Dmitrii V. Vavilin

Multiple deletions of small cab-like proteins in the cyanobacterium Synechocystis sp. PCC 6803: COnsequences for pigment biosynthesis and accumulation

Deletion of the genes for four or five small Cab-like proteins (SCPs) in photosystem (PS) I-less and PS I-less/PS II-less strains of Synechocystis sp. PCC 6803 caused a large decrease in the chlorophyll and carotenoid content of the cells without accumulation of early intermediates in the chlorophyll biosynthesis pathway, suggesting limited chlorophyll availability. The PS II/PS I ratio increased upon deletion of multiple SCPs in a wild type background, similar to what is observed in the presence of subsaturating concentrations of gabaculin, an inhibitor of an early step in the tetrapyrrole biosynthesis pathway. Upon deletion of multiple SCPs, neither 77 K fluorescence emission properties of phycobilisomeless thylakoids from the PS I-less/PS II-less strain nor the energy trapping efficiency of PS II were affected, indicating that under steady-state conditions SCPs do not bind much chlorophyll and do not serve as PS II antenna. Under conditions where protochlorophyllide reduction and thus chlorophyll synthesis were inhibited, chlorophyll disappeared quickly in a mutant lacking all five SCPs. This implies a role of SCPs in stabilization of chlorophyll-binding proteins and/or in reuse of chlorophylls. Under these conditions of inhibited reduction of protochlorophyllide, the accumulation kinetics of this intermediate were greatly altered in the absence of the five SCPs. This indicates an alteration of tetrapyrrole biosynthesis kinetics by SCPs. Based on this and other evidence, we propose that SCPs bind carotenoids and transiently bind chlorophyll, aiding in the supply of chlorophyll to nascent or reassembling photosynthetic complexes, and regulate the tetrapyrrole biosynthesis pathway as a function of the demand for chlorophyll.

Chlorophyll b can serve as the major pigment in functional photosystem II complexes of cyanobacteria

An Arabidopsis thaliana chlorophyll(ide) a oxygenase gene (cao), which is responsible for chlorophyll b synthesis from chlorophyll a, was introduced and expressed in a photosystem I-less strain of the cyanobacterium Synechocystis sp. PCC 6803. In this strain, most chlorophyll is associated with the photosystem II complex. In line with observations by Satoh et al. [Satoh, S., Ikeuchi, M., Mimuro, M. & Tanaka, A. (2001) J. Biol. Chem. 276, 4293–4297], chlorophyll b was made but accounted for less than 10% of total chlorophyll. However, when lhcb encoding light-harvesting complex (LHC)II from pea was present in the same strain (lhcb+/cao+), chlorophyll b accumulated in the cell to levels exceeding those of chlorophyll a, although LHCII did not accumulate. In the lhcb+/cao+ strain, the total amount of chlorophyll, the number of chlorophylls per photosystem II center, and the oxygen-evolving activity on a per-chlorophyll basis were similar to those in the photosystem I-less strain. Furthermore, the chlorophyll a/b ratio of photosystem II core particles (retaining CP47 and CP43) and of whole cells of the lhcb+/cao+ strain was essentially identical, and PS II activity could be obtained efficiently by chlorophyll b excitation. These data indicate that chlorophyll b functionally substitutes for chlorophyll a in photosystem II. Therefore, the availability of chlorophylls, rather than their binding specificity, may determine which chlorophyll is incorporated at many positions of photosystem II. We propose that the transient presence of a LHCII/chlorophyll(ide) a oxygenase complex in the lhcb+/cao+ strain leads to a high abundance of available chlorophyll b that is subsequently incorporated into photosystem II complexes. The apparent LHCII requirement for high chlorophyll(ide) a oxygenase activity may be instrumental to limit the occurrence of chlorophyll b in plants to LHC proteins.

Membrane lipid peroxidation, cell viability and Photosystem II activity in the green alga Chlorella pyrenoidosa subjected to various stress conditions

Abstract The unicellular green alga Chlorella pyrenoidosa was subjected to a variety of stress conditions (strong illumination, incubation with Cu 1+ or Zn 2+ , exposure to high temperatures). The amplitude of thermoluminescence (TL) peak at 125°C, accumulation of thiobarbituric acid reactive substances (TBARS), which indicate an accumulation of lipid peroxidation products, efficiency of Photosystem II reactions ( F v / F M ratio) and the percentage of viable cells were measured in stressed culture. Exposure of algae to strong (5000 μmol photons m 2 s 1 ) or to low (60 μmol photons m −2 s −1 ) light combined with the addition of 1.6 μM Cu 2+ or 30 μM Zn 2+ inactivated Photosystem II, decreased the viability of Chlorella cells, and, finally, significantly enhanced TL and the accumulation of TBARS, which was accompanied by chlorophyll bleaching. TL emission started to rise after a lag-period of about 30 min in algae subjected to strong illumination, 2–3 h in copper-treated algae, and 10 h in zinc-treated algae. A vast majority of cells were nonviable to the end of the lag-period. The addition of Cu 2+ or ZN 2+ in darkness caused a slight decrase in the F v / F M ratio without significant changes in TL emission. Incubation of algae at 50°C for 10 min did not affect the F v / F M ratio nd cell viability, whereas no viable cells and Photosystem II activity were detected in the culture incubated at 55°C. Heat stress at temperatures above 55°C significantly enhanced the amplitude of the 125°C TL peak and the accumulation of TBARS when the algae were further incubated at low light at room temperature. We conclude that, under the stress conditions used in this study, (i) lipid peroxides and products of their degradation are not responsible for the cytolethal effect in Chlorella and (ii) lipid peroxidation arises mainly upon illumination of dead cells.

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA–scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of δ-aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/chlL−/scpE− strain, whereas PChlide accumulated in the PS I-less/chlL−/scpB− strain. In the PS I-less/chlL− control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.

Small Cab-like proteins retard degradation of photosystem II-associated chlorophyll in Synechocystis sp. PCC 6803: kinetic analysis of pigment labeling with 15N and 13C.

Isotope (Na15NO3, (15NH4)SO4 or [13C]glucose) labeling was used to analyze chlorophyll synthesis and degradation rates in a set of Synechocystis mutants that lacked single or multiple small Cab-like proteins (SCPs), as well as photosystem I or II. When all five small Cab-like proteins were inactivated in the wild-type background, chlorophyll stability was not affected unless the scpABCDE- strain was grown at a moderately high light intensity of 100–300 μmol photons m-2 s-1. However, the half-life time of chlorophyll was 5-fold shorter in the photosystem I-less/scpABCDE- strain than in the photosystem I-less strain even when grown at low light intensity (∼3 μmol photons m-2 s-1) (32 ± 5 and 161 ± 25 h, respectively). In other photosystem I-less mutants that lacked one to four of the scp genes the chlorophyll lifetime was in between these two values, with the chlorophyll lifetime generally decreasing with an increasing number of inactivated scps. In contrast, the chlorophyll biosynthesis rate was only marginally affected by inactivation of scps except when all five scp genes were deleted. Small Cab-like protein deficiency did not significantly affect photoinhibition or turnover of photosystem II-associated β-carotene. It is concluded that SCPs do not alter the stability of functional photosystem II complexes but retard the degradation of photosystem II-associated chlorophyll, consistent with the proposed involvement of SCPs in photosystem II re-assembly or/and repair processes by temporarily binding chlorophyll while photosystem II protein components are being replaced.

The Origin of115–130°C Thermoluminescence Bands in Chlorophyll‐Containing Material

High‐temperature thermoluminescence (TL) emitted in the temperature region from +50 to +150°C has been studied in a variety of chlorophyll‐containing samples that were allowed to dry during the TL measurement. Analysis of the recorded traces by a multicomponent‐fit‐ting procedure revealed the existence of up to three bands of nonphotosynthetic origin with peak positions at62–75,114–128 and151–157°C and apparent activation energies of 27.0‐28.8, 14.1‐15.4 and 22.1‐23.3 kcal/mol (the bands are denoted as HT1 HT2 and HT3, respectively). Low‐temperature treatment of leaves, incubation of algae in the presence of paraquat, exposure of algae or isolated thylakoids to a strong light, all conditions known to stimulate oxidative damage to membrane lipids, caused appearance of a small HT1, band and significant rise in the intensity of the HT2 band. The increase in the HT2 component correlated positively with accumulation of conjugated dienes and malondialdehyde in thylakoids illuminated with a strong light. Different quenchers of active oxygen species and scavengers of free radicals added to preilluminated thylakoids or thylakoid lipid extracts before the TL measurements, as well as injection of argon into the TL measuring chamber, caused no changes in the intensity of the HT2 emission. The HT2 band in the thylakoids increased strongly upon addition of linoleate peroxidized by hydroxyradicals generated in the Fenton reaction but remained unchanged if the linoleate was oxidized with the use of lipoxygenase. We suggest that the HT2 band arises due to thermal decomposition of lipid cyclic peroxides present in the samples. In turn, the decomposition reaction leads to formation of carbonyls in triplet state with following migration of excitation energy toward chlorophyll. Contrary to the HT1, and HT2 bands, the HT3 band of TL cannot be associated with the thermolysis of lipid peroxidation products already present in the samples before starting the TL gradient.

Sublethal concentrations of copper stimulate photosystem II photoinhibition in Chlorella pyrenoidosa

Summary The effect of copper (CuSO 4 , 0.16-1.56 μM) on photosystem II (PS II) was investigated by measuring the chlorophyll fluorescence in Chlorella pyrenoidosa cells exposed to different irradiance levels and different temperatures. The algal culture remained viable only if less than 0.7-0.8 μM Cu 2+ was added. In darkness, PS II inactivation was indicated by a decrease in F v /F m ratio and was detected only at Cu 2+ concentrations higher than 0.7 μM. In the light, Cu 2+ stimulated PS II photoinhibition throughout the whole range of copper concentrations. Low temperature significantly enhanced the PS II photoinhibition in the presence of Cu 2+ . Kinetics of PS II inactivation monitored at algal growth light intensity in the presence of chloramphenicol were only slightly affected by the addition of Cu 2+ . However, Cu 2+ markedly slowed down (0.16 μM) or completely suppressed (0.7 μM) the recovery of PS II activity after the photoinhibitory treatment of the algae with a strong light. The results suggest that sublethal concentrations of copper can nonspecifically retard synthesis of the PS II D 1 protein in Chlorella cells , thus causing PS II inactivation under light.

Model for the Fluorescence Induction Curve of Photoinhibited Thylakoids

The fluorescence induction curve of photoinhibited thylakoids measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea was modeled using an extension of the model of Lavergne and Trissl (Biophys. J. 68:2474-2492), which takes into account the reversible exciton trapping by photosystem II (PSII) reaction centers and exciton exchange between PSII units. The model of Trissl and Lavergne was modified by assuming that PSII consists of photosynthetically active and photoinhibited (inactive in oxygen evolution) units and that the inactive PSII units can efficiently dissipate energy even if they still retain the capacity for the charge separation reaction. Comparison of theoretical and experimental fluorescence induction curves of thylakoids, which had been subjected to strong light in the presence of the uncoupler nigericin, suggests connectivity between the photoinhibited and active PSII units. The model predicts that photoinhibition lowers the yield of radical pair formation in the remaining active PSII centers. However, the kinetics of PSII inactivation in nigericin-treated thylakoids upon exposure to photoinhibitory light ranging from 185 to 2650 micromol photons m-2 s-1 was strictly exponential. This may suggest that photoinhibition occurs independently of the primary electron transfer reactions of PSII or that increased production of harmful substances by photoinhibited PSII units compensates for the protection afforded by the quenching of excitation energy in photoinhibited centers.

In search of a reversible stage of photoinhibition in a higher plant: No changes in the amount of functional Photosystem II accompany relaxation of variable fluorescence after exposure of lincomycin-treated Cucurbita pepo leaves to high light

Pumpkin (Cucurbita pepo L.) leaves in which chloroplast protein synthesis was inhibited with lincomycin were exposed to strong photoinhibitory light, and changes in FO, FM, FV/FM and in the amount of functional Photosystem II (O2 evolution induced by saturating single-turnover flashes) were monitored during the high-light exposure and subsequent dark or low-light incubation. In the course of the photoinhibitory illumination, FM, FV/FM and the amount of functional PS II declined continuously whereas FO dropped rapidly to some extent and then slowly increased. If the experiments were done at room temperature, termination of the photoinhibitory illumination resulted in partial relaxation of the FV/FM ratio and in an increase in FO and FM. The relaxation was completed in 10–15 min after short-term (15 min) photoinhibitory treatment but continued 30–40 min if the exposure to high light was longer than 1 h. No changes in the amount of functional PS II accompanied the relaxation of FV/FM in darkness or in low light, in the presence of lincomycin. Transferring the leaves to low temperature (+4°C) after the room-temperature illumination (2 h) completely inhibited the relaxation of FV/FM. Low temperature did not suppress the relaxation if the photoinhibitory illumination had also been done at low temperature. The results indicate that illumination of lincomycin-poisoned pumpkin leaves at room temperature does not lead to accumulation of a reversibly photoinactivated intermediate.

Isolation of functional photosystem II core particles from the Cyanobacterium synechocystis sp. PCC 6803.

This chapter contains the description of several methods used for the isolation of functional photosystem (PS)II core particles from wild-type (wt), PSI-less, and CP47 histidine-tagged cells of the cyanobacterium Synechocystis sp. PCC 6803. These protocols discuss the cultivation of PSI-containing and PSI-less cells, isolation of thylakoid membranes, purification of PSII core particles using a weak cation exchange or metal affinity column chromatography, and characterization of the final preparation. The described isolation procedures, which normally yield PSII particles highly active in oxygen evolution, can be easily adapted for obtaining preparations from different types of Synechocystis mutants with modified PSII.