Two independent, light-sensing two-component systems in a filamentous cyanobacterium.

Two ORFs, cphA and cphB, encoding proteins CphA and CphB with strong similarities to plant phytochromes and to the cyanobacterial phytochrome Cph1 of Synechocystis sp. PCC 6803 have been identified in the filamentous cyanobacterium Calothrix sp. PCC7601. While CphA carries a cysteine within a highly conserved amino-acid sequence motif, to which the chromophore phytochromobilin is covalently bound in plant phytochromes, in CphB this position is changed into a leucine. Both ORFs are followed by rcpA and rcpB genes encoding response regulator proteins similar to those known from the bacterial two-component signal transduction. In Calothrix, all four genes are expressed under white light irradiation conditions, albeit in low amounts. For heterologous expression and convenient purification, the cloned genes were furnished with His-tag encoding sequences at their 3' end and expressed in Escherichia coli. The two recombinant apoproteins CphA and CphB bound the chromophore phycocyanobilin (PCB) in a covalent and a noncovalent manner, respectively, and underwent photochromic absorption changes reminiscent of the P(r) and P(fr) forms (red and far-red absorbing forms, respectively) of the plant phytochromes and Cph1. A red shift in the absorption maxima of the CphB/PCB complex (lambda(max) = 685 and 735 nm for P(r) and P(fr), respectively) is indicative for a noncovalent incorporation of the chromophore (lambda(max) of P(r), P(fr) of CphA: 663, 700 nm). A CphB mutant generated at the chromophore-binding position (Leu246-->Cys) bound the chromophore covalently and showed absorption spectra very similar to its paralog CphA, indicating the noncovalent binding to be the only cause for the unexpected absorption properties of CphB. The kinetics of the light-induced P(fr) formation of the CphA-PCB chromoprotein, though similar to that of its ortholog from Synechocystis, showed differences in the kinetics of the P(fr) formation. The kinetics were not influenced by ATP (probing for autophosphorylation) or by the response regulator. In contrast, the light-induced kinetics of the CphB-PCB complex was markedly different, clearly due to the noncovalently bound chromophore.     

Chromophore selectivity in bacterial phytochromes: dissecting the process of chromophore attachment.

Bacterial phytochromes (Bphs) are ancestors of the well characterized plant photoreceptors. Whereas plant phytochromes perform their photoisomerization exclusively via a covalently bound bilin chromophore, Bphs are variable in their chromophore selection. This is demonstrated in the cyanobacterium Calothrix PCC7601 that expresses two Bphs, CphA and CphB. CphA binds phycocyanobilin (PCB) covalently, whereas CphB, lacking the covalently binding cysteine of the plant phytochromes, carries biliverdin IXalpha (BV) as the chromophore. Our experiments elucidate the different modes of chromophore-protein interaction in CphA and CphB and offer a rationale for their chromophore selectivity. The tight binding of BV by CphB prevents PCB from competing for the binding cavity. Even when the chromophore-binding cysteine has been inserted (CphB-mutant L266C), PCB replaces BV very slowly, indicating the tight, but not irreversible binding of BV. The mutant CphB L266C showed a redox-sensitivity with respect to its PCB binding mode: under reducing conditions, the chromoprotein assembly leads to spectra indicative for a covalent binding, whereas absence of dithiothreitol or its removal prior to assembly causes spectra indicative for noncovalent binding. Regarding the CphB-type Bphs lacking the covalently binding cysteine, our results support the involvement of the succeeding histidine residue in chromophore fixation via a Schiff base-like bond between the bilin A-ring carbonyl and the histidine imidazole group. The assembly process and the stability of the holo-proteins were strongly influenced by the concentration of added imidazole (mimicking the histidine side-chain), making the attachment of the chromophore via the histidine more likely than via another cysteine of the protein.     

A Non-hydrolyzable ATP Derivative Generates a Stable Complex in a Light-inducible Two-component System

Isothermal calorimetry (ITC) measurements yielded the binding constants during complex formation of light-inducible histidine kinases (HK) and their cognate CheY-type response regulators (RR). HK-RR interactions represent the core function of the bacterial two-component system, which is also present in many bacterial phytochromes. Here, we have studied the recombinant forms of phytochromes CphA and CphB from the cyanobacterium Tolypothrix PCC7601 and their cognate RRs RcpA and RcpB. The interaction between the two reaction partners (HK and RR) was studied in the presence and absence of ATP. A complex formation was observable in the presence of ATP, but specific interactions were only found when a non-hydrolyzable ATP derivative was added to the mixture. Also, the incubation of the HK domain alone (expressed as a recombinant protein) with the RR did not yield specific interactions, indicating that the HK domain is only active as a component of the full-length phytochrome. Considering also previous studies on the same proteins (Hübschmann, T., Jorissen, H. J. M. M., Börner, T., Gärtner, W., and de Marsac, N. (2001) Eur. J. Biochem. 268, 3383–3389) we now conclude that the HK domains of these phytochromes are active only when the chromophore domain is in its Pr form. The formerly documented phosphate transfer between the HK domain and the RR takes place via a transiently formed protein-protein complex, which becomes detectable by ITC in the presence of a non-hydrolyzable ATP derivative. This finding is of interest also in relation to the function of some (blue light-sensitive) photoreceptors that carry the HK domain and the RR fused together in one single protein.     

Phosphorylation of proteins in the light-dependent signalling pathway of a filamentous cyanobacterium.

The genome of the filamentous cyanobacterium Calothrix sp. PCC7601 contains two genes, cphA and cphB, encoding proteins with similarity to plant phytochromes and bacterial histidine kinases. In vitro, CphA and CphB readily attach a tetrapyrrole chromophore to develop spectrally active holoproteins that are photointerconvertible between a red light-absorbing and a far-red light-absorbing form. Together with the putative response regulators, RcpA and RcpB, the putative histidine kinases, CphA and CphB, are suggested to constitute two two-component systems of light-dependent signal transduction. In this report, we demonstrate the kinase activity of both CphA and CphB. In vitro experiments carried out on the purified proteins show that CphA and CphB are autophosphorylated in the presence of ATP and that phospho-CphA is capable of efficient phosphotransfer to RcpA as is phospho-CphB towards RcpB. The autophosphorylation and the phosphorelay are dependent on light. Both activities are reduced under red light vs. far-red light irradiation. No phosphoryl transfer occurred between phospho-CphA and RcpB or between phospho-CphB and RcpA. The response regulators RcpA and RcpB can receive a phosphoryl moiety also from the small phospho-donor acetyl phosphate. The stability of the phosphorylated regulators is not affected by CphA and CphB or light.     

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

Phytochromes are photoreceptors using a bilin tetrapyrrole as chromophore, which switch in canonical phytochromes between red (P_r) and far red (P_fr) light-absorbing states. Cph2 from Synechocystis sp., a noncanonical phytochrome, harbors besides a cyanobacteriochrome domain a second photosensory module, a P_r/P_fr-interconverting GAF-GAF bidomain (SynCph2(1-2)). As in the canonical phytochromes, a unique motif of the second GAF domain, the tongue region, seals the bilin-binding site in the GAF1 domain from solvent access. Time-resolved spectroscopy of the SynCph2(1-2) module shows four intermediates during P_r → P_fr phototransformation and three intermediates during P_fr → P_r back-conversion. A mutation in the tongue''s conserved PRXSF motif, S385A, affects the formation of late intermediate R3 and of a P_fr-like state but not the back-conversion to P_r via a lumi-F-like state. In contrast, a mutation in the likewise conserved WXE motif, W389A, changes the photocycle at intermediate R2 and causes an alternative red light-adapted state. Here, back-conversion to P_r proceeds via intermediates differing from SynCph2(1-2). Replacement of this tryptophan that is ∼15 Å distant from the chromophore by another aromatic amino acid, W389F, restores native P_r → P_fr phototransformation. These results indicate large scale conformational changes within the tongue region of GAF2 during the final processes of phototransformation. We propose that in early intermediates only the chromophore and its nearest surroundings are altered, whereas late changes during R2 formation depend on the distant WXE motifs of the tongue region. Ser-385 within the PRXSF motif affects only late intermediate R3, when refolding of the tongue and docking to the GAF1 domain are almost completed.     

Femtosecond kinetics of photoconversion of the higher plant photoreceptor phytochrome carrying native and modified chromophores

The photoprocesses of native (phyA of oat), and of C-terminally truncated recombinant phytochromes, assembled instead of the native phytochromobilin with phycocyanobilin (PCB-65 kDa-phy) and iso-phycocyanobilin (iso-PCB-65 kDa-phy) chromophores, have been studied by femtosecond transient absorption spectroscopy in both their red absorbing phytochrome (P_r) and far-red absorbing phytochrome (P_fr) forms. Native P_r phytochrome shows an excitation wavelength dependence of the kinetics with three main picosecond components. The formation kinetics of the first ground-state intermediate I₇₀₀, absorbing at ∼690 nm, is mainly described by 28 ps or 40 ps components in native and PCB phytochrome, respectively, whereas additional ∼15 and 50 ps components describe conformational dynamics and equilibria among different local minima on the excited-state hypersurface. No significant amount of I₇₀₀ formation can be observed on our timescale for iso-PCB phytochrome. We suggest that iso-PCB-65 kDa-phy either interacts with the protein differently leading to a more twisted and/or less protonated configuration, or undergoes P_r to P_fr isomerization primarily via a different configurational pathway, largely circumventing I₇₀₀ as an intermediate. The isomerization process is accompanied by strong coherent oscillations due to wavepacket motion on the excited-state surface for both phytochrome forms. The femto- to (sub-)nanosecond kinetics of the P_fr forms is again quite similar for the native and the PCB phytochromes. After an ultrafast excited-state relaxation within ∼150 fs, the chromophores return to the first ground-state intermediate in 400-800 fs followed by two additional ground-state intermediates which are formed with 2-3 ps and ∼400 ps lifetimes. We call the first ground-state intermediate in native phytochrome I_fr·750, due to its pronounced absorption at that wavelength. The other intermediates are termed I_fr·675 and pseudo-P_r. The absorption spectrum of the latter already closely resembles the absorption of the P_r chromophore. PCB-65 kDa-phy shows a very similar kinetics, although many of the detailed spectral features in the transients seen in native phy are blurred, presumably due to wider inhomogeneous distribution of the chromophore conformation. Iso-PCB-65 kDa-phy shows similar features to the PCB-65 kDa-phy, with some additional blue-shift of the transient spectra of ∼10 nm. The sub-200 fs component is, however, absent, and the picosecond lifetimes are somewhat longer than in 124 kDa phytochrome or in PCB-65 kDa-phy. We interpret the data within the framework of two- and three-dimensional potential energy surface diagrams for the photoisomerization processes and the ground-state intermediates involved in the two photoconversions.     

Conserved tyrosine in phytochromes controls the photodynamics through steric demand and hydrogen bonding capabilities

Using ultrafast spectroscopy and site-specific mutagenesis, we demonstrate the central role of a conserved tyrosine within the chromophore binding pocket in the forward (P_r → P_fr) photoconversion of phytochromes. Taking GAF1 of the knotless phytochrome All2699g1 from Nostoc as representative member of phytochromes, it was found that the mutations have no influence on the early (<30 ps) dynamics associated with conformational changes of the chromophore in the excited state. Conversely, they drastically impact the extended protein-controlled excited state decay (>100 ps). Thus, the steric demand, position and H-bonding capabilities of the identified tyrosine control the chromophore photoisomerization while leaving the excited state chromophore dynamics unaffected. In effect, this residue operates as an isomerization-steric-gate that tunes the excited state lifetime and the photoreaction efficiency by modulating the available space of the chromophore and by stabilizing the primary intermediate Lumi-R. Understanding the role of such a conserved structural element sheds light on a key aspect of phytochrome functionality and provides a basis for rational design of optimized photoreceptors for biotechnological applications.     

Phytochrome of the green alga Mougeotia: cDNA sequence, autoregulation and phylogenetic position

A cDNA clone encoding phytochrome (apoprotein) of the zygnematophycean green alga Mougeotia scalaris has been isolated and sequenced. The clone consisted of 3372 bp, encoded 1124 amino acids, and showed strain-specific nucleotide exchanges for M. scalaris, originating from different habitats. No indication was found of multiple phytochrome genes in Mougeotia. The 5 non-coding region of the Mougeotia PHY cDNA harbours a striking stem-loop structure. Homologies with higher-plant phytochromes were 52–53% for PHYA and 57–59% for PHYB. Highest homology scores were found with lower-plant phytochromes, for example 67% for Selaginella (Lycopodiopsida), 64% for Physcomitrella (Bryopsida) and 73% for Mesotaenium (Zygnematophyceae). In an unrooted phylogenetic tree, the position of Mougeotia PHY appeared most distant to all other known PHYs. The amino acids Gly-Val in the chromophore-binding domain (-Arg-Gly-Val-His-Gly-Cys-) were characteristic of the zygnematophycean PHYs known to date. There was no indication of a transmembrane region in Mougeotia phytochrome in particular, but a carboxyl-terminal 16-mer three-fold repeat in both, Mougeotia and Mesotaenium PHYs may represent a microtubule-binding domain. Unexpected for a non-angiosperm phytochrome, its expression was autoregulated in Mougeotia in a red/far-red reversible manner: under P_r conditions, phytochrome mRNA levels were tenfold higher than under P_fr conditions.     

A Polarity Probe for Monitoring Light-induced Structural Changes at the Entrance of the Chromophore Pocket in a Bacterial Phytochrome

Light-induced structural changes at the entrance of the chromophore pocket of Agp1 phytochrome were investigated by using a thiol-reactive fluorescein derivative that is covalently attached to the genuine chromophore binding site (Cys-20) and serves as a polarity probe. In the apoprotein, the absorption spectrum of bound fluorescein is red-shifted with respect to that of the free label suggesting that the probe enters the hydrophobic chromophore pocket. Assembly of this construct with the chromophores phycocyanobilin or biliverdin is associated with a blue-shift of the fluorescein absorption band indicating the displacement of the probe out of the pocket. The probe does not affect the photochromic and kinetic properties of the noncovalent bilin adducts. Upon photoconversion to P_fr, the probe spectrum undergoes again a bathochromic shift and a strong rise in CD indicating a more hydrophobic and asymmetric environment. We propose that the environmental changes of the probe reflect conformational changes at the entrance of the chromophore pocket and are indicative for rearrangements of the chromophore ring A. Flash photolysis measurements showed that the absorption changes of the probe are kinetically coupled to the formation of Meta-R_C and P_fr. In the biliverdin adduct, an additional component occurs that probably reflects a transition between two Meta-R_C substates. Analogous results to that of the noncovalent phycocyanobilin adduct were obtained with the mutant V249C in which probe and chromophore are covalently attached. The conformational changes of the chromophore are correlated to proton transfer to the protein surface.Phytochromes are red-light photoreceptors occurring in plants, bacteria, and fungi where they control important developmental processes (1–6). The discovery of microbial phytochromes from genome sequencing (7–9) provided new prospects for biochemical, spectroscopic and structural analyses of this light sensor family. Agp1 (AtBphP1)³ from the soil bacterium Agrobacterium tumefaciens is a typical member of the widespread family of proteobacterial phytochromes (10, 11) and is the subject of the present study.The domain arrangement of canonical phytochromes consists of an N-terminal photosensory domain, including PAS, GAF, and PHY domains and a C-terminal regulatory kinase domain (see, e.g. Ref. 3). Bacterial phytochromes lack the N-terminal extension, and the PAS module insertion of plant phytochromes (3). In most of the bacterial phytochromes, the C-terminal regulatory domain is a histidine kinase (4). These kinases form homodimers as functional units (12) where the subunits transphosphorylate each other (13). The cofactors are linear tetrapyrroles that are covalently attached via a thioether linkage (14) to the side chains of specific conserved cysteine residues. The native chromophore of plant phytochromes is phytochromobilin (PΦB) (14), some cyanobacterial phytochromes incorporate phycocyanobilin (PCB) (15, 16), and all other bacterial phytochromes bind biliverdin (BV) (10, 11). Whereas the chromophore binding site of the more reduced bilins PΦB and PCB is located in the GAF domain, the binding site of BV is close to the N terminus upstream of the PAS domain (4, 11). The two distinct binding sites apparently require a specific substituent at the C3 carbon of pyrrole ring A, either an ethylidene (PΦB and PCB) or a vinyl (BV) group, for covalent attachment of the bilin chromophore (4). The holophytochrome assembly that includes covalent attachment of the chromophore is an autocatalytic process implying an intrinsic bilin C-S lyase activity of the apophytochrome (17). Kinetic studies of the autoassembly in vitro showed that ligation of the chromophore is the ultimate step following incorporation in the binding pocket and internal protonation (18).Phytochromes display photochromicity involving two either thermally stable or long-lived states, P_r and P_fr (red and far-red absorbing forms), that can be reversibly converted by light of appropriate wavelengths. The P_r to P_fr photoconversion is initiated by a rapid Z/E isomerization of the C-D methine bridge of the bilin chromophore (19–22) leading within picoseconds to the formation of the Lumi-R intermediate (23, 24). The following thermal relaxations via Meta-R_A and Meta-R_C intermediates to P_fr proceed on the time scale of microseconds and milliseconds (25–28).Assembly of Agp1 with locked BV derivatives showed that the geometry of the C-D methine bridge is 15Z_anti in P_r and 15E_anti in P_fr (29) suggesting that this methine bridge remains in the anti conformation during photoconversion. The crystal structures of the chromophore binding domains of the bacteriophytochromes from Deinococcus radiodurans and Rhodopseudomonas palustris revealed that the BV chromophore adopts a 5Z_syn,10Z_syn,15Z_anti configuration/conformation in the P_r state (30–32). The 5Z_syn geometry of the A-B methine bridge in the P_r state was confirmed by assembly of Agp1 with the corresponding locked BV chromophore (33). Recently, heteronuclear NMR investigations and crystallographic studies on the complete photosensory domain of the cyanobacterial phytochrome Cph1 from Synechocystis showed that the PCB chromophore is also in the 5Z_syn,10Z_syn,15Z_anti geometry in P_r (34, 35).Because the locked 5Z_syn adduct of Agp1 did not show a P_fr-like photo-product, conformational changes of the A-B methine bridge in the thermal relaxation cascade have been predicted (33). Flash photolysis experiments with this adduct suggested that these changes occur in the Meta-R_A to Meta-R_C transition (36). The stereochemistry of the A-B methine bridge in the P_fr state and in the preceding intermediates could not be determined unambiguously yet. Recent studies with doubly locked chromophores suggest that the C5–C6 single bond undergoes a thermal rotation from syn to anti in the photoconversion of Agp1, whereas an additional Z/E isomerization around the C4C5 double bond (hula-twist mechanism) was postulated for Agp2 (37). However, the crystal structure of the photosensory domain of the bacteriophytochrome PaBphP in its P_fr-enriched dark-adapted state favors the 5Z_syn conformation of the BV chromophore (38). Structural changes of the A-B methine bridge were excluded for the PCB chromophore of Cph1 on the basis of heteronuclear NMR (34), whereas low temperature Fourier transform IR studies on plant phytochrome suggested an environmental change of the ring A carbonyl group and/or a twist of the A-B methine bridge (39).The mechanism by which the signal is transmitted from the bilin chromophore to the protein is still obscure. The recent three-dimensional structures of the complete photosensory domains of Cph1 (35) and PaBphP (38) reveal key interactions between GAF and PHY domains in the corresponding dark states reflecting P_r and P_fr, respectively. In view of the intrinsic differences between the two phytochromes, it is not trivial to differentiate which of the numerous structural differences arise from light-induced conformational changes and are thus potentially important for signal transmission. We note that many approaches to provide a clue on the mechanism of signal transmission from the bilin chromophore to its proximate environment imply that this process is exclusively coupled to the photo-isomerization localized at ring D and its environment and that the chromophore then remains a passive element in the thermal relaxation cascade. This point of view is supported by recent results from femtosecond stimulated Raman spectroscopy suggesting that the chromophore structures in Lumi-R and P_fr are very similar (24). On the other hand, size exclusion chromatography experiments demonstrated that the global conformational changes observed for the P_fr state of Agp1 WT are absent in constructs (locked 5Zs adduct and mutants D197A and H250A), where the formation of P_fr is inhibited but the primary photoreaction proceeds (33, 40). These results are difficult to explain in terms of an ultra-fast signal transmission from the chromophore to the surrounding residues in its pocket.Light-induced conformational changes at the surface of plant phytochrome were observed by using covalently attached labels that are sensitive to the polarity of the microenvironment (41, 42). Due to the accessibility of several binding sites (i.e. the sulfhydryl groups of cysteines) in these experiments, the labeling was unspecific preventing further assignment of the observed changes to particular regions of the protein. Time-resolved absorption measurements with a covalently attached fluorescein derivative showed that the changes occur in the Meta-R_C to P_fr transition (41). In the present work with Agp1 phytochrome, we take advantage of the highly reactive sulfhydryl group of Cys-20, the genuine binding site of the BV chromophore, to specifically attach a fluorescein derivative. We observed that this construct assembles with PCB and BV forming noncovalent photochromic adducts, spectrally and kinetically undisturbed by the fluorescein label. Upon photo-conversion, the absorption band of the label displays a bathochromic shift and increase in ellipticity suggesting that the label moves in a more hydrophobic and asymmetric environment in the P_fr state. The label thus serves as a polarity probe at the entrance of the binding pocket. We postulate that these polarity changes reflect conformational changes of the A-B methine of the bilin chromophore and/or the microenvironment of ring A at the entrance of the binding pocket. Time-resolved measurements reveal that the changes occur in the Meta-R_A to Meta-R_C and Meta-R_C to P_fr transitions. Analogous results were obtained with the V249C mutant of Agp1 in which both the fluorescein probe and the PCB chromophore are covalently attached.     

Die Photomodulation der Akkumulationsrate von Ascorbinsäure beim Senfkeimling (Sinapis alba L.) durch Phytochrom

Summary Ascorbic acid accumulation in the mustard seedling is controlled by P _fr(active phytochrome). Kinetic studies demonstrate that P _frexerts a rapid and fully reversible control over the steady state rate of ascorbic acid accumulation. Following the terminology of Weisz (1967) for this type of metabolic control the term photomodulation by P _fr is used.—The control by P _fris independent of RNA synthesis. Therefore regulation of gene activity is probably not involved in photomodulation of the rate of ascorbic acid accumulation.—There is only a limited period within which P _frcan control ascorbic acid accumulation. This period is fixed by the time pattern of primary differentiation in the seedling. There is no interaction between photomodulation by P _frand control by primary differentiation of ascorbic acid accumulation.     

Phytochrome behaves as a dimer in vivo

Abstract It is well established that phytochrome exists as a dimer in vitro. A comparison of the relative photoequilibrium concentrations of P_rP_r, P_rP_fr and P_frP_fr, with the relative sizes of the P_fr-pools which undergo dark reversion in the intact plant, leads to the hypothesis that phytochrome also exists as a dimer in vivo, This hypothesis is in accordance with kinetic properties of the phytochrome system under continuous irradiation. Additional support for this view is provided by the observation that P_fr-destruction after a red light flash, which should favour the formation of P_rP_fr dimers, is paralleled by a decay of P_r, even if the presence of P_r cycled through P_fr can be excluded. Preliminary observations could indicate an interaction of the subunits of a phytochrome dimer during the process of phototransformation.     

Phytochrome in seeds of Amaranthus caudatus

Summary Dry seeds of Amaranthus caudatus show little or no photoreversible absorption changes, attributable to phytochrome. During imbibition phytochrome appears in two phases, one immediately after sowing and the second after about 8 hr. Experiments at different temperatures and under continuous illumination with red, far-red and blue light suggest that there are two pools of phytochrome. The first phase in the appearance of phytochrome could be due to the change in optical properties of the sample on hydration or to rehydration of inactive phytochrome, or both. The second phase probably represents phytochrome synthesis. It is absent at 0° and precedes the water uptake associated with germination by some 10 hr. This second pool of phytochrome does not accumulate in red and blue illuminated seeds indicating that the rate of P _fr decay is more rapid than the rate of phytochrome synthesis. The difference spectra of phytochrome in both 2 hr imbibed seeds and 72 hr old seedlings show peaks of absorption at 663 and 735 nm. The presence of P _fr in dark imbibed seeds and the process of inverse reversion of P _r to P _fr in darkness have been demonstrated. The results are discussed in relation to previous hypotheses for the mechanism of photocontrol of Amaranthus seed germination.     

The photoactivation energy of the visual pigment in two spectrally different populations of <Emphasis Type="Italic">Mysis relicta</Emphasis> (Crustacea,Mysida)

We report the first study of the relation between the wavelength of maximum absorbance (λ_max) and the photoactivation energy (E _a) in invertebrate visual pigments. Two populations of the opossum shrimp Mysis relicta were compared. The two have been separated for 9,000 years and have adapted to different spectral environments (“Sea” and “Lake”) with porphyropsins peaking at λ_max=529 nm and 554 nm, respectively. The estimation of E _a was based on measurement of temperature effects on the spectral sensitivity of the eye. In accordance with theory (Stiles in Transactions of the optical convention of the worshipful company of spectacle makers. Spectacle Makers’ Co., London, 1948), relative sensitivity to long wavelengths increased with rising temperature. The estimates calculated from this effect are E _a,529=47.8±1.8 kcal/mol and E _a,554=41.5±0.7 kcal/mol (different at P<0.01). Thus the red-shift of λ_max in the “Lake” population, correlating with the long-wavelength dominated light environment, is achieved by changes in the opsin that decrease the energy gap between the ground state and the first excited state of the chromophore. We propose that this will carry a cost in terms of increased thermal noise, and that evolutionary adaptation of the visual pigment to the light environment is directed towards maximizing the signal-to-noise ratio rather than the quantum catch.     

Etude du phytochrome des graines de Pinus nigra Arn par spectrophotométrie bichromatique in vivo

Summary Phytochrome photoconversions P_rP_fr and P_frP_r can be measured by differential spectrophotometry in dry seeds (6% water content) of Pinus nigra Arn. A red light irradiation given before imbibition induces germination when the seeds are subsequently wetted and kept in darkness.In continuous darkness the phytochrome content shows a drastic increase at the beginning of moistening.The detectable pigment is entirely in the P_r form. The normal P_frP_r dark reversion is observed. P_fr destruction does not take place.     

The role of phytochrome in photoperiodic time measurement and its relation to rhythmic timekeeping in the control of flowering in Chenopodium rubrum

Summary To follow changes in the status of phytochrome in green tissue and to relate these changes to the photoperiodic control of flowering, we have used a null response technique involving 1.5-min irradiations with mixtures of different ratios of R and FR radiation.Following a main photoperiod of light from fluorescent lamps that was terminated with 5 min of R light, the proportion of P_fr in Chenopodium rubrum cotyledons was high and did not change until the 3rd hour in darkness; at this time, P_fr disappeared rapidly. When the dark period began with a 5-min irradiation with BCJ or FR light to set the proportion of P_fr low P_fr gradually reappeared during the first 3 h of darkness and then disappeared again.The timing of disappearance of P_fr is consistent with the involvement of phytochrome in photoperiodic time measurement. Reappearance of P_fr after an initial FR irradiation explains why FR irradiations sometimes fail to influence photoperiodic time measurement or only slightly hasten time measurement. A R light interruption to convert P_r to P_fr delayed, the timer by 3 h but only for interruptions after and not before the time of P_fr disappearance. Such 5-min R-light interruptions did not influence the operation of the rhythmic timekeeping mechanism. Continuous or intermittent-5 min every 1.5 h-irradiations of up to 6 h in duration were required to rephase the rhythm controlling flowering. A skeleton photoperiod of 6 h that was began and terminated by 5 or 15 min of light failed to rephase the rhythm.The shape of the curves for the rhythmic response of C. rubrum to the length of the dark period are sometimes suggestive of clocks operating on the principle of a tension-relaxation mechanism. Such a model allows for separate timing action of a rhythm and of P_fr disappearance over the early hours of darkness. Separate timing action does not, however, preclude an interaction between the rhythm and phytochrome in controlling flowering.Abbreviations FR far-red - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - R red - BCJ photographic ruby-red irradiation A grant in aid of research from the National Research Council of Canada to B. G. Cumming is gratefully acknowledged.     

Structure of the Cyanobacterial Phytochrome 2 Photosensor Implies a Tryptophan Switch for Phytochrome Signaling

Phytochromes are highly versatile photoreceptors, which occur ubiquitously in plants as well as in many light-responsive microorganisms. Here, photosynthetic cyanobacteria utilize up to three different phytochrome architectures, where only the plant-like and the single-domain cyanobacteriochromes are structurally characterized so far. Cph2 represents a third group in Synechocystis species and affects their capability of phototaxis by controlling c-di-GMP synthesis and degradation. The 2.6-Å crystal structure of its red/far-red responsive photosensory module in the P_r state reveals a tandem-GAF bidomain that lacks the figure-of-eight knot of the plant/cph1 subfamily. Its covalently attached phycocyanobilin chromophore adopts a highly tilted ZZZssa conformation with a novel set of interactions between its propionates and the GAF1 domain. The tongue-like protrusion from the GAF2 domain interacts with the GAF1-bound chromophore via its conserved PRXSF, WXE, and W(G/A)G motifs. Mutagenesis showed that the integrity of the tongue is indispensable for P_r → P_fr photoconversion and involves a swap of the motifs'' tryptophans within the tongue-GAF1 interface. This “Trp switch” is supposed to be a crucial element for the photochromicity of all multidomain phytochromes.     

Photoperiodism and rhythmic response to light

Abstract. Seedlings of Pharhitis nil show a circadian rhythm in the capacity to flower in response to the timing of a second red light pulse given at various times after a first saturating exposure to red when this is given together with a benzyladeninc spray. There are also changes in the photon irradiance required for half maximum response to the second red pulse. The photochemical properties of phytochrome in the photoperiodically sensitive cotyledons were also shown to change rhythmically. Oscillations in both p_r→ P_fr and P_fr→ P_r photoconversion characteristics persisted over at least two circadian cycles with a periodicity of about 12 h. There were, however, no significant oscillations in either P_fr peak absorbance or in Δ(ΔA). The changes in sensitivity for the photoconversion of P_r→ P_fr did not parallel the much larger changes in sensitivity of the flowering response to red light. The amplitude of the P_r→ P_fr rhythm was at least as great as that for P_r→ P_fr, but the flowering response to far-red light was not rhythmic, nor was there any large change in sensitivity. The changes in photoconversion properties may reflect a basic biochemical oscillation which affects both photoreceptor properties and sensitivity to photoreceptor input. There was also a marked rhythm in the P_fr/P ratio that would be established by a saturating pulse of red light and this too may have affected the flowering response to such a pulse. Far-red light inhibited flowering when given at any time during the inductive night. After 14 h in darkness, P_fr could still be measured in the cotyledons and it was concluded that far-red light inhibited flowering by removing P_fr As red light also inhibited flowering at this time, there may be two pools of phytochrome with different kinetic properties.     

The phytochrome system in light-grown Zea mays L.

The phytochrome system is analyzed in light-grown maize (Zea mays L.) plants, which were prevented from greening by application of the herbicide SAN 9789. The dark kinetics of phytochrome are not different in the first, second or third leaf. It is concluded that in light-grown maize plants phytochrome levels are regulated by P_r formation and P_fr and P_r destruction, rather than by P_frP_r dark reversion. P_r undergoes destruction after it has been cycled through P_fr. The consequences of this P_r destruction on the phytochrome system are discussed.Abbreviations SAN 9789 4-chloro-5-(methylamino)-2-(,,-trifluoro-m-tolyl)-3(2H) pyridazinone - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot P_fr+P_r     

A detailed analysis of phytochrome decay and dark reversion in mustard cotyledons

Summary During the first 10 min after a saturating dose of red light, 72 h dark-grown mustard cotyledons show no phytochrome decay. Within the same time interval there exists a transient form of P _fr (=P _fr ^T ) which is no longer photoconvertible at 0°C, but is at 25°C. This P _fr ^T converts in the dark to P _fr and P _r . These dark reversions take about 10 min. After a lag phase of 10 min the P _fr decay can be described by a single, first order kinetic curve. The time courses of these reactions are functions of the time of etiolation.Research supported by DAAD and by Deutsche Forschungsgemeinschaft (SFB 46).