EVIDENCE FOR THE EXISTENCE OF MEMBRANE-ASSOCIATED PHYTOCHROME IN THE CELL

Abstract Comparative fluorescence and photochemical studies of phytochrome in etiolated seedlings of maize and in soluble and membrane-containing fractions isolated from them were camed out. The membrane fractions prepared in the absence of Mg²⁺ from etiolated coleoptiles contained 13% of total photoreversible phytochrome, which was readily solubilized by mild detergents. Its molecular size was indistinguishable from soluble phytochrome and equal to nondegraded maize phytochrome. Low-temperature fluorescence studies with intact tissue found that the position of the emission maximum at 85 K (λ_max) and the extent of the phototransformation of the red-absorbing form (Pr) into the first stable photoproduct, lumi-R, at 85 K (γ₁), varied in different parts of etiolated seedlings: λ_max and γ₁ reached their maximum values in the tips of coleoptiles and roots, 686 nm and 0.30–0.40, whereas the lowest values, 682 nm and ca 0.05, were observed in the root base. These parameters correlated well with those obtained for the pigment in the soluble and membrane-containing fractions: 684 and 680 nm, and 0.33 and 0.06, respectively. The extent of the Pr phototransformation into the far red-absorbing form (Pfr) (γ₂) did not differ much: values of 0.80–0.85 and 0.70–0.75 correlated with the high and low values of γ₁. These variations of the parameters were interpreted in agreement with our previous observations in terms of two phytochrome A species whose relative concentrations vary depending on the experimental conditions—the longer wavelength bulk light-labile species with high γ₁ (Pr″), and the shorter wavelength minor light-stable species with low γ₁ (Pr″). Close similarity between Pr’and the soluble phytochrome and between Pr″ and the membrane-bound phytochrome points to the possible origin of the native Pr’and PrPrime; species, thus providing evidence for the existence of membrane-bound pigment in the cell.     

PHYTOCHROME STATES IN ETIOLATED PEA SEEDLINGS: FLUORESCENCE AND PRIMARY PHOTOREACTIONS AT LOW TEMPERATURES

Emission spectra of the red phytochrome form (Pr) and fluence time-response curves of the Pr fluorescence intensity changes were measured in etiolated pea seedlings at low temperatures (80–150 K) in connection with its phototransformations into the initial photoproduct (Lr) and back upon actinic red (667 nm) and far-red (696 nm) illumination. The variable fluorescence reaches 45% at 85 K and decreases with the rise of temperature. Three kinetic components of the changes were found in the direct (Pr→Lr) and back (Lr→Pr) photoreactions belonging to three states of phytochrome: “slow”, “fast” and “very fast” (respective indices: s, f and vf). The amplitudes of the components and rate constants to reach photoequilibrium were determined in the direct and back photoreactions at different temperatures, and from this, their quantum yields, extent of the Pr?Lr phototransformation and activation energy of the reactions were evaluated for the three Pr and Lr states. The yields differ from each other by approximately a factor of 10 and those for the direct and back photoreactions are close to each other. The proportion of the amplitudes of the variable fluorescence of the three phytochrome states changes with temperature and upon the Pr→Lr photo-transformation and the Pr states differ in the position of their emission spectra by 3–5 nm. A close similarity between the Pr and Lr properties was observed, which implies a symmetrical scheme of their photoreactions. It is suggested that the three phytochrome species may originate in different conformational states of the chromophore and they independently transform in parallel photoreactions into the respective photoproducts: Prvf?Lrvf, Prf?Lrf and Prs?Lrs.     

Fluorescence and Photochemistry of Recombinant Phytochrome from the Cyanobacterium Synechocystis

Fluorescence and photochemical properties of phytochrome from the cyanobacterium Synechocystis were investigated in the temperature interval from 293 to 85 K. The apoprotein was obtained by overexpression in Escherichia coli and assembled to a holophytochrome with phycocyanobilin (PCB) and phytochromobilin (PφB), Syn(PCB)phy and Syn(PφB)phy, respectively. Its red-absorbing form, Pr, is characterized at 85 K by the emission and excitation maxima at 682 and 666 nm in Syn(PCB)phy and at 690 and 674 nm in Syn(PφB)phy. At room temperature, the spectra are blue shifted by 5–10 nm. The fluorescence intensity dropped down by ?15–20-fold upon warming from 85 to 293 K and activation energy of the fluorescence decay was estimated to be ca 5.4 and 4.9 kJ mol^?1 in Syn(PCB)phy and Syn(PφB)phy, respectively. Phototransformation of Pr upon red illumination was observed at temperatures above 160–170 K in Syn(PCB)phy and above 140–150 K in Syn(PφB)phy with a 2–3 nm shift of the emission spectrum to the blue and increase of the intensity of its shorter wavelength part. This was interpreted as a possible formation of the photoproduct of the meta-Ra type of the plant phytochrome. At ambient temperatures, the extent of the Pr phototransformation to the far-red-absorbing form, Pfr, was ca 0.7–0.75 and 0.85–0.9 for Syn(PCB)phy and Syn(PφB)phy, respectively. Fluorescence of Pfr and of the photoproduct similar to lumi-R was not observed. With respect to the photochemical parameters, Syn(PCB)phy and Syn(PφB)phy are similar to each other and also to a small fraction of phyA (phyA″) and to phyB. The latter were shown to have low photochemical activity at low temperatures in contrast to the major phyA pool (phyA″), which is distinguished by the high extent (ca 50%) of Pr photoransformation at 85 K. These photochemical features are interpreted in terms of different activation barriers for the photoreaction in the Pr excited state.     

THE EFFECT OF ACIDS ON THE INFRARED SPECTRA OF SCHIFF BASES—II. IMINES CONTAINING THE C=C-C=N AND C=C-C=C-C=N UNITS

Abstract— The Fourier-transform infrared spectra of chloroform-d solutions of conjugated imines CH₃CH=CHCH=NCH(CH₃)₂ and CH₃CH₂CH=CHCH=CHCH=NCH(CH₃)₂ and the related protonated species with HCl, HBr, HI, trichloro, dichloro, monobromo and monochloroacetic acids or propionic acid are presented. The effects of conjugation and protonation are examined. The results show that conjugation slightly increases the basicity of the Schiff bases. HCl, HBr and HI protonate the Schiff bases completely. The carboxylic acids protonate partially depending on their p K _a, values. When the Schiff base contains two (or more) C=C bonds conjugated with C=N, the main C=C stretching band undergoes a strong intensification showing that sizeable dipole moment variations occur along the conjugated chain.     

Diversity of Chlamydomonas channelrhodopsins

Channelrhodopsins act as photoreceptors for control of motility behavior in flagellates and are widely used as genetically targeted tools to optically manipulate the membrane potential of specific cell populations ("optogenetics"). The first two channelrhodopsins were obtained from the model organism Chlamydomonas reinhardtii (CrChR1 and CrChR2). By homology cloning we identified three new channelrhodopsin sequences from the same genus, CaChR1, CyChR1 and CraChR2, from C. augustae, C. yellowstonensis and C. raudensis, respectively. CaChR1 and CyChR1 were functionally expressed in HEK293 cells, where they acted as light-gated ion channels similar to CrChR1. However, both, which are similar to each other, differed from CrChR1 in current kinetics, inactivation, light intensity dependence, spectral sensitivity and dependence on the external pH. These results show that extensive channelrhodopsin diversity exists even within the same genus, Chlamydomonas. The maximal spectral sensitivity of CaChR1 was at 520 nm at pH 7.4, about 40 nm redshifted as compared to that of CrChR1 under the same conditions. CaChR1 was successfully expressed in Pichia pastoris and exhibited an absorption spectrum identical to the action spectrum of CaChR1-generated photocurrents. The redshifted spectra and the lack of fast inactivation in CaChR1- and CyChR1-generated currents are features desirable for optogenetics applications.     

Extreme dehydration of plant tissues irreversibly converts the major and variable phyA' into the minor and conserved phyA'

Effect of dehydration of plant tissues on the two native phenomenological phytochrome A (phyA) pools - major, variable and soluble phyA' and minor, relatively conserved and presumably membrane(protein)-associated phyA' - was investigated on etiolated seedlings of barley and maize. With the use of in situ low-temperature fluorescence spectroscopy and photochemistry, it was found that even a considerable loss of water (up to 75-85% of the initial fresh weight) by coleoptiles does not bring about noticeable alterations of the spectroscopic and photochemical parameters of phytochrome pointing to a relative stability of the phyA'/phyA' system in this regard. However, extreme dehydration (loss of weight 90%) of plant tissues including freeze-drying caused dramatic changes of the phytochrome properties - blue shift of the emission maximum and its widening and reduction in the extent of the Pr photoconversion into lumi-R at 85 K and into Pfr at 273 K. Rehydration of the dried tissues did not reverse the spectroscopic changes and did not recover the Pr-->lumi-R phototransformation at 85 K but restored the ability of Pr to photoconvert into Pfr at ambient temperatures. At the same time, the total phytochrome content was not affected by these treatments. These effects were interpreted as an irreversible transformation of phyA' into phyA' upon extreme loss of water by plant tissues suggesting that water may play a role in stabilizing the conformation of the major and soluble phyA' species. The data also imply that phyA in dry and imbibing seeds is likely represented primarily by its phyA' isoform.     

Tyrosine 263 in Cyanobacterial Phytochrome Cph1 Optimizes Photochemistry at the prelumi‐R→lumi‐R Step

We report a low‐temperature fluorescence spectroscopy study of the PAS‐GAF‐PHY sensory module of Cph1 phytochrome, its Y263F mutant (both with known 3D structures) as well as Y263H and Y263S to connect their photochemical parameters with intramolecular interactions. None of the holoproteins showed photochemical activity at low temperature, and the activation barriers for the Pr→lumi‐R photoreaction (2.5–3.1 kJ mol^?1) and fluorescence quantum yields (0.29–0.42) were similar. The effect of the mutations on Pr→Pfr photoconversion efficiency (Φ_Pr→Pfr) was observed primarily at the prelumi‐R S₀ bifurcation point corresponding to the conical intersection of the energy surfaces at which the molecule relaxes to form lumi‐R or Pr, lowering Φ_Pr→Pfr from 0.13 in the wild type to 0.05–0.07 in the mutants. We suggest that the E_a activation barrier in the Pr* S₁ excited state might correspond to the D‐ring (C19) carbonyl – H290 hydrogen bond or possibly to the hindrance caused by the C13¹/C17¹ methyl groups of the C and D rings. The critical role of the tyrosine hydroxyl group can be at the prelumi‐R bifurcation point to optimize the yield of the photoprocess and energy storage in the form of lumi‐R for subsequent rearrangement processes culminating in Pfr formation.     

EFFECT OF CALCIUM ON DARK ADAPTATION IN Phycornyces PHOTOTROPISM

Adaptation processes enable phototropism of Phycomyces to operate over a 10-decade range of blue-light intensity (1 nW m-2-10 W m-2). To investigate the influence of calcium on dark adaptation, the phototropic latency method was employed with the modification that sporangiophores were temporarily immersed in solutions containing CaCl2 or LaCl3. Following such treatment, the time course of bending was found to have two components with distinct latencies and bending rates. After immersion in darkness for 30 min in LaCl3 solution or 1 h in a solution of CaCl2, MgCl2, or the calcium chelator EGTA, each sporangiophore was adapted to a blue light beam (1 W m-2) for 45 min by rotation around its vertical axis. Cessation of rotation defined the onset of the phototropic stimulus, at which time the intensity was reduced by as much as 10(3)-fold. For a 10(2)-fold reduction (to 10(-2) W m-2), immersion in CaCl2 (10-100 microM) reduces the latency 13 min for the early bending component and 18 min for the late component, whereas treatment with the calcium-channel blocker lanthanum (0.1-11 microM LaCl3) increases the latency 12 min for the early component and 13 min for the late component. EGTA (10 microM) also had an inhibitory effect, increasing the latency of the first and the second components by 7 and 10 min, respectively. In experiments performed similarly, but without the light adaptation treatment after immersion, no differences between calcium-treated and control sporangiophores were found. The bending rates of both components show only a weak dependence on calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photosensory Functions of Channelrhodopsins in Native Algal Cells†

Photomotility responses in flagellate alga are mediated by two types of sensory rhodopsins (A and B). Upon photoexcitation they trigger a cascade of transmembrane currents which provide sensory transduction of light stimuli. Both types of algal sensory rhodopsins demonstrate light‐gated ion channel activities when heterologously expressed in animal cells, and therefore they have been given the alternative names channelrhodopsin 1 and 2. In recent publications their channel activity has been assumed to initiate the transduction chain in the native algal cells. Here we present data showing that: (1) the modes of action of both types of sensory rhodopsins are different in native cells such as Chlamydomonas reinhardtii than in heterologous expression systems, and also differ between the two types of rhodopsins; (2) the primary function of Type B sensory rhodopsin (channelrhodopsin‐2) is biochemical activation of secondary Ca²⁺‐channels with evidence for amplification and a diffusible messenger, sufficient for mediating phototaxis and photophobic responses; (3) Type A sensory rhodopsin (channelrhodopsin‐1) mediates avoidance responses by direct channel activity under high light intensities and exhibits low‐efficiency amplification. These dual functions of algal sensory rhodopsins enable the highly sophisticated photobehavior of algal cells.     

Fluorescence investigation of the recombinant cyanobacterial phytochrome (Cph1) and its C-terminally truncated monomeric species (Cph1Delta2): implication for holoprotein assembly,chromophore-apoprotein interaction and photochemistry

Recombinant dimeric full-length Cph1 holophytochrome and its C-terminally-truncated monomeric species [Cph1Delta2, comprising the chromophore-bearing N-terminal sensory module (residues 1 to 514)] from the cyanobacterium Synechocystis expressed in E. coli and reconstituted in vitro with phycocyanobilin (PCB) were investigated with the use of fluorescence spectroscopy and photochemistry in the temperature range from 85 to 293 K. Holoprotein assembly in Cph1 apparently proceeds via intermediate states with the emission maximum at 680-690 nm (I685) and 700 nm (I700) and a half-life time, at room temperature, of < or =5 s. Conversion of the putative I685 into mature Cph1 involves relaxation of the chromophore into a more flexible conformation. Cph1 and Cph1Delta2 were closely similar in their spectroscopic and photochemical characteristics (position of the emission band and its width, character of the temperature dependence of the fluorescence and activation energy of the fluorescence decay, kinetics and extent of the Pr conversion at low and ambient temperatures), suggesting that there is no immediate effect of the C-terminus on the photochemical properties of the chromophore in Cph1 and that chromophore-chromophore interactions in the dimer are not significant. The latter is also supported by the lack of energy transfer from the phycoerythrobilin (PEB) to PCB in the mixed PEB/PCB adduct of Cph1. At the same time, certain variations in the fluorescence and photochemical parameters of Cph1 with temperature of the sample and intensity of the excitation light and dependence of the emission spectra on excitation wavelength were observed. These variations are interpreted as a manifestation of the Cph1 heterogeneity which may be due to the existence of different conformers of the chromophore and photoproduct formation under excitation light.