Prokaryotes and Phytochrome. The Connection to Chromophores and Signaling

CYANOBACTERIA AND PLANTS...

Based on the first spectral measurements, it was correctly argued that the phytochrome chromophore might be a bilin similarto those of phycobiliproteins in cyanobacteria and red algae (Butleret al., 1959). Phycobiliproteins bear four types of bilin, namelyphycocyanobilin (PCB), the chromophore of phycocyanin (PC, whichis thus generally the most abundant), phycoerythrobilin (PEB),phycoviolobilin (PVB), and phycourobilin (PUB) (see Fig. 1). Apophytochromesautoassemble with PCB to form holophytochrome photoreceptors $---$ auseful feature, as PCB can be prepared rather easily from commerciallyavailable cyanobacteria, enabling the preparation of functionalphytochromes by recombinant methods (Wahleithner et al., 1991).However, in all land-plant phytochromes examined so far, anothermember of the family, phytochromobilin (P $Phi$ B), is the natural chromophore(Rüdiger and Thümmler, 1994). Phytochrome chromophores undergoa characteristic Z $right-arrow$ E isomerization around the C15==C16 doublebond between rings C and D during Pr $right-arrow$ Pfr conversion. PEB alsoassembles with apophytochrome in vitro, but the product is notphotochromic because the C15-C16 bond is saturated (Li and Lagarias,1992). The five bilins (PCB, P $Phi$ B, PVB, PEB, and PUB) above differonly in single double bonds and are derived from biliverdin, thefirst open-chain tetrapyrrole in this biosynthetic pathway (Fig.1).

View larger version (22K):
[in this window]
[in a new window]

Figure 1. Structure of heme and the natural bilins biliverdin (BV), P $Phi$ B, PCB, PEB, PVB, and PUB. Heme oxygenase converts heme to bilivirdin by cleaving between rings A and D at the positions marked. Differences in the other bilins with respect to bilivirdin are also indicated.

	ONLY THE FIRST GENE FOR THE BILIN SYNTHETIC PATHWAY IS KNOWN

TOP

ONLY THE FIRST GENE...

In seed plants, the enzymes for bilin synthesis are located in the plastids but are nuclear encoded. The pathway begins withthe opening of the heme tetrapyrrole ring between pyrroles A andD by heme oxygenase to form the linear tetrapyrrole (bilin) biliverdinIX $alpha$ . Genes for this enzyme are known from the cyanobacterium SynechocystisPCC6803 (Cornejo et al., 1998), from the plastome of red algae,and from the genomes of Arabidopsis and several animals. In Arabidopsisheme oxygenase is encoded by the nuclear gene HY1 (Muramoto etal., 1999). The ptr116 phototropic mutant of the moss Ceratodoncan be rescued by exogenous biliverdin by microinjecting cellswith mammalian heme oxygenase enzyme or by overexpressing HY1,showing that all three functionally complement the defective ptr116gene (Brücker et al., 1999).

The subsequent steps are not fully understood biochemically, nor have any further associated genes been described, althoughthe Arabidopsis HY2 locus is a likely candidate. Further sequencesfrom the Synechocystis genome must also be involved in the productionof PCB and otherbilins.

	CYANOBACTERIAL MODELS FOR PHYTOCHROME

TOP

CYANOBACTERIAL MODELS FOR...

Early physiological studies indicated that cyanobacteria might harbor useful information about the phytochrome system. Actionspectroscopy revealed photoreversible effects analogous to thoseof plant phytochrome but with the interesting distinction that,while in plants the responses are maximally induced by red lightand reverted by far-red light, most photoreversible effects incyanobacteria respond to red light ( $lambda$ _max $approx$ 650 nm) and green light( $lambda$ _max $approx$ 520 nm) (Vogelman and Scheibe, 1978).

The most intensely studied effect here is CCA. Unlike plants, cyanobacteria possess phycobilisome structures that funnel energyinto the photosynthetic system. The principle accessory pigmentsinvolved are the blue-green (red-absorbing) PC and allophycocyaninand the red (blue-green-absorbing) phycoerythrin (PE). Some speciesare able to use CCA to adjust the ratio of these pigments accordingto environmental conditions. In green light PE dominates, whereasin red light PC dominates. In this way, the $lambda$ _max of photosynthesisis shifted to the $lambda$ _max of the light environment (Gaidukov, 1902).

When a series of green ( $approx$ 540 nm) and red ( $approx$ 650 nm) pulses was given to a culture of the cyanobacterium Fremyella diplosiphonand the culture kept in darkness, the last light pulse determinedthe dominant accessory pigment formed (Vogelman and Scheibe, 1978).This kind of photoreversibility points to a photoreceptor withphotochromic properties, an unusual spectral feature that allowedplant phytochrome to be isolated and characterized. However, thisapproach was less successful in cyanobacteria, principally because,unlike angiosperms, they do not etiolate. Although a photoreversiblepigment showing difference maxima at 520 and 650 nm has been described(Scheibe, 1972), it was not characterizedfurther.

Later, the $alpha$ -subunit of the minor phycobilisome component phycoerythrocyanin (PEC) was shown to be photochromic $---$ but with differencemaxima at 500 and 570 nm. The physiological role of PEC is unknown,although it might have a role as a photoreceptor as well as actingas a photosynthesis antenna. The PEC $alpha$ chromophore is PVB, whichundergoes a Z $right-arrow$ E photoisomerization analogous to phytochrome(Zhao et al., 1995). Moreover, the PEC $alpha$ and PC $alpha$ sequences areapproximately 65% identical and the molecules have similar three-dimensionalstructures $---$ yet only the former is photochromic. While one mighttherefore suppose that the differences between these biliproteinscould provide a master key to unlock the secrets of photochromicityat the atomic level, whether this key would fit phytochrome isquestionable. First, there is no sequence homology between phycobiliproteinsand phytochrome. Second, isolated phycobiliproteins show far strongerfluorescence than phytochrome, implying that their chromophoresare much more tightly held and/or have a very different photochemistry.Third, relative to the dark state, the conformational changesin PEC $alpha$ are associated with a blue shift, whereas a red shiftis seen withphytochrome.

Interestingly, although phycobiliprotein apoproteins are generally capable of autocatalytically attaching bilin chromophoresin vitro, it has been shown that specific lyases mediate the assemblyin vivo, accelerating the reaction and ensuring correct bilinattachment. Phytochrome autoassembles in vitro too, but whethera discrete phytochrome bilin lyase exists is simply notknown.

	TWO-COMPONENT SIGNALING ENTERS THE FRAY

TOP

TWO-COMPONENT SIGNALING ENTERS...

A quite different line of investigation also connects plant phytochromes with prokaryotic systems. Schneider-Poetsch et al.(1991) drew attention to a significant amphiphilic sequence similaritybetween the phytochrome C terminus and the transmitter moduleof bacterial sensory His protein kinases (HPKs). HPKs are a groupof proteins responsible for the first step in the so-called two-componentsignal transduction pathways (see Fig. 2A) that provide the prokaryoticcell with its capacity for perception and response.

View larger version (28K):
[in this window]
[in a new window]

Figure 2. Two-component signal transduction. A, Basic scheme. The HPK dimer is activated by conformation changes induced by stimuli perceived by the sensor module, usually N-terminal. The transmitter module of each subunit then transfers a phosphate (red dot) from ATP to a conserved His residue (circle) of the other subunit. The phosphate group is transferred to a conserved Asp residue (square) of the cognate response regulator, which is thereby activated. An autophosphatase activity returns the response regulator to its inactive state in the phosphorylation cycle (PC) $---$ the rate of hydrolysis is sometimes regulated by interactions with the "inactive" form of the same HPK or with another molecule. B, Cph1/Rcp1 system. Pr, The ground state of the phytochrome in darkness or far-red light is the active HPK; irradiation with red light converts the molecule to Pfr, in which the HPK activity of the transmitter module is inhibited. Following autophosphorylation at H538_#995, Pr transfers the phosphate to the response regulator Rcp1. The biochemical functions of Pfr and the response regulator are unknown.

The activity of each HPK is regulated by an associated sensory module whose conformation changes in response to an environmentalsignal such as an interaction with a specific ion or molecule.The HPK is a dimer and, upon sensor activation, each subunit phosphorylatesthe other at a conserved H target residue (H_#995) within thetransmitter module. The phosphate is then transmitted to a conservedD residue in the receiver module on the second component of thetransduction system, the response regulator. This then does asits name suggests, either by activating transcription of specificgenes itself or by interacting with other proteins to bring aboutspecific physiological changes in the cell appropriate to theenvironmental signal. These two-component signal transductionsystems seem to be the primary regulatory connections betweenprokaryotic metabolism and theenvironment.

Schneider-Poetsch's suggestion that phytochrome might represent a plant sensory HPK was enhanced by the discovery of eukaryoticHPK homologs SLN1 in yeast and ETR1 in plants a year or so later,and indeed the idea that phytochrome might be a light-dependentkinase was nothing new. A seductive aspect was that it providedphytochrome with its long-sought reaction partner: This shouldbe a response regulator homolog. Many were not convinced by theHPK/phytochrome homology, however, and there was one very largeproblem: although all of the functional subdomains characteristicof HPK transmitters are recognizable in phytochromes, the all-importantH_#995 target residue itself is poorly conserved (see alignment).It seemed that the affair was over when Boylan and Quail (1996)showed that even in phytochromes in which H_#995 was conserved,it could be mutated without noticeable physiologicaleffect.

	POSSIBLE PHOTORECEPTORS IN CYANOBACTERIA

TOP

POSSIBLE PHOTORECEPTORS IN...

Work with the cyanobacterial CCA perception system, however, continued independently. Complementation methods were used toclone genes involved in the regulation of chromatic adaptationin F. diplosiphon. RcaE (Kehoe and Grossman, 1996) encodes a 74-kDpolypeptide with an approximately 150-residue portion toward theN terminus showing homologies to several regions around the chromophore-bindingdomain of plant phytochromes, as well as C-terminal motifs typicalof transmitter modules. Intriguingly, RcaE also bears two subdomains(T2L and R2L, T105_#265-L129_#289 and R241_#436-L268_#463,see alignment) showing homology to the plant ethylene receptorETR1. Although the biochemistry has yet to be demonstrated, RcaEprobably phosphorylates the response regulator RcaF, which inturn phosphorylates RcaC. The latter bears a transmitter- andtwo receiver-like modules, as well as a DNA-binding motif thoughtto mediate differential transcription of the PC and PE gene complexes.In the similar cyanobacterium Calothrix, RcaD and RcaA act asphosphorylation-dependent activators of the PC and PE gene clusters,respectively.

The 155-kD conceptual gene product of PlpA (Wilde et al., 1997; sll1124 in CyanoBase) in Synechocystis PCC6803 also showsregions of similarity to phytochromes (hence the name Plp forphytochrome-like protein) and two-component modules (see alignment).Indeed, BLAST searches show that RcaE has approximately 25% aminoacid identity (40% similarity) to PlpA, although the latter hasa long N-terminal extension. This particular Synechocystis straindoes not show CCA. It does, however, change the stochiometry betweenPS1 and PS2 according to the irradiance and spectral distributionof the light environment: In plpA knockouts the balance between the photosystems is disturbed.Furthermore, in contrast to the wild type, the plpA mutant cannot grow photoautotrophically in bluelight.

Although both PlpA and RcaE are clearly important in cyanobacterial photoperception, it has proven difficult to demonstratethat they are photoreceptors. The sensory function could be fulfilledby a separate molecule, as in many two-component systems. At leaston the basis of homology to the phytochrome N terminus, thereis little reason to expect either RcaE or PlpA gene products tobe bona fide phytochromes: If one assumes that the chromophoreis thioether-linked to a C residue, as in phycobiliproteins andplant phytochromes, the alignment in this region is constrainedto C198_#380 and C784_#380 for RcaE and PlpA, respectively.The surrounding subdomain is quite different from that seen inphytochromes, where it is well conserved and changes generallylead to a complete loss of function. However, although difficultieswith overexpressing PlpA and RcaE in Escherichia coli have hamperedin vitro studies, it has now been reported that both do seem tobe capable of attaching bilins (A. Wilde, T. Börner, D. Kehoe,and A. Grossman, unpublisheddata).

	A PROKARYOTIC PHYTOCHROME CREATES EXCITEMENT IN THE FIELD

TOP

A PROKARYOTIC PHYTOCHROME...

Quite separately, the entire 3.57-Mbp chromosome of Synechocystis PCC6803 was sequenced in a singularly efficient projectat the Kazusa Institute in Japan (CyanoBase: http://www.kazusa.or.jp),providing the scientific community with a wealth of valuable newdata. Among the 3,168 open reading frames identified, a phytochrome-likesequence (slr0473) was recognized (Kaneko et al., 1995; Hugheset al., 1996). The N-terminal moiety showed patchy but unmistakablesimilarity to phytochromes, including the all-important chromophorebinding region, whereas the 30-kD C-terminal moiety was clearlyhomologous to typical two-component transmitter modules with characteristicallyconserved H-, N-, G1-, F-, and G2-boxes (see alignment and Fig.3).

View larger version (23K):
[in this window]
[in a new window]

Figure 3. Synechocystis phytochrome (Cph1) in relation to plant phytochromes and sensory His protein kinases (HPK's). Residue numbering is that from the alignment (http://www.plantphysiol.org/cgi/content/full/121/4/1059/DC2 and http://www.biologie.fu-berlin.de/phytochrome/align2x.htm). The N-terminal bilin-bearing sensor module (blue) is recognizable in all phytochromes, while the C-terminal transmitter module (yellow) is common to phytochromes and most HPKs of the two-component type. The sensory modules of other HPKs (brown) are different and are sometimes carried on separate polypeptides. A PAS module (green), important for plant phytochrome signal transduction, is absent from Cph1. The chromophore attachment site, two PAS repeats, the signal transduction core (STC or Quail box), and the H-, N-, G1-, F-, and G2-boxes are highly conserved. Plant B-type phytochromes generally have an N-terminal extension, otherwise the N terminus of all phytochromes is Ser/Thr rich.

The question nevertheless remained: is it a phytochrome? The 85-kD gene product was further analyzed simultaneously by Lagarias'sgroup at University of California-Davis and by our laboratory(Hughes et al., 1997; Lamparter et al., 1997; Yeh et al., 1997).The apoprotein overexpressed in E. coli autocatalytically attachedPCB chromophore in vitro to form a blue-green photochromic pigment,clearly establishing that it encodes a bona fide cyanobacterialphytochrome,Cph1.

The discovery of this phytochrome caused an immediate paradigm shift in the field. Schneider-Poetsch's suggestion that theunknown mechanism of primary signal transduction could be relatedto the well-established two-component system in bacteria suddenlybecame a very hot topic. Moreover, the utility of a prokaryoticphytochrome system in biochemical and molecular-genetic studiesopens new experimental possibilities. In particular, the efficiencywith which highly soluble recombinant phytochrome can be preparedfrom E. coli overexpressors offers fresh hope that the three-dimensionalstructure of this class of photoreceptors could be resolved viaNMR and x-ray diffraction analysis of phytochromecrystals.

	Cph1 IN VITRO

TOP

Cph1 IN VITRO

Cph1 has been subjected to a range of optical and biophysical studies in our laboratory (Lamparter et al., 1997) and in thoseof our colleagues. Recombinant apoprotein (Cph1°) is expressedremarkably efficiently in E. coli, accumulating to approximately30% of cytosolic protein. Moreover, equipped with a C-terminaloligohistidine tag, it can be purified almost to homogeneity ina single Ni²⁺-affinity chromatographic step. E. coli does not support bilinsynthesis, and thus autoassembly does not occur in vivo. If Cph1°is added to PCB in vitro, however, a dramatic blue to blue-greencolor change occurs within seconds. This results from two processes.Initially, recombinant holoprotein (Cph1*) is formed as Pr, wherebythe PCB red absorbance peak at 610 nm is shifted to 658 nm asthe helical form of the free bilin becomes unwound in the proteinenvironment. Thereafter, if observed in daylight, the Pr is photoconvertedto Pfr, whose absorbance peak is shifted even further to 702nm.

Both photochromic forms are quite stable in darkness. The yield of pure Cph1* is routinely about 20 mg per liter of culture.It can be concentrated to above 15 mg/mL quite easily, satisfyingan important further precondition for many biophysical and physicochemicalstudies, including crystallization. Like other HPKs and plantphytochromes, Cph1* behaves as a dimer in solution. As one wouldexpect for a photoreceptor, the extinction coefficient of Cph1*is very high (approximately 100 mM¹ cm¹ for Pr at $lambda$ _max) and the quantum conversion efficiency is about0.16 in both directions $---$ values similar to those for plant phytochromes.Cph1° can also be assembled with other chromophores: The P $Phi$ B adductshows a red shift of about 15 nm for both Pr and Pfr, as seenwith plant phytochromes. PEB adducts cannot photoconvert becauseof their missing C15==C16 double bond (see Fig. 1); the quantumenergy is released as fluorescence and their absorbance maximumis blue-shifted to 579nm.

Fourier-transform Raman-resonance (FTRR) and flash photolysis (Remberg et al., 1997), low-temperature fluorescence (Sineshchekovet al., 1998), and Fourier-transform IR absorbance (FTIR, H. Förstendorfand F. Siebert, unpublished data) spectroscopic methods have alsobeen used. Despite the considerable differences in the peptidesequences, Cph1* shows remarkably similar physicochemical propertiesto those of B-type phytochromes. FTRR is a sensitive probe forthe status of the chromophore in biliproteins, revealing in thiscase many similarities between the chromophores of native oatphytochrome A and the equivalent P $Phi$ B adduct of Cph1*. For bothphytochromes, spectral differences between the Pr and the Pfrform reflect the Z $right-arrow$ E isomerization of the chromophore and changesin its hydrogen bonding with the protein. Moreover, as in plantphytochromes, subtle differences between the PCB and the P $Phi$ B adductof Cph1* can be attributed to the ring D side chain (vinyl groupfor P $Phi$ B versus ethyl group forPCB).

FTRR also indicated different torsions around methine bridges within the chromophore and differences in chromophore/proteininteractions between Cph1* and oat phytochrome. As for other phytochromes,the formation of intermediates during Pr $right-arrow$ Pfr photoconversionof Cph1* was readily observed by flash photolysis and fast spectroscopy.The first photoproduct detected (lumi-R) of Cph1* appeared substantiallymore quickly than that for plant phytochromes and was followedby a novel intermediate whose kinetics were delayed almost 2-foldby ²H exchange, implying that a protonation/deprotonation is involvedat this point. FTIR difference spectra also indicate ²H effects, and a photoreversible pH shift (J. Hughes and J. vanThor, unpublished data) seems to confirm that proton extrusionaccompanies Pfrformation.

Fluorescence measurements at low temperature address the photoconversion from a different point of view. Whereas at ambienttemperature phytochrome fluorescence yields are very low, theserise dramatically upon cooling; Pr $right-arrow$ Pfr photoconversion is inhibited,although photoconversion into intermediate forms is sometimespossible. For plant PHYA at 70 K, up to 50% of the Pr can convertinto lumi-R, whereas this conversion is not possible for plantPHYB. PCB and P $Phi$ B Cph1* adducts are also unable to form lumi-Rat this temperature, implying that Cph1 is more related to PHYBthan to PHYA photochemically. Different activation barriers forthe photoreaction are thought to explain the differences betweenphytochrome types. The only intermediate photoproduct found afterallowing the temperature to rise seemed to be rather differentfrom the lumi-R of plantphytochromes.

	Cph1* IS A LIGHT-DEPENDENT HIS PROTEIN KINASE

TOP

Cph1* IS A LIGHT-DEPENDENT...

The Lagarias group (Yeh et al., 1997) analyzed the biochemistry of Cph1* regarding its apparent homology to two-componentsystems (see Fig. 2B). Cph1* autophosphorylates at the expectedH538_#995, but it was a great surprise that the active kinasewas not Pfr but Pr. This flew in the face of most plant physiologicaldata, which implied that Pfr was the active form. There was moreto come,however.

Unlike eukaryotes, prokaryotes often group biochemically related genes together in a single cistron, thereby keeping the jobof coordinating expression simple while obligingly providing thescientist with clues to unknown biochemical associations. Whilebiochemists had long sought the primary reaction partner(s) forplant phytochrome, the likely reaction partner for Synechocystisphytochrome was clear from the Kazusa chromosome map. Fifteenbases downstream of the Cph1 stop codon begins a short open readingframe, slr0474, unmistakably coding for a 17-kD response regulatorof the two-component type (Lamparter et al., 1997). Yeh et al.(1997) overexpressed this gene in yeast and demonstrated thatthe autophosphorylated Pr form of Cph1* promptly transmitted itsphosphate to the expected Asp residue D68 of the putative responseregulator. slr0474 was thus the first primary reaction partnerfor phytochrome to be identified and was named response regulatorfor cyanobacterial phytochrome, Rcp1. Here again, Pr was moreactive than Pfr. All of the known enzymatic activities of phytochrome(bilin ligase, His autokinase, His-Asp transphosphorylase, and,as we shall see, Ser/Thr kinase) were first described by the Lagariaslaboratory.

Although many response regulators are DNA-binding proteins and act as transcriptional activators, the Synechocystis modelis not quite so simple. Rcp1 has no DNA-binding motifs and thuspresumably acts as an intermediate phosphocarrier in a more complexrelay. This is also seen in other two-component systems such asSpo and Rca. The epithet refers to the transmitter module of thekinase and the receiver module of the response regulator, whereasthe transduction system as a whole can be considerably more extensive,with pathways converging and branching to form a sophisticatedcontrol network. So with what does Rcp1 interact? Unfortunately,in Synechocystis only Cph1 and Rcp1 are co-transcribed, the flankinggenes being read in the opposite direction. As the next partnercannot simply be deduced from the genome map, finding the restof the transduction chain will prove moredifficult.

	Cph1 IN VIVO REMAINS A MYSTERY

TOP

Cph1 IN VIVO REMAINS...

Although Synechocystis certainly contains abundant PCB, the native chromophore of Cph1 is not known. CCA in other cyanobacteriashows well-separated maxima in the blue-green and red regions,whereas the absorbance maxima of PCB and P $Phi$ B adducts of Cph1*are poorly separated and are at longer wavelengths (see above).If a Cph1 homolog is indeed the photoreceptor for CCA, then theblue shift might result from the use of a different chromophore.Alternatively, a quite separate photoreceptor might be involved.Measuring difference spectra in extracts of Synechocystis wasunsuccessful because of masking pigments, even when using PC^- deletion mutants. In an attempt to overcome this, homologousrecombination was used to replace the wild-type chromosomal genewith a sequence extended to provide an oligohistidine-tagged translationproduct similar to that in the E. coli overexpression clones,thereby allowing the photoreceptor to be purified by affinitymethods. However, despite this technology, the extracts have yieldedonly tiny amounts of the modified native Cph1, indicating a verylow expression level. Interestingly, the purified fraction showednot only the expected red/far-red difference spectrum with maximaaround 650 and 700 nm, but also a red/green difference with maximaat 530 and 650 nm, close to the maxima for CCA. Whether the red/greenreversible signal relates to a co-purified protein or directlyto Cph1 remains to be determined (T. Lamparter, A. Wilde, andT. Hübschmann, unpublisheddata).

Ironically, despite all the studies of Cph1 in vitro, its physiological function is unknown: What aspect of the light environmentdoes it perceive and what response does it mediate? This gap inour knowledge is all the more surprising because the efficienthomologous recombination available in Synechocystis allows knockoutmutants to be created with some ease. Indeed, both Cph1 and Cph1/Rcp1 knockouts have been generated in several labs, but an associatedphenotype has yet to be found (D. Scanlan, A. Wilde, and T. Börner,unpublished data). Perhaps the effects are masked by another photoreceptorsystem, or the Cph1-Rcp1 pathway might lead to a physiologicaldead end in this particular strain. One might expect Cph1 to regulateCCA $---$ but, unfortunately, PCC6803 and most other strains of Synechocystislack PE entirely and thus could not show CCA even if they wantedto. However, Synechocystis PCC6701 shows classical CCA, carryinga PE gene cluster closely homologous to that in Fremyella andreplacing PC with PE in blue-green light. The Cph1 homolog inCCA-active Calothrix has also now been cloned (N. Tandeau de Marsac,unpublished data). It will be interesting to see if a cph1 knockout in one of the CCA-active types shows the Rcaphenotype.

	OTHER PROKARYOTIC PHYTOCHROMES HAVE ALSO BEEN IDENTIFIED

TOP

OTHER PROKARYOTIC PHYTOCHROMES...

Several other Synechocystis genes also show similarities to phytochromes. It seems that the true homolog of RcaE is not PlpA(sll1124), as implied above, but, rather, is represented in Cyanobaseby two pseudogenes separated by a transposon (sll11473-sll1475);in other PCC6803 cultures the RcaE homolog is intact (A. Wilde,unpublished data). sll0821 is also intriguing as it shows tworegions with homology to the phytochrome chromophore subdomain,both of which bind PCB in vitro (S.-H. Wu and J.C. Lagarias, unpublisheddata).

Even further removed from plant phytochromes are the BphP (bacterial phytochrome photoreceptor) genes recently found on thechromosomes of Deinococcus radiodurans and Pseudomonas aeruginosa.The former autoassembles with bilin chromophores in vitro to yielda phytochrome-like red/far-red light photochromic product (R.Vierstra and S. Davis, unpublished data). This result is surprisingbecause, although a region resembling the phytochrome chromophoresubdomain is apparent, residue #380 is M rather than C. Whilewe assume that the chromophore attachment site of Cph1 is C259_#380,this has yet to be demonstrated chemically. (See also "Note Addedin Proof")

	FROM PROKARYOTES TO PLANT PHYTOCHROME

TOP

FROM PROKARYOTES TO PLANT...

Most of the above discussion concerns phytochrome in prokaryotes, but how does that help the plant physiologist? Most importantly,it provides conceptual links. First, the alignment of Cph1 toplant phytochromes and HPKs provided a new and clearer view ofphytochrome molecular architecture. Second, while the initialcloning of phytochrome was a great technical achievement in itself,the sequence did not provide us with beguiling homologies to moleculesof known function. Cph1 provides a link not only to bacterialtwo-component systems, but also to several other eukaryotic homologsincluding SLN1, DHKA and DHKB, ETR1, and CKI1, all thought totake part in phosphorelay-mediated signaling. This rejuvenatedthe idea that phytochromes might be light-dependent proteinkinases.

	PLANT PHYTOCHROMES POSSESS A PAS MODULE INVOLVED IN SIGNAL TRANSDUCTION

TOP

PLANT PHYTOCHROMES POSSESS A...

Cph1 alignments revealed an additional approximately 300-residue module peculiar to plant phytochromes, placed between thesensory and transmitter modules (probably between A497_#648and L498_#954, see alignment and Fig. 3). The module is alsomissing from the Deinococcus and Pseudomonas homologs. This regionof the plant phytochrome sequence had already aroused interestsince it contains a repeated motif (#709-#751 and #846-#888) relatedto the PAS³ domain family (Jones and Edgerton, 1994; Lagarias et al., 1995).It would seem that this PAS module was added to a Cph1-like progenitorearly in plant evolution, perhaps even before eukaryotes arose,bringing with it a set of biochemical features probably includinga new signalingmechanism.

PAS domains (see Taylor and Zhulin, 1999) are found in diverse proteins throughout the living world; particularly interestingis the apparent PAS homology of the bacterial photoreceptor PYP(photoactive yellow protein) (Lagarias et al., 1995). PAS domainsoften bind ligands and are involved in protein-protein interactionsincluding signal transduction. There is ambivalent evidence thatthe PAS repeats S599_#675 to L683_#766 and L685_#768to R815_#901 are involved in the dimerization of phytochromeA (Edgerton and Jones, 1993; Quail, 1997b). Furthermore, randommutagenesis studies indicate that the PAS module is crucial tothe plant phytochrome signaling mechanism (Quail et al., 1995).Yeast two-hybrid studies identified several phytochrome interactingfactors that seem likely to bind to the PAS module. One of theseis involved in phytochrome signaling in vivo, is nuclear localized,and even possesses a DNA-binding domain (Ni et al., 1998; Hallidayet al., 1999), offering a remarkably $---$ if not deceptively $---$ simplepicture of plant phytochrome action, given that newly formed Pfrmigrates to the nucleus (Sakamoto and Nagatani, 1996).

Although the plant phytochrome PAS module is missing from Cph1, a Hidden Markov model (http://coot.embl-heidelberg.de/SMART/)detects PAS-domain-related structures in Cph1, RcaE, and PlpAat different positions. A weak but significant similarity betweenHPK modules and the PAS domain has also been pointed out (Yehand Lagarias, 1998), providing the latest twist to an unfinishedstory.

	KINASE AND KINASE-RELATED FUNCTIONS?

TOP

KINASE AND KINASE-RELATED...

As far as kinase function is concerned, the conceptual framework is not simple. As we have seen, athough the H538_#995target in Cph1 and its homologs in other sensory HPKs in bothprokaryotes and eukaryotes is essential for autokinase and phosphorelayfunction, the homologous residue in plant phytochromes is neitherconserved nor functional. While it is nevertheless possible thatplant phytochromes could act as HPKs (for example, some PHYAsshow an H-box-like [L/V][A/P]SHELQ[Q/H]AL_#961-#970 motifat the PAS/transmitter module boundary) and response-regulatorhomologs certainly exist in plants, we emphasize that there isno evidence that any plant phytochrome functions as an HPK. Nevertheless,as we shall see, the HPK transmitter domain seems to be very muchinvolved in signaltransduction.

The two-component paradigm might help us to understand plant phytochrome function independently of the prokaryotic kinaseaction; the H-box is by no means the best conserved of the two-componenttransmitter subdomains in plant phytochromes. The structures mighthave been retained for some purpose other than autophosphorylationand phosphotransfer. The unusual architecture of the chemotaxisHPK CheA suggests twopossibilities.

First, the three-dimensional structure of CheA shows a relict H-box in the conventional position, which, along with downstreamresidues, comprises the K290_#981 to R354_#1055 dimerizationsite (Bilwes et al., 1999). All HPKs seem to form stable dimerswith submicromolar dissociation constants as a result of subunitbinding in this region. Plant phytochromes are also dimers butthe domains involved are uncertain (see Quail, 1997b). The CheAdimerization domain is made up of two antiparallel, highly amphiphilic $alpha$ -helices with hydrophobic residues exposed on the subunit surface;PHD (http://www.embl-heidelberg.de/ predictprotein) predicts thatthis region in plant phytochromes is also largely helical withan amphiphilicpattern.

Second, recent studies (U. Sweere and K. Harter, unpublished data) in Arabidopsis indicate that the N-terminal 100-residuefragment of phytochrome B binds the response regulator homologARR4, whereas the equivalent phytochrome A fragment does not.B-type phytochromes generally bear a characteristic N-terminalextension (#1-#37, see alignment) relative to other family members,so it is possible that the extension mediates the interaction.This would be analogous to the unconventional H-target subdomainat the N terminus of CheA, although little sequence homology isapparent and there is no evidence that the phytochrome is involvedin a phosphotransfer. The system seems to connect to a two-componentsystem involved in hormonesignaling.

Missing a conserved H_#995-target residue in plant phytochromes, it was suggested that the perfectly conserved Y_#991nearby might have taken over the acceptor function (Schneider-Poetschet al., 1991). Tyr and Ser/Thr protein kinases (YPKs and S/TPKs,respectively) work differently from HPKs; after autophosphorylation,rather than donating their own single phosphates, they phosphorylatetheir substrates with phosphate groups from free ATP⁴. There are, however, precedents for protein kinases showing adifferent substrate specificity from that implied by their primarystructure. Moreover, immunological methods suggest a light-regulatedY phosphorylation of oat PHYA (Sommer et al., 1996). Althoughthe residue involved is not known, motifs around Y_#319 andY_#1055 in various phytochromes resemble the phosphotyrosine-bindingsite of SH2 domains. While this appears to be the sum of currentevidence for phytochrome YPK function, the possible relationshipbetween two-component systems and YPK/Ras GTPase signaling (Stockand Lukat, 1991) should encourage a careful search for relatedmechanisms in the case ofphytochrome.

	PLANT PHYTOCHROME IS A DIFFERENT KIND OF KINASE

TOP

PLANT PHYTOCHROME IS A...

Even before the sequence of oat PHYA was published, Quail and co-workers drew attention to its peculiarly S/T-rich N terminusas a possible kinase substrate. This was perhaps born of necessityas it was the only feature of the sequence that hinted at a function.While phytochrome N-terminal sequences are not well conserved,S and T residues predominate $---$ also in Cph1. Indeed, S8_#45 inoat PHYA is phosphorylated; however, the physiological significanceof this is unclear, as the level of phosphorylation is similarfor Pr and Pfr (Lapko et al., 1997). Interestingly, mutation ofthe N-terminal Ser residues in PHYA increases rather than decreasesits physiological potency in transgenic plants, so the phospho-Sermodification might serve to attenuate phytochrome action, analogouslyto arrestin-mediated quenching of rhodopsin (Elich and Chory,1997). In the case of phytochrome it is uncertain whether thisis an autophosphorylation event or whether a separate kinase isinvolved.

The idea that phytochrome might be a S/T kinase bothered biochemists for many years, but recent evidence using recombinantsystems in vitro and in vivo indicates that plant phytochromescan indeed autophosphorylate S/T residues and phosphorylate otherproteins, including Rcp1, in a light-dependent manner (Yeh andLagarias, 1998; Fankhauser et al., 1999; Lapko et al., 1999).Major differences from the Cph1 system should be made clear, however.First, plant Pfr becomes more strongly labeled than Pr, implyingthat the assembled sensory module in its ground state repressesthe autokinase activity $---$ the opposite of Cph1*. Second, althoughplant phytochrome phosphorylated the Rcp1 response regulator invitro, the target was not the D68 used by Cph1*. As histones toowere effective substrates, the relevance of this observation mightbe called into question. However, it seems that the phytochromekinase substrate PKS1 is phosphorylated by Pfr both in vitro andin vivo, with overexpression leading to repression of phytochromeaction. Third, rather than H538_#995, one or more unknown S/Tresidue(s) in plant phytochrome are autophosphorylated. S599_#675of oat PHYA $---$ within the PAS module but N-terminal of the firstrepeat $---$ shows Pfr-enhanced phosphorylation in vivo and would thusseem to be an obvious candidate. However, it is not conservedand the S599K_#675 mutant still autophosphorylates and phosphorylatesPKS1 $---$ although light regulation is lost. In relation to domainfunction it is interesting to note that, while it is probablynot a functional HPK, the plant phytochrome transmitter modulealone is sufficient for PSK1binding.

Three-dimensional structural studies with kinases and their allies are already advanced (for example, Bilwes et al., 1999),providing useful background information regarding possible functionsin phytochrome. Most of the residues directly responsible forATP binding in HPKs and gyrases (GXG_#1166-#1168, GLGL_#1196-#1199and G_#1213) are well conserved in phytochromes-interestinglydeviant are N_#1118 and D_#1168. We look forward to theday when phytochrome will contribute to studies of kinase functioningeneral.

	Pr VERSUS Pfr

TOP

Pr VERSUS Pfr