Applications of fluorescence spectroscopy in the investigation of plant phytochrome invivo.

Fluorometry is an effective research tool in biology and medicine; it is widely used in the study of the photosynthetic pigment apparatus in vivo. This method can be applied to the key plant photoreceptor phytochrome (phy). The fluorescence of phytochrome in plants was recorded for the first time in the group of the author, and a spectrofluorometric technique for its in vivo study was developed. The photophysical and photochemical properties of the pigment were described, and the photoreceptor was shown to be present in plants as two phenomenological types-active (at cryogenic temperatures) and water-soluble (Pr') and inactive and amphiphilic (Pr″). The scheme of the photoreaction explaining their photochemical distinctions was proposed. Phytochrome A was shown to comprise both types (phyA' and phyA″), whereas phytochrome B was only the second type. For phyA', distinct conformers have been detected. phyA' and phyA″ differ by the N-terminus of the molecule, possibly by serine phosphorylation. They mediate, respectively, the very low fluence and high irradiance photoresponses. Light, internal factors (kinase/phosphatase balance, pH), and hormones (jasmonate) were shown to affect the content and functions of the two phyA pools. All this points to the effectiveness of the developed method for invivo investigations of the phytochrome system. The data obtained can be applied in practical terms in agrobiology and light culture, as well as in the use of phytochrome as a new nanotool and a fluorescent probe.

Phytochrome A in plants comprises two structurally and functionally distinct populations - water-soluble phyA' and amphiphilic phyA″.

Photoreceptor phytochrome A (phyA) plays a key role both in the individual development and in the evolution of higher plants. It acts in three distinct modes - far-red light-induced very low fluence responses (VLFRs), high irradiance responses (HIRs), and red/far-red-reversible low fluence responses (LFRs). Signal transduction from phyA includes its transportation from the cytoplasm into the nucleus and activation of light-responsive genes there. It is also active in the cytoplasm. Two types of phyA speckles were detected upon its light-induced nucleocytoplasmic partitioning and a fraction remained in the cytoplasm. In this review, we present a concept that this complex picture of the phyA action is due, at least partially, to the existence of two phyA types in the cell differing by the structure of the N-terminus, probably, by its serine phosphorylation. These are phosphorylated water-soluble phyA' and underphosphorylated ambiquitous phyA″ represented by two fractions - water-soluble and membrane-associated. From the analysis of the phyA pools' activity in the regulation of phyA synthesis, seed germination, seedling establishment, and (proto)chlorophyll biosynthesis it is concluded that phyA″ is responsible for the regulation of seed germination, whereas in seedlings phyA' mediates the VLFRs, and the water-soluble phyA″ fraction, the HIRs. The membrane-associated phyA″ is likely to be active in cytoplasmic photoregulatory events. Functional interaction between phyA and the defense-related hormone jasmonic acid is also considered.

Regulation of Chlorophyll Biogenesis by Phytochrome A.

The photosynthetic apparatus accomplishes two major functions in plants - solar energy conversion and protection of the plant from photodestruction. Its highly orchestrated formation includes coordinated biosynthesis of chlorophyll (Chl) and of its binding to matrix proteins. Light plays here the central role driving both metabolic and regulatory processes. The regulation is achieved via operation of sophisticated photoreceptor machinery with the phytochrome system as its main component. This review concentrates on Chl a biosynthesis and the role of phytochrome A (phyA) in this process. The mechanism of action of phyA and the specificity of its state in the plant has been described, in particular, the existence of two native types with different modes of action. This review touches upon the dependence of the effects of phyA on tissues and organs of the plant and its species, genetic modifications, and hormonal status.

The dephosphorylated S8A and S18A mutants of (oat) phytochrome A comprise its two species, phyA' and phyA'', suggesting that autophosphorylation at these sites is not involved in the phyA differentiation.

Phytochrome A (phyA) is represented in plants by two species, phyA' and phyA'', with different properties and modes of action (Sineshchekov, Funct. Plant Biol., 2019, 46, 118-135). They differ by the modification of a serine(s) residue at the N-terminus, possibly, by phosphorylation. To verify if these serines could be the Ser8 and Ser18 (in Avena sativa phyA, AsphyA), whose autophosphorylation modulates AsphyA stability and sensitivity as shown with the use of the serine-to-alanine substitution AsphyA mutants (S8A, S18A and S8/18A) (Han et al., Plant Cell Physiol., 2010, 51, 596-609), we have undertaken low-temperature (85 K) fluorescence investigations of phyA in these transgenic lines. The content and proportion of phyA' and phyA'' were essentially the same in wild-type AsphyA and its mutants, and in endogenous Arabidopsis phyA. All the lines revealed a higher phyA'/phyA'' proportion upon longer germination-inducing preillumination (3 h vs. 15 min white light) supporting our earlier finding that the phyA differentiation into the subpools is light-regulated. These observations and our earlier data imply that this process involves N-terminal serine(s) different from the autophosphorylated Ser8 and Ser18 (in AsphyA) narrowing down the area of further search for the exact site(s) of the phyA modification.

Two molecular species of phytochrome A with distinct modes of action.

Adaptation of plants to environmental light conditions is achieved via operation of a highly complex photoreceptor apparatus. It includes the phytochrome system comprising phytochromes A and B (phyA and phyB) as the major components. phyA differs from phyB by several properties, including its ability to mediate all three photoresponse modes - the very low and low fluence responses (VLFR and LFR respectively) and the high irradiance responses (HIR), whereas phyB is responsible for LFR. This review discusses the uniqueness of phyA in terms of its structural and functional heterogeneity. The photoreceptor is presented in monocots and dicots by two native molecular species, phyA' and phyA'', differing by spectroscopic, photochemical and phenomenological properties. phyA differentiation into substates includes post-translational phosphorylation of a serine residue(s) at the N-terminal extension of the molecule with phyA' being the phosphorylated species and phyA'', dephosphorylated. They differ also by their mode of action, which depends on the cellular context. The current working hypothesis is that phyA' mediates VLFR and phyA'', HIR and LFR. The content and functional activity of the two pools are regulated by light and by phosphatase/kinase equilibrium and pH in darkness, what contributes to the fine-tuning of the phytochrome system. Detection of the native pools of the cryptogamic plant fern Adiantum capillus-veneris phy1 (phy1' and phy1'') similar to those of phyA suggests that the structural and functional heterogeneity of phyA is not a unique phenomenon and may have arisen earlier in the molecular evolution of the phytochrome system than the appearance of the angiosperm phytochromes.

Fern Adiantum capillus-veneris phytochrome 1 comprises two native photochemical types similar to seed plant phytochrome A.

Phytochrome (phy) in etiolated seedlings of wild-type (WT) Arabidopsis (Ler) and its transgenic lines (TL) L15 and L20 transformed with Adiantum capillus-veneris PHY1 cDNA (Okamoto et al., 1997) was investigated using low-temperature (85K) fluorescence spectroscopy and photochemistry. It was found that while WT seed germination requires stimulation by light, the TL germinated equally well with or without pre-illumination. Phytochrome content [Ptot] was 2-fold higher in TL whereas the level of Pr→lumi-R phototransformation at 85K (γ1) was similar between WT (0.25) and TL (0.27). When seeds germinated with pre-illumination, the proportion of the photochemical types Pr' active and Pr″ inactive at 85K was 50/50 in WT and 54/46 in TL, respectively. Dark-germinated TL had a γ1 value of 0.16 and the proportion of Pr' and Pr″ was 32/68, respectively, without changes in [Ptot]. Evaluations based on these data revealed that phy1 has Pr' and Pr″, designated phy1' and phy1″, akin to phyA, which comprises both Pr photochemical types (phyA' and phyA″), and in contrast to phyB that possesses only Pr″. The proportion of phy1' and phy1″ depends on pre-illumination for induction of germination. The pigment most likely accumulated in the seeds and was active in promoting Arabidopsis seed germination.

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.

Two modes of the light-induced phytochrome A decline--with and without changes in the proportion of its isoforms (phyA' and phyA''): evidence from fluorescence investigations of mutant phyA-3D pea.

Different modes of the phytochrome function are connected with its polymorphism, the major isoforms being phytochromes A and B (phyA and phyB). In its turn, phyA comprises two native species, phyA' and phyA'', whose precise nature and functions remain obscure. With the use of in situ fluorescence spectroscopy, we investigated their properties in a mutant of pea, phyA-3D, characterized by exaggerated photoresponses and impaired photodestruction of phyA. The mutation is a substitution of alanine by valine at the position 194 in phyA. The phyA-3DphyB and phyB mutants were also investigated. In dark-grown plants, all the lines had the content and properties of the two phyA species very similar to the wild type. However, a considerably more intense reduction in [phyA] without changes in the phyA'/phyA'' equilibrium was found in far-red grown mutant plants suggesting a hypersensitivity of phyA-3D with regard to its autoregulation. On the contrary, under red illumination, a higher stability of phyA-3D was observed confirming our earlier findings. This allows a conclusion that the A194V substitution in phyA-3D not only impairs its destruction but also enhances its signaling ability, suggesting a role of this locus in modulation of its activity.

Phytochrome A: functional diversity and polymorphism.

Phytochrome (phy), a 124 kDa biliprotein, mediates plants' perception of environmental light conditions including quantity, quality and duration of light. The complex phenomenology of phy function is connected with its polymorphism, the major phys being phyA and phyB. PhyA mediates irreversible photoresponses in the very low and high fluence ranges (VLFR and HIR) primarily in the far-red (FR) spectral region, whereas phyB mediates the 'classical' R/FR reversible responses in the low fluence range (LFR). This phyA specificity is determined at the level of (i) intramolecular events, (ii) turnover, phyA being light-labile, and (iii) nuclear-cytoplasmic partitioning and interaction with partner proteins. A unique feature of phyA is that two native isoforms, phyA' and phyA'', comprise it, distinguished by spectroscopic and photochemical properties, localization and abundance in plant tissues, light stability, and other properties. They differ by the post-translational modification at the 6 kDa N-terminus, possibly phosphorylation, phyA' being phosphorylated and phyA'' dephosphorylated. Both species participate in the light-induced nuclear-cytoplasmic partitioning. The light-labile phyA' is responsible for de-etiolation (VLFR and HIR modes), whereas the relatively more light-stable phyA'' could be active throughout the whole life cycle. PhyA'' interferes with the action of phyA' and this interaction may be part of the fine tuning mechanism of the phyA function. Finally, within the phyA' pool there are different conformers in thermal equilibrium, that differ by the activation and kinetic parameters of the Pr-->lumi-R photoreaction. This heterogeneity of phyA may account, at least partially, for the complex dynamics of its photoprocesses and the phenomenology of photoresponses.

Up-regulation by phytochrome A of the active protochlorophyllide, Pchlide655, biosynthesis in dicots under far-red light.

It is well-documented that phytochrome A (phyA) down-regulates the synthesis of NADPH:protochlorophyllide (Pchlide) oxidoreductase and active Pchlide(655) under far-red light (FR). In this work, we demonstrate that phyA can up-regulate the synthesis of Pchlide(655) under FR as well and that its sign and extent depend on plant species and tissue. With the use of fluorescence spectroscopy, it was found that [Pchlide(655)] in the upper stems of FR-grown seedlings of pea and tobacco increased > or =10-fold and much lower in cotyledons or leaves as compared with the dark-grown. In the upper stems of Arabidopsis and tomato, the positive effect of FR was low, 1.2- to 1.5-fold, and the negative effect of FR was seen in cotyledons. In stems of wild-type (WT) tobacco and its line overexpressing full-length oat phyA (FL), we observed gross stimulating effect of FR while in its line overexpressing N-terminally truncated (Delta7-69) oat phyA (NA) it was low. Because WT and FL comprise both native phyA forms, phyA' and phyA", while NA, only phyA", the regulation under FR can be associated with phyA', while phyA" inhibits the action of phyA'. In etiolated seedlings of the NA line, [Pchlide(655)] was much higher than in those of WT and FL suggesting that phyA" may have relation to this enhancement. The regulation of Pchlide(633) in contrast to Pchlide(655) was positive independent of the plant species and tissue.

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.

Recombinant phytochrome A in yeast differs by its spectroscopic and photochemical properties from the major phyA' and is close to the minor phyA": evidence for posttranslational modification of the pigment in plants.

Previously, two pools of phytochrome A (phyA' and phyA") have been detected by in situ low-temperature fluorescence spectroscopy and photochemistry; it was suggested that they might differ in the nature of their posttranslational modification. In order to verify this possibility Arabidopsis and rice (Oryza) phyA were expressed in yeast and the pigments were assembled in vivo with phycocyanobilin (PCB) and phytochromobilin (P phi B). The resulting recombinant phytochromes in the red-light-absorbing form (Pr) were characterized in the yeast cell by (1) the fluorescence emission spectra; (2) the temperature dependence of Pr fluorescence intensity and activation energy of fluorescence decay; and (3) the extent of photoconversion of Pr into photoproduct lumi-R (gamma 1) or far-red-light absorbing form (Pfr) (gamma 2). Both Arabidopsis phyA/PCB and Oryza phyA/P phi B had low gamma 1 of ca 0.05, allowing their attribution to the Pr" phenomenological type of phytochrome comprising phyA", phyB and cryptogam phytochromes. The spectroscopic properties of Oryza phyA/P phi B were also very close to phyA". However, both investigated holoproteins differed from phyA", both with respect to the character of temperature dependence of the fluorescence yield and activation energy. Thus, recombinant Oryza phyA/P phi B is similar but not identical to phyA". The data demonstrate that the low-abundance-fraction plant phyA (phyA") comes from the same gene as the major (phyA') fraction. Because both endogenous phyA fractions differ from the phytochrome expressed in yeast, they appear to be posttranslationally modified and/or bound to partner proteins or cellular substructures. However, the character of the presumed chemical modification is different in phyA' and phyA" and its extent is more profound in the case of the former.

Recombinant phytochrome of the moss Ceratodon purpureus (CP2): fluorescence spectroscopy and photochemistry.

The recombinant phytochrome of the moss Ceratodon purpureus (CP2) expressed in Saccharomyces cerevisiae and reconstituted with phycocyanobilin (PCB) was investigated using fluorescence spectroscopy. The pigment had an emission maximum at 670 nm at low temperature (85 K) and at 667 nm at room temperature (RT) and an excitation maximum at 650-652 nm at 85 K (excitation spectra could not be measured at RT). Both spectra had a half-band width of approx. 30-35 nm at 85 K. The fluorescence intensity revealed a steep temperature dependence with an activation energy of fluorescence decay (Ea) of 5.9-6.4 and 12.6-14.7 kJ mol(-1) in the interval from 85 to 210 K and from 210 to 275 K, respectively. The photochemical properties of CP2/PCB were characterised by the extent of the red-induced (lambda(a) = 639 nm) Pr conversion into the first photoproduct lumi-R at 85 K (gamma1) of approximately 0.07 and into Pfr at RT (gamma2) of approximately 0.7. From these characteristics, CP2/PCB can be attributed to the Pr" photochemical type with gamma1 < or = 0.05, which comprises the minor phyA fraction (phyA"), phyB, Adiantum phy1 and Synechocystis Cph1 in contrast to the major phyA' fraction (Pr' type with gamma1 = 0.5). Within the Pr" type, it is closer to phyA" than to phyB and Cph1.

Fluorescence and photochemical characterization of phytochromes A and B in transgenic potato expressing Arabidopsis phytochrome B.

Phytochrome in etiolated sprouts of wild type (WT) potato and its transgenic strains (DARA5 and DARA12) expressing Arabidopsis thaliana phytochrome B (phyB) was investigated using low-temperature (85 K) fluorescence spectroscopy and photochemistry. Phytochrome content, [Ptot], position of the Pr emission and excitation spectra, lambda(max), and extent of the Pr-->lumi-R, gamma1, and Pr-->Pfr, gamma2, phototransformations (at 85 and 273 K, respectively) were shown to vary in the transgenic lines and WT depending on tissue used (upper vs. lower parts of etiolated sprouts) and light-induced phytochrome depletion. Differences in the parameters between the transgenic lines and WT were detected which were interpreted in terms of the two phenomenological Pr types: a labile Pr' with gamma1 approximately 0.5 consisting of a major phytochrome A (phyA) fraction (phyA') and a relatively conserved Pr" with gamma1 = 0 comprising a minor phyA fraction (phyA") and phyB. Both DARA lines had higher [Pr"] as compared with WT in the lower parts of etiolated stems, especially after light-induced phytochrome depletion (residual phytochrome in DARA5 and DARA12 made up to one-third of its initial level vs. <5% in WT). These differences were associated with the expression of Arabidopsis phyB in the DARA lines and its higher light stability than that of phyA. Arabidopsis phyB expressed in potato was characterised by lambda(max) = 683/669 nm in the emission/excitation (absorption) spectra and gamma1 = 0. PhyB also revealed a relatively low gamma2 (approx. 0.5) and its early red drop as compared with the gamma2 wavelength dependence for phyA. This is believed to contribute to the lower signalling ability of phyB and to confine the region (red) of its physiological activity.

Fluorescence spectroscopy and photochemistry of phytochromes A and B in wild-type, mutant and transgenic strains of Arabidopsis thaliana.

Phytochrome (P) was characterized in etiolated seedlings of wild-type, mutant and transgenic strains of Arabidopsis with the use of low-temperature (85 K) fluorescence spectroscopy and photochemistry. The position (lambda max) of the Pr emission spectrum, its intensity (F0) proportional to [P tot] and the extent of the Pr-->lumi-R phototransformation at 85 K (gamma 1) were shown to vary depending on the plant strains and tissues used, while the extent of the Pr-->Pfr transformation at 273 K (gamma 2) remained relatively constant. Depletion of phyA (fre1-1 in Nagatani et al., Plant Physiol. 102 (1993) 269-277, and fhy2-2 in Whitelam et al., Plant Cell 5 (1993) 757-768) resulted in a steep decrease of F0 to approximately equal to 10%. The phyB mutant (hy3-B064 in Reed et al., Plant Cell 5 (1993) 147-157) revealed a slight reduction (by approximately equal to 20%) of F0 while lambda max and gamma 1 remained practically unaffected. In phyAphyB mutuant no P emmission was observed. Overexpression of oat phyA (13k7 and 21k15 in Boylan and Quail, Proc. Natl. Acad. Sci. USA 88 (1991) 10806-10810) brought about an increase of F0 by two or three times, a shift of lambda max to 685 nm and an increase of gamma 1 to 0.3-0.4. On the contrary, an increase of F0 (up to 40%) in Arabidopsis and rice phyB overexpressors (ABO and RBO in Wagner et al., Plant Cell 3 (1991) 1275-1288) was followed by a decrease of gamma 1 values to 0.13-0.14. These data together with the results on phyB (lh) mutant of cucumber prove the existence of the two phyA populations with high (phyA') and low (phyA") photochemical activity at low temperatures. PhyB emits maximally in the same region as phyA in Arabidopsis (approximately equal to 683 nm) and at shorter wavelength (< 680 nm) in rice. It is characterized by low photochemical activity at 85 K (gamma 1 < or = 0.05) and can be attributed in this respect to the same pigment type as phyA".

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 (P phi B), Syn(PCB)phy and Syn(P phi B)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(P phi B)phy. At room temperature, the spectra are blue shifted by 5-10 nm. The fluorescence intensity dropped down by approximately 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(P phi B)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(P phi B)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(P phi B)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(P phi B)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 phototransformation at 85 K. These photochemical features are interpreted in terms of different activation barriers for the photoreaction in the Pr excited state.

The system of phytochromes: photobiophysics and photobiochemistry in vivo.

Phytochrome is a key photoregulation pigment in plants which determines the strategy of their development throughout their life cycle. The major achievement in the recent investigations of the pigment is the discovery of its structural and functional heterogeneity: existence of a family of phytochromes (phyA-phyE) differing by the apoprotein was demonstrated. We approach this problem by investigating the chromophore component of the pigment with the use of the developed method of in vivo low-temperature fluorescence spectroscopy of phytochrome. In etiolated plants, phytochrome fluorescence was detected and attributed to its red-light absorbing form (Pr) and the first photoproduct (lumi-R), and a scheme of the photoreaction in phytochrome, a distinction of which is the activation barrier in the excited state, was put forward. It was found that the spectroscopic and photochemical characteristics of Pr depend on the plant species and phytochrome mutants and overexpressors used, on localization of the pigment in organs and tissues, plant age, effect of preillumination and other physiological factors. This variability of the parameters was interpreted as the existence of at least two phenomenological Pr populations, which differ by their spectroscopic characteristics and activation parameters of the Pr --> lumi-R photoreaction (in particular, by the extent of the Pr --> lumi-R photoconversion at low temperatures, gamma1): the longer-wavelength major and variable by its content in plant tissues Pr' with gamma1 = 0.5 and the shorter-wavelength minor relatively constant Pr" with gamma1 < or = 0.05. The analysis of the phytochrome mutants and overexpressors allows a conclusion that phytochrome A (phyA), which dominates in etiolated seedlings, is presented by two isoforms attributed to Pr' and Pr" (phyA' and phyA", respectively). Phytochrome B (phyB) accounts for less than 10% of the total phytochrome fluorescence and belongs to the Pr" type. It is also characterized by the relatively low extent of the Pr photoconversion into the far-red-light absorbing physiologically active phytochrome form, Pfr. Fluorescence of the minor phytochromes (phyC-phyE) is negligible. The recently discovered phytochrome of the cyanobacterium Synechocystis also belongs to the phenomenological Pr" type. PhyA' is a light-labile and soluble fraction, while phyA" is a relatively light-stable and, possibly, membrane (protein)-associated. Experiments with transgenic tobacco plants overexpressing full-length and C- and N-terminally truncated oat phytochrome A suggest that phyA' and phyA" might differ by the post-translational modification of the small N-terminal segment (amino acid residues 7-69) of the pigment. PhyA' is likely to be active in the de-etiolation processes while phyA" together with phyB, in green plants as revealed by the experiments on transgenic potato plants and phytochrome mutants of Arabidopsis and pea with altered levels of phytochromes A and B and modified phenotypes. And finally, within phyA', there are three subpopulations which are, possibly, different conformers of the chromophore. Thus, there is a hierarchical system of phytochromes which include: (i) different phytochromes; (ii) their post-translationally modified states and (iii) conformers within one molecular type. Its existence might be the rationale for the multiplicity of the photoregulation reactions in plants mediated by phytochrome.

Evidence for the existence of membrane-associated phytochrome in the cell.

Comparative fluorescence and photochemical studies of phytochrome in etiolated seedlings of maize and in soluble and membrane-containing fractions isolated from them were carried 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 (lambda max) and the extent of the phototransformation of the red-absorbing form (Pr) into the first stable photoproduct, lumi-R, at 85 K (gamma 1), varied in different parts of etiolated seedlings: lambda max and gamma 1 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) (gamma 2) did not differ much: values of 0.80-0.85 and 0.70-0.75 correlated with the high and low values of gamma 1. 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 gamma 1 (Pr'), and the shorter wavelength minor light-stable species with low gamma 1 (Pr").(ABSTRACT TRUNCATED AT 250 WORDS)

[Fluorescence of rhodopsins and its relation to primary processes of light energy transformation]. / Fluorestsentsiia rodopsinov i ee sviaz' s pervichnymi protsessami prevrashcheniia énergii sveta.

A new area of the investigation of visual and bacterial rhodopsins--fluorescence spectroscopy of the pigments is discussed. Fluorescence properties are considered in relation to photochemical transformations of the pigments at low temperatures. A number of fluorescent states of the pigments are described. It is shown that the excited states of bacteriorhodopsin and visual rhodopsin are characterized by a series of common features. The analysis of general properties of the pigments excited states allows a conclusion that the singlet excited states take part in the photoreaction. The photoreaction scheme is discussed in which structural changes of the chromophore take place already in the excited state.