Spectral Dependence of Chlorophyll Biosynthesis Pathways in Plant Leaves.

This review covers studies on the dependence of chlorophyll photobiosynthesis reactions from protochlorophyllide on the spectral composition of actinic light. A general scheme of the reaction sequence for the photochemical stage in chlorophyll biosynthesis for etiolated plant leaves is presented. Comparative analysis of the data shows that the use of light with varied wavelengths for etiolated plant illumination reveals parallel transformation pathways of different protochlorophyllide forms into chlorophyllide, including a pathway for early photosystem II reaction center P-680 pigment formation.

Mechanisms of phototransformation of protochlorophyllide into chlorophyllide.

The purpose of this review is to summarize and discuss data obtained in studies on the mechanisms of the primary photophysical and photochemical reactions of protochlorophyllide photoreduction in plant materials (etiolated leaves and leaf homogenates) and in model systems. Based on the results of numerous studies, it can be stated that the reduction of active forms of the chlorophyll precursor is a multistep process comprising two or three short-lived intermediates characterized by a singlet ESR signal. The first intermediate is probably a complex with charge transfer between protochlorophyllide and the hydride ion donor NADPH. The conserved tyrosine residue Tyr193 of protochlorophyllide oxidoreductase is the donor of the second proton.

Pathways of formation of pigment forms at the terminal photobiochemical stage of chlorophyll biosynthesis.

The pathways of transformation of the chromophore of pigment-protein complexes have been studied at the terminal light-dependent stage of chlorophyll biosynthesis in plant leaves. The overall scheme of the sequence of photochemical and dark reactions of the pigment chromophore initiated by the reaction of photochemical hydration of a molecule of the precursor (protochlorophyllide) is presented. Schemes of the transformations of the components of the photoactive protochlorophyllide-oxidoreductase complex are discussed. Data are presented of features of the process at different stages of the formation of the pigment apparatus of plants.

Photoactive pigment-enzyme complexes of chlorophyll precursor in plant leaves.

This review summarizes contemporary data on structure and function of photoactive pigment--enzyme complexes of the chlorophyll precursor that undergoes photochemical transformation to chlorophyllide. The properties and functions of the complex and its principal components are considered including the pigment (protochlorophyllide), the hydrogen donor (NADPH), and the photoenzyme protochlorophyllide oxidoreductase (POR) that catalyzes the photochemical production of chlorophyllide. Chemical variants of the chlorophyll precursor are described (protochlorophyllide, protochlorophyll, and their mono- and divinyl forms). The nature and photochemical activity of spectrally distinct native protochlorophyllide forms are discussed. Data are presented on structural organization of the photoenzyme POR, its substrate specificity, localization in etioplasts, and heterogeneity. The significance of different POR forms (PORA, PORB, and PORC) in adaptation of chlorophyll biosynthesis to various illumination conditions is considered. Attention is paid to structural and functional interactions of three main constituents of the photoactive complex and to possible existence of additional components associated with the pigment-enzyme complex. Historical aspects of the problem and the prospects of further investigations are outlined.

Biosynthesis of chlorophyll from protochlorophyll(ide) in green plant leaves.

Using spectral methods, the biosynthesis of protochlorophyll(ide) and chlorophyll(ide) in green plant leaves was studied. The main chlorophyll precursors in the green leaves (as in etiolated leaves) were photoactive photocholorophyll(ide) forms Pchl(ide)655/650(448) and Pchl(ide)653/648(440). The contributions into Chl biosynthesis of the shorter-wavelength precursor forms, which were accumulated in darkened green leaves as well, were completely absent (of Pchl(ide) 633/628(440)) or insignificant (of Pchl(ide) 642/635(444)).

Participation of free radicals in photoreduction of protochlorophyllide to chlorophyllide in an artificial pigment-protein complex.

The primary stages of protochlorophyllide phototransformation in an artificially formed complex containing heterologously expressed photoenzyme protochlorophyllide-oxidoreductase (POR), protochlorophyllide, and NADPH were investigated by optical and ESR spectroscopy. An ESR signal (g = 2.002; H = 1 mT) appeared after illumination of the complex with intense white light at 77 K. The ESR signal appeared with simultaneous quenching of the initial protochlorophyllide fluorescence, this being due to the formation of a primary non-fluorescent intermediate. The ESR signal disappeared on raising the temperature to 253 K, and a new fluorescence maximum at 695 nm belonging to chlorophyllide simultaneously appeared. The data show that the mechanism of protochlorophyllide photoreduction in the complex is practically identical to the in vivo mechanism: this includes the formation of a short-lived non-fluorescent free radical that is transformed into chlorophyllide in a dark reaction.

Biosynthesis of chlorophyll of photosystem II reaction centers in plant leaves in early stages of etiolation.

Spectral methods were used to study the sequences of chlorophyll biosynthesis reactions in etiolated pea, bean, and maize plants in early stages (3-4 days) of growth. For these juvenile plants, along with the reaction chain known for mature (7-9 day-old) plants, a new reaction chain was found which started with phototransformation of the long-wavelength form PChld 686/676 into PChld 653/648. (PChld 653/648 differs from the main known precursor form PChld 655/650). The subsequent photoreduction of PChld 653/648 leads to the formation of Chld 684/676, which is transformed into Chl 688/680 in the course of a dark reaction. After completion of this reaction, fast (20-30 sec) quenching of the fluorescence of the reaction product is observed with the formation of non-fluorescent Chl 680. The reaction accompanied by pigment fluorescence quenching is absent in pea mutants with depressed function of Photosystem II reaction centers. This suggests that the newly found reaction chain leads to the formation of chlorophyll of the Photosystem II reaction center.

Nonpigment components of the photochlorophyllide photoactive complex: studies of low-temperature blue-green fluorescence spectra.

Fluorescence spectra in the blue-green region and excitation fluorescence spectra of green wheat leaves, etiolated wheat leaves and isolated inner etioplast membranes (prolamellar bodies and prothylakoids) were compared to specify the structure of the active protochlorophyllide pigment-protein complex of inner etioplast membranes. Three bands in the blue region at 420, 443 and 470 nm and a broader green band at 525 nm were found. Comparison of the emission and excitation spectra suggests that the main components responsible for the blue fluorescence of etioplast inner membranes are pyridine nucleotides and pterins. The green fluorescence (525 nm) excitation spectra of etiolated samples were identical to the excitation spectrum of flavin fluorescence. The fact confirms the suggestion that flavins are the constituents of the active protochlorophyllide-protein complex.

The final stage of chlorophyll biosynthesis and formation of reaction centres of photosystems.

The survey reviews Russian studies of the final stage of chlorophyll biosynthesis and the role of A. A. Krasnovsky (Sr.) in the development of this direction. The current state of the problem is considered. The results of studies carried out by Krasnovsky's followers at Moscow State University are summarized. Schemes of the pathways of chlorophyll and pheophytin biosynthesis, biogenesis of pigment complexes of the two photosystems of photosynthesis proposed by those scientists are presented.

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15.

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650→Chlide 695/684 and Pchl (ide) 640/635→Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203-233 K).

Light-regulated pigment interconversion in pheophytin/chlorophyll-containing complexes formed during plant leaves greening.

Upon illumination of etiolated maize leaves the photoconversion of protochlorophyllide Pchlide 655/650 into chlorophyllide Chlide 684/676 was observed. It was shown that chlorophyllide Chlide 684/676 in the dark is transformed into pheophytin Pheo 679/675 and chlorophyll Chl 671/668 by means of two parallel reactions, occurring at room temperature: Chlide 684/676. The formed pheophytin Pheo 679/675 was unstable and in the dark was transformed into chlorophyll Chl 671/668 in a few seconds: Pheo 679/675 → Chl 671/668. The last reaction is reversed by the light: Chl/668 Pheo 679/675. Thus, on the whole in the greening etiolated leaves this process occurs according to the following scheme:The observed light-regulated interconversion of Mg-containing and Mg-free chlorophyll analogs is activated by ATP and inhibited by AMP.

Photoinduced formation of pheophytin/chlorophyll-containing complexes during the greening of plant leaves.

Illumination of etiolated maize leaves with low-intensity light produces a chlorophyll/pheophytin-containing complex. The complex contains two native chlorophyll forms Chl 671/668 and Chl 675/668 as well as pheophytin Pheo 679/675 (with chlorophyll/pheophytin ratio of 2/1). The complex is formed in the course of two successive reactions: reaction of protochlorophyllide Pchlde 655/650 photoreduction resulted in chlorophyllide Chlde 684/676 formation, and the subsequent dark reaction of Chlde 684/676 involving Mg substitution by H2 in pigment chromophore and pigment esterification by phytol. Out data show that the reaction leading to chlorophyll/pheophytin-containing complex formation is not destructive. The reaction is in fact biosynthetic, and is competitive with the known reactions of biosynthesis of the bulk of chlorophyll molecules. The relationship between chlorophyll and pheophytin biosynthesis reactions is controlled by temperature, light intensity and exposure duration.The native complex containing pheophytin a and chlorophyll a is supposed to be a direct precursor of the PS II reaction centre in plant leaves.

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves.

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650→R668, P650→R688, and P650→R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643-644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658-711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.

Phosphorescence of protochlorophyll(ide) and chlorophyll(ide) in etiolated and greening bean leaves : Assignment of spectral bands.

The assignment is presented for the principal phosphorescence bands of protochlorophyll(ide), chlorophyllide and chlorophyll in etiolated and greening bean leaves measured at -196°C using a mechanical phosphoroscope. Protochlorophyll(ide) phosophorescence spectra in etiolated leaves consist of three bands with maxima at 870, 920 and 970 nm. Excitation spectra show that the 870 nm band belongs to the short wavelength protochlorophyll(ide), P627. The latter two bands correspond to the protochlorophyll(ide) forms, P637 and P650. The overall quantum yield for P650 phosphorescence in etiolated leaves is near to that in solutions of monomeric protochlorophyll, indicating a rather high efficiency of the protochlorophyll(ide) triplet state formation in frozen plant material. Short-term (2-20 min) illumination of etiolated leaves at the temperature range from -30 to 20°C leads to the appearance of new phosphorescence bands at about 990-1000 and 940 nm. Judging from excitation and emission spectra, the former band belongs to aggregated chlorophyllide, the latter one, to monomeric chlorophyll or chlorophyllide. This indicates that both monomeric and aggregated pigments are formed at this stage of leaf greening. After preillumination for 1 h at room temperature, chlorophyll phosphorescence predominates. The spectral maximum of this phosphorescence is at 955-960 nm, the lifetime is about 2 ms, and the maximum of the excitation spectrum lies at 668 nm. Further greening leads to a sharp drop of the chlorophyll phosphorescence intensity and to a shift of the phosphorescence maximum to 980 nm, while the phosphorescence lifetime and a maximum of the phosphorescence excitation spectrum remains unaltered. The data suggest that chlorophyll phosphorescence belongs to the short wavelength, newly synthesized chlorophyll, not bound to chloroplast carotenoids. Thus, the phosphorescence measurement can be efficiently used to study newly formed chlorophyll and its precursors in etiolated and greening leaves and to address various problems arising in the analysis of chlorophyll biosynthesis.

The N intermediate of bacteriorhodopsin at low temperatures: stabilization and photoconversion.

In aqueous suspensions of purple membranes (pH 10.2, 0.4 M KCl) an intermediate having an absorption maximum at 570-575 nm (at -196 degrees C) was produced by first heating the M intermediate up to -30 degrees C and then stabilizing it by subsequent cooling to -60 degrees C. We suggest that this species is the intermediate N (or P or R) found and characterized earlier near room temperature. Upon illumination at -196 degrees C N is transformed into a bathochromically absorbing species KN which has an absorption maximum near 605 nm and an extinction 1.35 times that of N. This light reaction is photoreversible. The quantum yield ratio for the forward and back reaction is 0.18 +/- 0.02. The maximum photo steady state concentration of KN is about 0.24. The N intermediate was also trapped in water suspensions of purple membranes at neutral pH and low salt concentration by illumination at lambda greater than 620 nm during cooling. In addition to N another intermediate absorbing in the red (maximum at 610-620 nm) was accumulated in smaller amounts. It is not photoactive at -196 degrees C and apparently is the O intermediate or a photoproduct of N.

Two components of photoreceptor potential in phototaxis of the flagellated green alga Haematococcus pluvialis.

The kinetics of the photoreceptor potential of phototaxis in biflagellated green alga Haematococcus pluvialis in response to a 10-ns laser pulse of three wavelengths (465, 550, and 590 nm) were measured in single cells with 30 mus time resolution. The rise and the decay of photoinduced potential are both at least biphasic. The first component of the rise is very stable and has no measurable (<30 mus) time delay. The second component is triggered after a 120-400-mus lag period, depending on flash intensity. Its appearance is sensitive to the physiological state of the cell and the amplitude can be increased by phototactically ineffective red background illumination. The electrical generators for both components are localized in the same region of the cell membrane (on the stigma-bearing side) and these components have the same depolarizing sign. The results indicate that the photoreceptor potential in phototaxis comprises two components, which could be interpreted as light-induced charge movement within the photoreceptor molecules and changes in ion permeability of the cell membrane.

The primary reactions in the protochlorophyll(ide) photoreduction as investigated by optical and ESR spectroscopy.

It has been found that at low temperatures (77K-153K) a long-lived (at these temperatures) singlet ESR signal induced by intensive light appears in etiolated leaves of plants and in model systems including both the monomeric and aggregated protochlorophyll.Comparison of the results of ESR, fluorescence and absorption spectra measurements made it possible to suggest that at the initial stages of the protochlorophyll(ide) photoreduction process at least two paramagnetic non-fluorescent intermediates are formed, one of which seems to be identical to the previously found intermediate with absorption maximum at 690 nm. On the strength of the obtained results a conclusion can be drawn that photoreduction of the semi-isolated double-c=c-bond of the chlorophyll precursor molecule in etiolated leaves and in model systems is actualized via at least two stages of free radicals formation. A scheme of the primary reactions of chlorophyllide biosynthesis has been proposed.

[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.

The role of a long-wavelength pigment form in the chlorophyll biosynthesis.

Photoconversion of protochlorophyllide650 form was observed in etiolated leaves illuminated with long-wavelength-690 nm-light. This process showed Shibata shift and was found to have a strong temperature dependence between 20 and -40°C. The low rate of reaction, the strong temperature dependence and calculations on the spectral overlap integral of absorption and fluorescence bands in this spectral region indicate that the phototransformation of the 650 nm form of protochlorophyllide may be caused by a back energy migration from a long-wavelength pigment form absorbing around 690 nm; this pigment form is probably a long-wavelength form of protochlorophyll/ide.