosynthesis directly. Thishowever, remains to be conclusively shown. The situation ishowever, more complicated because cytokinin addition cancause a det2 phenotype in dark grown wild type Arabidopsisseedlings (Chory et al., 1994) and moreover the CPD geneis down-regulated by cytokinin, suggesting that cytokinin



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negatively controls brassinosteroid biosynthesis (Szekereset al., 1996).

Cytokinins Cytokinins regulate a number of developmentalprocesses including activation of cell division, suppression ofapical dominance and senescence, inhibition of root growthand stimulation of de-etiolation. A major difficulty in theelucidation of the cytokinin signalling pathway is the scarcityof target genes that are induced specifically by the hormone.To date, three genes, IBC6, IBC7 (Brandstatter & Kieber,1998) and CycD3 (Riou-Khamlichi et al., 1999), appear to beprimary targets of cytokinin since their induction can occurwithout new protein synthesis. Cytokinin and light canclearly elicit similar physiological and biochemical responsesand recent progress has started to untangle the complexity ofthis interaction.

In 1989 Chory and colleagues isolated the det1 mutant,which constitutively displays many light-dependent charac-teriztics in darkness including leaf and chloroplast develop-ment, anthocyanin accumulation, and induction of severallight-regulated nuclear and chloroplast genes (Chory et al.,1989). It was subsequently shown that by adding cytokininsto wild-type plants in darkness the normal etiolation processwas disrupted resulting in a det1 phenotype including thepresence of intact chloroplasts and induction of light-regulated genes (Chory et al., 1991). These results probablyprovided the first good link between cytokinin and lightsignalling, suggesting that cytokinin is important during thegreening process in Arabidopsis.

More recently Sano and colleagues (Ikeda et al., 1999)demonstrated that WPK4 is a protein kinase induced by lightand cytokinin but down-regulated by sucrose. This furtherimplies that sucrose overrides the effect of cytokinin in termsof WPK4 expression similar to the antagonistic effect sucrosehas on wild-type Arabidopsis seedlings in response to far-redlight irradiation. Another example illustrating that cytokinincan override light signals is the fact that red light-inducedloss of hypocotyl gravitropism in Arabidopsis is restoredupon addition of cytokinin (Golan et al., 1996). In addition,cytokinin also replaces light in the inhibition of hypocotylelongation.

The finding that chloroplasts contain a wide range ofcytokinins and the enzymes needed for their metabolism(Benkova et al., 1999) has also provided a link between lightand cytokinin signalling. Very recently, it has been shown thatcytokinin can rescue photomorphogenic responses in the pealip1 mutant of pea (Seyedi et al., 2001). By adding cytokininto both the lip1 mutant and wild type seedlings in darkness,phyA levels were increased in the lip1 mutants and the highlevels of POR were reduced in both lip1 and wild type.

Taken together the results to date suggest a close connec-tion between cytokinin and phytochrome signalling. Withthe recent identification of the cytokinin receptor inArabidopsis (Inoue et al., 2001) and the concerted effort

in identifying more cytokinin-specific genes, additionalcytokinin–mediated interactions between light and thishormone will undoubtedly surface.

Gibberellic acid There is a wealth of literature regardinggibberellic acid (GA). However, the role of this plant growthregulator in signalling and moreover its interaction withphytochrome signal transduction, remains somewhat unclear.Probably the best known GA signalling mutant, spy (spindly),was isolated based on its light green leaves, increasedinternode length and spindly growth phenotypes whichis reminiscent of wild type plants after repeated GA3applications. The spy phenotype also resembles the phyBmutant phenotype. SPY has been cloned and encodes aTRP repeat protein with sequence homology to a serine(threonine)-O-linked N-acetylglucosamine transferase( Jacobsen et al., 1996) and SPY is most likely a repressor ofthe GA response.

Conversely, the signalling mutant, gai (GA-insensitive),shows reduced GA sensitivity (Peng et al., 1997a). GAIencodes a protein with sequence homology to SCARE-CROW (SCR) (Di Laurenzio et al., 1996) at the C-terminalregion. However, the N-terminus of GAI contains a putativenuclear localization signal, which is absent in SCR. Becausea null allele of gai confers a weak spy-like phenotype, it ispossible that GAI acts as a repressor of GA signalling. Also,the suppression of gai, by strong alleles of spy, suggeststhat SPY and GAI participate in the same pathway with SPYacting upstream of GAI.

Silverstone et al. (1998) isolated a recessive suppressor ofthe Arabidopsis ga 1–3 mutant that can restrain the effects ofGA deficiency. The disrupted gene is named RGA (repressorof ga 1–3) and the RGA locus encodes a member of the GRAS(VHIID) protein family, which includes SCR and GAI. RGAis also shown to localize to the nucleus. Interestingly, the pres-ence of leucine heptad repeats in these two proteins and thesequence homology between them does suggest that theymay interact to form a regulatory complex to repress GAsignalling. In addition, PAT1, which is a member of theGRAS (VHIID) protein family involved in phyA signalling,has been isolated (Bolle et al., 2000) implying that the GRASprotein family may have a global role in plants signal trans-duction. All members of the GRAS family also harbour struc-tural motifs suggestive of protein–protein interactions, whichindicate that different pathways may intercept at the level ofcommon interacting partners as part of a signalling network.

Recent studies have shown more directly that phytochromeand GA are indeed interlinked. For instance, the genes GA4and GA4H, both encoding GA 3β-hydroxylase, are rapidlyinduced in imbibed seeds within 1 h after a red light pulse(Yamaguchi et al., 1998). Indeed, phyB promotes seed germi-nation by increasing GA biosynthesis. Interestingly, GH4 butnot GA4H is induced in a phyB-deficient mutant, indicatingthat the induction of different GA hydroxylases is mediated



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through different phytochromes. Similarly, double mutantstudies have shown that a functional GA signalling system isnecessary for the elongated hypocotyl phenotype observed inphytochrome mutants; GA inhibits hypocotyl elongation byreducing GA responsiveness (Peng & Harberd, 1997). It hasalso been shown that endoreduplication levels in Arabidopsishypocotyls are under negative control of phytochrome(Gendreau et al., 1998) whilst GA has a positive effect on theendoreduplication (Gendreau et al., 1999).

GA also affects flowering, but in contrast to the phyB-induced delay in flowering, GA promotes flowering. How-ever, phyB does of course also mediate flowering through aGA-independent pathway (Blazquez & Weigel, 1999).

At this point in time the interaction between phytochromeand GA signalling is somewhat hard to interpret. Neverthe-less, it is clear that phytochrome and GA signalling pathwaysdo interact in order for plants to control such diverse develop-mental processes as seed germination and flowering.

Ethylene In darkness ethylene inhibits hypocotyl elongationwhilst in light ethylene promotes hypocotyl elongation(Smalle et al., 1997). The cloning and characterization of theArabidopsis HOOKLESS1 (HLS1) gene has demonstratedthat ethylene promotes cell elongation in specific cells in theapical hook region (Lehman et al., 1996). Interestingly, HLS1is thought to control this elongation by regulating either thetransport or chemical modification of auxin (Smalle et al.,1997). Moreover, auxin resistant mutants are ethyleneinsensitive (Hobbie & Estelle, 1995). These results suggestthat there is a three-way interconnected pathway betweenlight, auxin and ethylene signalling.

By contrast to GA, ethylene has a positive effect onendoreduplication events in the hypocotyl in the light and indarkness (Gendreau et al., 1999). Although the ethylenesignal transduction pathway is probably the best understoodhormone pathway ( Johnson & Ecker, 1998) there is stillalong way to go before we fully understand the integration ofethylene and phytochrome signalling in plants.

2. Light and sugars

Sugars clearly have opposing effects on a broad range of genes.For instance sugars stimulate the expression of genes involvedin anthocyanin biosynthesis, glycolysis, and defence responsesbut at the same time inhibit the expression of genes involvedin chlorophyll biosynthesis, photosynthetic functions,gluconeogenesis, starch degradation, and the glyoxylate cycle.As end products of photosynthesis it is not surprising thatthere is a plethora of interactions between light signalling andsugars. In WT seedlings, sucrose antagonizes the effect of far-red light with respect to inhibition of hypocotyl elongationand cotyledon opening (Barnes et al., 1996). Also, theaddition of sucrose overrides the far-red light induced killingeffect (Barnes et al., 1996). Similarly, high levels of sucrose

suppress the expression of photosynthetic genes duringdevelopment in the dark. A class of mutants (sun, sucrose-uncoupled) have been isolated that show reduced repressionof photosynthetic genes in the presence of sucrose (Dijkwelet al., 1997). More detailed analysis has revealed that sun7displays a reduced response of sucrose-mediated repressionof cotyledon opening in far-red light whilst sun6 shows areduced response of the sucrose-mediated block of far-redlight-induced killing (Barnes et al., 1996). These resultsclearly illustrate the myriad of interactions between sugarsensing mechanisms and phytochrome signal transduction.

Recent studies have identified at least two sugar signallingpathways: one appears to be regulated by hexokinase ( Janget al., 1997) whilst the other by SNF1-like protein kinases(Nemeth et al., 1998). Arabidopsis seed germination, cotyle-don expansion and greening, and the emergence of true leavesare all arrested by 6% glucose. Sheen and colleagues havedemonstrated that the Arabidopsis hexokinases, HXK1 and 2,play a key role in sensing this glucose signal ( Jang et al., 1997).By overexpressing HXK1/2, plants become more sensitivetowards sugar, whereas antisense HXK1/2 plants are less sen-sitive compared to wild type.

The Arabidopsis AKIN10 and 11 genes can complementthe yeast snf1 deletion mutant, demonstrating that these genesmost likely encode SNF1 homologs (Bhalerao et al., 1999).Using the yeast two hybrid system, the C-terminal domainsof AKIN10/11 can interact with the N-terminal domain ofPRL−1 and these interactions are sensitive to glucose. Konczand colleagues (Nemeth et al., 1998) have isolated the mutantprl-1 (pleiotropic root locus), based on its glucose hypersensi-tivity and PRL-1, a WD-40 protein, most probably acts as anegative regulator of glucose responsive genes. Sucrose phos-phate synthase is a target of sugar regulation in plants and isinactivated by phosphorylation at high sugar concentrations.Purified AKIN10/11, were, able, to, phosphorylatea, peptide,of, sucrose, phosphate, synthase, in vitro and this specific pro-tein kinase activity was further inhibited by purified PRL-1.Bhalerao et al. (1999) showed further that the kinase activityin plants was stimulated by sucrose confirming the in vitrofinding that PRL-1 functions as a negative regulator of AKIN10/11-kinase activity.

The cross talk between sugar sensing and phytochrome sig-nalling can also be seen as an indirect interaction through theaction of ethylene. Glucose-mediated developmental arrestand greening can be overcome by ethylene treatment (Zhouet al., 1998) and this opposing effect of ethylene is not seen inetr1–1, a receptor mutant that is insensitive to ethylene. Con-versely, ctr1–1, a constitutive ethylene triple response mutant,is not affected by glucose. These results indicate that the sugarand ethylene signalling pathways work in opposition tomodulate the early stages of Arabidopsis seedling development,maybe through the action of phytochrome.

In terms of positive sugar signalling components, Zhouet al. (1998) isolated gin-1, a glucose-insensitive recessive



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mutant, which escapes the developmental arrest imposedby 6% glucose. GIN1 is most probably a positive regulatoryelement in transmitting the glucose signal for repression ofdevelopmental processes such as seed germination, cotyledonexpansion and greening, and true leaf development. Glucoseis also known to delay flowering, which is relieved by thegin-1 mutation, indicating that the glucose repressive effecton flowering is mediated by GIN-1.

The interactions that take place between phytochromesignalling and other signalling pathways are fascinating. Oneway of gaining more insight into the interpathway regulationwill be to use microarray analysis to monitor global transcriptchanges in response to specific pathway cues. For instance,microarray technology has very recently shown that a numberof auxin-, GA-, and ethylene-regulated genes are repressedafter far-red irradiation (Tepperman et al., 2001). Clearly, theinteractions that take place play a very important role in coor-dinating the execution of key developmental processes and itwill be interesting to learn more about the multiple regulatoryaspect of this communication network.

VI. Conclusions and the future

The recent progress in phytochrome research has beentremendous and at times it is hard to reconcile and integrateall the incoming data into an overall picture. The view thatphytochrome signal transduction represents a linear chainof events is now outdated and it is increasingly clear thatphytochrome signalling should be viewed as a multi-dimensional network. In this review we have attempted toconceptionalise this complex network in terms of cellbiology, shedding light on recent and exciting findings withparticular emphasis on the role of the different subcellularcompartments in controlling phytochrome signalling cascades.

One emerging theme in phytochrome signalling isundoubtedly that a wealth of signalling activities take place inthe nucleus. This is of course not entirely surprising given thatphotomorphogenic responses, as for many physiologicalresponses, are usually underpinned by alterations in nucleargene expression dynamics. However, the demonstration thatphyA and phyB translocate to the nucleus in response to lightand that photoactive phytochrome can interact with nuclearDNA-binding proteins to regulate gene expression, demon-strate that at least some branches of phytochrome signallingare exceedingly short (Ni et al., 1998, 1999; Martinez-Garciaet al., 2000). Evidence also shows that a number of signallingintermediates interact directly with phytochrome, suggestingthat phytochrome may act as a nuclear ‘workhorse’, formingprotein complexes that execute appropriate signalling sub-pathways by physical contact to other nuclear proteins.Maybe these complexes represent the observed subnuclearfoci or speckles? However, there are also a number of phyto-chrome signalling intermediates that are localized to thenucleus that do not interact with phytochrome directly

(Hudson et al., 1990; Fairchild et al., 2000; Huq et al., 2000;Ballesteros et al., 2001).

How is the nuclear signalling network controlled? One ele-gant way of controlling the signalling responses is at the levelof nuclear translocation. For instance, the inactive Pr form ofphyA and phyB are retained in the cytosol until activated bylight upon which they translocate to the nucleus (Sakamoto& Nagatani, 1996; Kircher et al., 1999a; Yamaguchi et al.,1999; Gil et al., 2000; Kim et al., 2000). Similarly, COP1 hasbeen shown to be cytosolic in the light but to translocate to thenucleus in darkness (von Arnim & Deng, 1996; Osterlund& Deng, 1998; Stacey et al., 1999). How are constitutivelynuclear proteins controlled? One mechanism clearly involvesthe 26S proteasome whereby signalling components arespecifically targeted for degradation providing yet anotherelegant way of controlling the signal flux (Wei et al., 1994;Chamovitz et al., 1996; Wei & Deng, 1998; Wei et al., 1998;Dieterle et al., 2001). In addition, the 26S proteasome mayalso be involved in phyA degradation itself.

Although the nucleus seems to be the centre of attention,the cytoplasm also plays an important role in phytochromesignal transduction. The light-dependent nuclear transloca-tion of phyA, phyB and COP1 clearly depends on the cytosolfor accurate reversible retention mechanisms. Likewise,several phytochrome signalling intermediates are cytosolicdemonstrating that not all signalling events take place exclus-ively in the nucleus (Bolle et al., 2000; Hsieh et al., 2000;Guo et al., 2001). In addition, the plethora of interactionsbetween phytochrome signalling and other signallingpathways require both nuclear and cytoplasmic intersections(Møller & Chua, 1999).

The chloroplast has often been on the sideline in phyto-chrome research, which is somewhat puzzling since activeholophytochrome depends on phytochromobibilin, gener-ated in the chloroplast. However, it is becoming increasinglyclear that the chloroplast plays an important role, not only inregulating nuclear gene expression via retrograde signals, butalso in controlling photomorphogenic responses (Mochizukiet al., 2001; Møller et al., 2001).

Although our knowledge of phytochrome research isexpanding rapidly there are still numerous questions thatremain to be answered. Why do only some phytochromesshow light-dependent nuclear translocation? Why do somenuclear components form speckles whilst others do not andwhy are the speckles different between various classes of pro-teins? Are most bona fide phytochrome signalling cascadesshort and nuclear localized and is the cytoplasm merely aperipheral support component? How are retrograde signalstransduced from the chloroplast to the nucleus and what is theprecise role of chloroplasts in terms of photomorphogenicresponses? These are only some of the interesting questionsthat beg an answer.

It is perfectly obvious that each cellular organelle has itsown specific role in terms of phytochrome signal transduction.



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It is also clear that these multiple organelles are inter-connected in order to execute the appropriate molecular andphysiological responses. Moreover, the extensive subcellularnetwork of interactions and intersections between lightsignalling and other signalling pathways is very impressive.How the numerous signals and pathways are coordinatedbetween cellular organelles and how this network is fine-tuned in response to changes in the light environment remainexciting future challenges. One thing is however, clear: thefuture is light!

Acknowledgements

We would like to thank Nam-Hai Chua and Stefan Kircherfor sharing results prior to publication.

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