Nanodiamond Particles Reduce Oxidative Stress Induced by Methyl Viologen and High Light in the Green Alga Chlamydomonas reinhardtii.

Widely used in biomedical and bioanalytical applications, the detonation nanodiamonds (NDs) are generally considered to be biocompatible and non-toxic to a wide range of eukaryotic cells. Due to their high susceptibility to chemical modifications, surface functionalisation is often used to tune the biocompatibility and antioxidant activity of the NDs. The response of photosynthetic microorganisms to redox-active NDs is still poorly understood and is the focus of the present study. The green microalga Chlamydomonas reinhardtii was used to assess the potential phytotoxicity and antioxidant activity of NDs hosting hydroxyl functional groups at concentrations of 5-80 µg NDs/mL. The photosynthetic capacity of microalgae was assessed by measuring the maximum quantum yield of PSII photochemistry and the light-saturated oxygen evolution rate, while oxidative stress was assessed by lipid peroxidation and ferric-reducing antioxidant capacity. We demonstrated that hydroxylated NDs might reduce cellular levels of oxidative stress, protect PSII photochemistry and facilitate the PSII repair under methyl viologen and high light associated stress conditions. Factors involved in this protection may include the low phytotoxicity of hydroxylated NDs in microalgae and their ability to accumulate in cells and scavenge reactive oxygen species. Our findings could pave the way for using hydroxylated NDs as antioxidants to improve cellular stability in algae-based biotechnological applications or semi-artificial photosynthetic systems.

Acclimation Response of Green Microalgae Chlorella Sorokiniana to 2,3',4,4',6-Pentachlorobiphenyl.

The effect of the toxicant 2,3',4,4',6-pentachlorobiphenyl (PCB-119) on the growth, chlorophyll content, and PSII activity of C. sorokiniana cells was investigated. A strong negative effect of the toxicant was observed at PCB concentration of 0.05 µg mL-1 , when culture growth ceased, chlorophyll strongly bleached, and cell death occurred. The use of original highly sensitive fluorimeter to measure three types of high-resolution chlorophyll fluorescence kinetics allowed us to detect an initial dramatic decrease in the activity of primary photosynthetic reactions, followed by their almost complete recovery at the end of the incubation period when most cells were dead. The study of the distribution of individual cells in culture in terms of Fv /Fm parameter, which reflects the quantum yield of PSII photochemistry, revealed the existence of 2-3% of cells retaining high Fv /Fm (>0.7) in the presence of the toxicant. The treated cultures were able to resume growth after prolonged incubation in fresh medium. The high sensitivity fluorescence methods used made it possible to identify stress-resistant cells which maintain high photosynthetic activity in the presence of lethal doses of toxic substances; these cells provide recovery of the population after stress.

Single-walled carbon nanotubes protect photosynthetic reactions in Chlamydomonas reinhardtii against photoinhibition.

Single-walled carbon nanotubes (SWCNTs) are among the most exploited carbon allotropes in nanosensing, bioengineering, and photobiological applications, however, the interactions of nanotubes with the photosynthetic process and structures are still poorly understood. We found that SWCNTs are not toxic to the photosynthetic apparatus of the model unicellular alga Chlamydomonas reinhardtii and demonstrate that this carbon nanomaterial can protect algal photosynthesis against photoinhibition. The results show that the inherent phytotoxicity of the nanotubes may be overcome by an intentional selection of nanomaterial characteristics. A low concentration (2 µg mL-1) of well-dispersed, purified and small SWCNTs did not alter the growth and pigment accumulation of the cultures. Indeed, under the photoinhibitory conditions of our experiments, SWCNT-enriched samples were characterized by a lower rate of PSII inactivation, reduced excitation pressure in PSII, a higher rate of photosynthetic electron transport, and an increased non-photochemical quenching in comparison with the controls. In addition, SWCNTs change the distribution of energy between the photosystems in favour of PSII (state 1). The underlying mechanism of this action is not yet understood but possibly, electrons or energy can be exchanged between the redox active nanotubes and photosynthetic components, and probably other redox active intra-chloroplast constituents. Alternatively, nanotubes may promote the formation of an NPQ conformation of PSII. Our results provided evidence for such electron/energy transfer from photosynthetic structures toward the nanotubes. The discovered photoprotective effects can potentially be used in photobiotechnology to maintain the photosynthetic activity of microorganisms under unfavourable conditions.

Mathematical Simulation of Electron Transport in the Primary Photosynthetic Processes.

Summarized results of investigation of regulation of electron transport and associated processes in the photosynthetic membrane using methods of mathematical and computer modeling carried out at the Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, are presented in this review. Detailed kinetic models of processes in the thylakoid membrane were developed using the apparatus of differential equations. Fitting of the model curves to the data of spectral measurements allowed us to estimate the values of parameters that were not determined directly in experiments. The probabilistic method of agent-based Monte Carlo modeling provides ample opportunities for studying dynamics of heterogeneous systems based on the rules for the behavior of individual elements of the system. Algorithms for simplified representation of Big Data make it possible to monitor changes in the photosynthetic apparatus in the course of culture growth in a photobioreactor and for the purpose of environmental monitoring. Brownian and molecular models describe movement and interaction of individual electron carrier proteins and make it possible to study electrostatic, hydrophobic, and other interactions leading to regulation of conformational changes in the reaction complexes. Direct multiparticle models explicitly simulate Brownian diffusion of the mobile protein carriers and their electrostatic interactions with multienzyme complexes both in solution and in heterogeneous interior of a biomembrane. The combined use of methods of kinetic and Brownian multiparticle and molecular modeling makes it possible to study the mechanisms of regulation of an integral system of electron transport processes in plants and algae at molecular and subcellular levels.

Molecular, Brownian, kinetic and stochastic models of the processes in photosynthetic membrane of green plants and microalgae.

The paper presents the results of recent work at the Department of Biophysics of the Biological Faculty, Lomonosov Moscow State University on the kinetic and multiparticle modeling of processes in the photosynthetic membrane. The detailed kinetic models and the rule-based kinetic Monte Carlo models allow to reproduce the fluorescence induction curves and redox transformations of the photoactive pigment P700 in the time range from 100 ns to dozens of seconds and make it possible to reveal the role of individual carriers in their formation for different types of photosynthetic organisms under different illumination regimes, in the presence of inhibitors, under stress conditions. The fitting of the model curves to the experimental data quantifies the reaction rate constants that cannot be directly measured experimentally, including the non-radiative thermal relaxation reactions. We use the direct multiparticle models to explicitly describe the interactions of mobile photosynthetic carrier proteins with multienzyme complexes both in solution and in the biomembrane interior. An analysis of these models reveals the role of diffusion and electrostatic factors in the regulation of electron transport, the influence of ionic strength and pH of the cellular environment on the rate of electron transport reactions between carrier proteins. To describe the conformational intramolecular processes of formation of the final complex, in which the actual electron transfer occurs, we use the methods of molecular dynamics. The results obtained using kinetic and molecular models supplement our knowledge of the mechanisms of organization of the photosynthetic electron transport processes at the cellular and molecular levels.

Simulating the Interplay between the Uptake of Inorganic Phosphate and the Cell Phosphate Metabolism under Phosphorus Feast and Famine Conditions in Chlorella vulgaris.

Using a mathematical simulation approach, we studied the dynamics of the green microalga Chlorella vulgaris phosphate metabolism response to shortage and subsequent replenishing of inorganic phosphate in the medium. A three-pool interaction model was used to describe the phosphate uptake from the medium, its incorporation into the cell organic compounds, its storage in the form of polyphosphates, and culture growth. The model comprises a system of ordinary differential equations. The distribution of phosphorous between cell pools was examined for three different stages of the experiment: growth in phosphate-rich medium, incubation in phosphate-free medium, and phosphate addition to the phosphorus-starving culture. Mathematical modeling offers two possible scenarios for the appearance of the peak of polyphosphates (PolyP). The first scenario explains the accumulation of PolyP by activation of the processes of its synthesis, and the decline in PolyP is due to its redistribution between dividing cells during growth. The second scenario includes a hysteretic mechanism for the regulation of PolyP hydrolysis, depending on the intracellular content of inorganic phosphate. The new model of the dynamics of P pools in the cell allows one to better understand the phenomena taking place during P starvation and re-feeding of the P-starved microalgal cultures with inorganic phosphate such as transient PolyP accumulation. Biotechnological implications of the observed dynamics of the polyphosphate pool of the microalgal cell are considered. An approach enhancing the microalgae-based wastewater treatment method based on these scenarios is proposed.

State of the phycobilisome determines effective absorption cross-section of Photosystem II in Synechocystis sp. PCC 6803.

Quenching of excess excitation energy is necessary for the photoprotection of light-harvesting complexes. In cyanobacteria, quenching of phycobilisome (PBS) excitation energy is induced by the Orange Carotenoid Protein (OCP), which becomes photoactivated under high light conditions. A decrease in energy transfer efficiency from the PBSs to the reaction centers decreases photosystem II (PS II) activity. However, quantitative analysis of OCP-induced photoprotection in vivo is complicated by similar effects of both photochemical and non-photochemical quenching on the quantum yield of the PBS fluorescence overlapping with the emission of chlorophyll. In the present study, we have analyzed chlorophyll a fluorescence induction to estimate the effective cross-section of PS II and compared the effects of reversible OCP-dependent quenching of PBS fluorescence with reduction of PBS content upon nitrogen starvation or mutations of key PBS components. This approach allowed us to estimate the dependency of the rate constant of PS II primary electron acceptor reduction on the amount of PBSs in the cell. We found that OCP-dependent quenching triggered by blue light affects approximately half of PBSs coupled to PS II, indicating that under normal conditions, the concentration of OCP is not sufficient for quenching of all PBSs coupled to PS II.

Mapping Single Walled Carbon Nanotubes in Photosynthetic Algae by Single-Cell Confocal Raman Microscopy.

Carbon nanotubes (CNTs) are among the most exploited carbon allotropes in the emerging technologies of molecular sensing and bioengineering. However, the advancement of algal nanobiotechnology and nanobionics is hindered by the lack of methods for the straightforward visualization of the CNTs inside the cell. Herein, we present a handy and label-free experimental strategy based on visible Raman microscopy to assess the internalization of single-walled carbon nanotubes (SWCNTs) using the model photosynthetic alga Chlamydomonas reinhardtii as a recipient. The relationship between the properties of SWCNTs and their biological behavior was demonstrated, along with the occurrence of excitation energy transfer from the excited chlorophyll molecules to the SWCNTs. The non-radiative deactivation of the chlorophyll excitation promoted by the SWCNTs enables the recording of Raman signals originating from cellular compounds located near the nanotubes, such as carotenoids, polyphosphates, and starch. Furthermore, the outcome of this study unveils the possibility to exploit SWCNTs as spectroscopic probes in photosynthetic and non-photosynthetic systems where the fluorescence background hinders the acquisition of Raman scattering signals.

Comparative analysis of plastocyanin-cytochrome f complex formation in higher plants, green algae and cyanobacteria.

Mechanisms of the complex formation between plastocyanin and cytochrome f in higher plants (Spinacia oleracea and Brassica rapa), green microalgae Chlamydomonas reinhardtii and two species of cyanobacteria (Phormidium laminosum and Nostoc sp.) were investigated using combined Brownian and molecular dynamics simulations and hierarchical cluster analysis. In higher plants and green algae, electrostatic interactions force plastocyanin molecule close to the heme of cytochrome f. In the subsequent rotation of plastocyanin molecule around the point of electrostatic contact in the vicinity of cytochrome f, copper (Cu) atom approaches cytochrome heme forming a stable configuration where cytochrome f molecule behaves as a rather rigid body without conformational changes. In Nostoc plastocyanin molecule approaches cytochrome f in a different orientation (head-on) where the stabilization of the plastocyanin-cytochrome f complex is accompanied by the conformational changes of the G188E189D190 loop that stabilizes the whole complex. In cyanobacterium P. laminosum, electrostatic preorientation of the approaching molecules was not detected, thus indicating that random motions rather than long-range electrostatic interactions are responsible for the proper mutual orientation. We demonstrated that despite the structural similarity of the investigated electron transport proteins in different photosynthetic organisms, the complexity of molecular mechanisms of the complex formation increases in the following sequence: non-heterocystous cyanobacteria - heterocystous cyanobacteria - green algae - flowering plants.

Multiparticle Brownian dynamics simulation of experimental kinetics of cytochrome bf oxidation and photosystem I reduction by plastocyanin.

A model of electron transport from cytochrome f to photosystem I mediated by plastocyanin was designed on the basis of the multiparticle Brownian dynamics method. The model combines events which occur over a wide time range, including protein diffusion along the thylakoid membrane, long-distance interactions between proteins, formation of a multiprotein complex, electron transfer within a complex and complex dissociation. Results of the modeling were compared with the experimental kinetics measured in chloroplast thylakoids. Computer simulation demonstrated that the complex interior of the photosynthetic membrane, electrostatic interactions and Brownian diffusion provide physical conditions for the directed electron flow along the photosynthetic electron transport chain.

Comparative analyses of H2 photoproduction in magnesium- and sulfur-starved Chlamydomonas reinhardtii cultures.

Magnesium (Mg)-deprived Chlamydomonas reinhardtii cells are capable to sustain hydrogen (H2 ) photoproduction at relatively high photosystem II (PSII) activity levels for an extended time period as compared with sulfur (S)-deprived cells. Herein, we present a comparative study of H2 photoproduction induced by Mg and S shortage to unravel the specific rearrangements of the photosynthetic machinery and cell metabolism occurring under the two deprivation protocols. The exhaustive analysis of photosynthetic activity and regulatory pathways, respiration and starch metabolism revealed the specific rearrangements of the photosynthetic machinery and cellular metabolism, which occur under the two deprivation conditions. The obtained results allowed us to conclude that the expanded time period of H2 production upon Mg-deprivation is due to the less harmful effects that Mg-depletion has on viability and metabolic performance of the cells. Unlike S-deprivation, the photosynthetic light and dark reactions in Mg-deprived cells remained active over the whole H2 production period. However, the elevated PSII activity in Mg-deprived cells was counteracted by the operation of pathways for O2 consumption that maintain anaerobic conditions in the presence of active water splitting.

Multiple regulatory mechanisms in the chloroplast of green algae: relation to hydrogen production.

A complex regulatory network in the chloroplast of green algae provides an efficient tool for maintenance of energy and redox balance in the cell under aerobic and anaerobic conditions. In this review, we discuss the structural and functional organizations of electron transport pathways in the chloroplast, and regulation of photosynthesis in the green microalga Chlamydomonas reinhardtii. The focus is on the regulatory mechanisms induced in response to nutrient deficiency stress and anoxia and especially on the role of a hydrogenase-mediated reaction in adaptation to highly reducing conditions and ATP deficiency in the cell.

Photosynthesis-related quantities for education and modeling.

A quantitative understanding of the photosynthetic machinery depends largely on quantities, such as concentrations, sizes, absorption wavelengths, redox potentials, and rate constants. The present contribution is a collection of numbers and quantities related mainly to photosynthesis in higher plants. All numbers are taken directly from a literature or database source and the corresponding reference is provided. The numerical values, presented in this paper, provide ranges of values, obtained in specific experiments for specific organisms. However, the presented numbers can be useful for understanding the principles of structure and function of photosynthetic machinery and for guidance of future research.

Antimycin A effect on the electron transport in chloroplasts of two Chlamydomonas reinhardtii strains.

The effects of antimycin A on the redox state of plastoquinone and on electron donation to photosystem I (PS I) were studied in sulfur-deprived Chlamydomonas reinhardtii cells of the strains cc406 and 137c. We found that this reagent suppresses cyclic electron flow around PS I in the cc406 strain, whereas this inhibitory effect was completely absent in the 137c strain. In the latter strain, antimycin A induced rapid reduction of plastoquinone in the dark and considerably enhanced the rate of electron donation to P700 (+) in the dark. Importantly, neither myxothiazol, an inhibitor of mitochondrial respiration, FCCP, a protonophore, nor propyl gallate, an inhibitor of the plastid terminal oxidase, induced such a strong effect like antimycin A. The results indicate that in the chloroplast of the 137c strain, antimycin A has a site of action outside of the machinery of cyclic electron flow.

Acclimation of green algae to sulfur deficiency: underlying mechanisms and application for hydrogen production.

Hydrogen is definitely one of the most acceptable fuels in the future. Some photosynthetic microorganisms, such as green algae and cyanobacteria, can produce hydrogen gas from water by using solar energy. In green algae, hydrogen evolution is coupled to the photosynthetic electron transport in thylakoid membranes via reaction catalyzed by the specific enzyme, (FeFe)-hydrogenase. However, this enzyme is highly sensitive to oxygen and can be quickly inhibited when water splitting is active. A problem of incompatibility between the water splitting and hydrogenase reaction can be overcome by depletion of algal cells of sulfur which is essential element for life. In this review the mechanisms underlying sustained hydrogen photoproduction in sulfur deprived C. reinhardtii and the recent achievements in studying of this process are discussed. The attention is focused on the biophysical and physiological aspects of photosynthetic response to sulfur deficiency in green algae.

Membrane potential is involved in regulation of photosynthetic reactions in the marine diatom Thalassiosira weissflogii.

High-intensity Chl fluorescence transients (OJIP transients) and light-induced kinetics of the delayed light emission were measured in diatom microalga Thalassiosira weissflogii in the presence of various uncouplers and photosynthetic inhibitors. The I step in the OJIP transients in T. weissflogii was essentially reduced or completely absent but was restored in the presence of uncouplers valinomycin, FCCP, and nigericin. Moreover, valinomycin enhanced ΔpH-dependent non-photochemical fluorescence quenching following the OJIP rise. In the presence of valinomycin, the transthylakoid membrane potential was significantly inhibited as evaluated by measurements of the delayed light emission. The results suggest a membrane potential control of the fluorescence yield in T. weissflogii. Possible mechanisms underlying the observed effects of uncouplers are discussed.

Two-electron reactions S2QB -->S0QB and S3QB -->S1QB are involved in deactivation of higher S states of the oxygen-evolving complex of Photosystem II.

The oxygen-evolving complex of Photosystem II cycles through five oxidation states (S(0)-S(4)), and dark incubation leads to 25% S(0) and 75% S(1). This distribution cannot be reached with charge recombination reactions between the higher S states and the electron acceptor Q(B)(-). We measured flash-induced oxygen evolution to understand how S(3) and S(2) are converted to lower S states when the electron required to reduce the manganese cluster does not come from Q(B)(-). Thylakoid samples preconditioned to make the concentration of the S(1) state 100% and to oxidize tyrosine Y(D) were illuminated by one or two laser preflashes, and flash-induced oxygen evolution sequences were recorded at various time intervals after the preflashes. The distribution of the S states was calculated from the flash-induced oxygen evolution pattern using an extended Kok model. The results suggest that S(2) and S(3) are converted to lower S states via recombination from S(2)Q(B)(-) and S(3)Q(B)(-) and by a slow change of the state of oxygen-evolving complex from S(3) and S(2) to S(1) and S(0) in reactions with unspecified electron donors. The slow pathway appears to contain two-electron routes, S(2)Q(B) -->S(0)Q(B), and S(3)Q(B) -->S(1)Q(B). The two-electron reactions dominate in intact thylakoid preparations in the absence of chemical additives. The two-electron reaction was replaced by a one-electron-per-step pathway, S(3)Q(B) -->S(2)Q(B) -->S(1)Q(B) in PS II-enriched membrane fragments and in thylakoids measured in the presence of artificial electron acceptors. A catalase effect suggested that H(2)O(2) acts as an electron donor for the reaction S(2)Q(B) -->S(0)Q(B) but added H(2)O(2) did not enhance this reaction.

Illumination with ultraviolet or visible light induces chemical changes in the water-soluble manganese complex, [Mn(4)O(6)(bpea)(4)]Br(4).

We measured the photosensitivity of an artificial tetranuclear oxo-Mn(IV) complex, [Mn(4)O(6)(bpea)(4)]Br(4), which has an adamantane-shaped {Mn(4)O(6)}(4+) core. Illumination caused changes in the absorption spectrum of the compound consistent with a one-electron reduction in the compound. Bromide appears to be the most probable electron donor in the reaction system. Chemical modification of the cluster appears to destabilize it, predisposing it to reductive degradation. UV light was more efficient than visible light in causing the changes. The data support the suggestion that the natural oxygen-evolving Mn complex is photosensitive and can oxidize components of the oxygen-evolving complex in its excited state causing photoinhibition, and that photostability is an important issue in designing Mn complexes for artificial photosynthesis. Furthermore, light-induced oxidation of bromide by [Mn(4)O(6)(bpea)(4)](4+) may suggest that oxidation of chloride is involved in natural water splitting or has been involved during the evolution of the water-splitting enzyme.

Hydrogen production by photoautotrophic sulfur-deprived Chlamydomonas reinhardtii pre-grown and incubated under high light.

We have previously demonstrated that Chlamydomonas reinhardtii can produce hydrogen under strictly photoautotrophic conditions during sulfur deprivation [Tsygankov et al. (2006); Int J Hydrogen Energy 3:1574-1584]. The maximum hydrogen photoproduction was achieved by photoautotrophic cultures pre-grown under a low light regime (25 microE m(-2) s(-1)). We failed to establish sustained hydrogen production from cultures pre-grown under high light (100 microE m(-2) s(-1)). A new approach for sustained hydrogen production by these cultures is presented here. Assuming that stable and reproducible transition to anerobiosis as well as high starch accumulation are important for hydrogen production, the influence of light intensity and dissolved oxygen concentration during the oxygen evolving stage of sulfur deprivation were investigated in cultures pre-grown under high light. Results showed that light higher than 175 microE m(-2) s(-1) during sulfur deprivation induced reproducible transition to anerobiosis, although the total amount of starch accumulation and hydrogen production were insignificant. The potential PSII activity measured in the presence of an artificial electron acceptor (DCBQ) and an inhibitor of electron transport (DBMIB) did not change in cultures pre-grown under 20 microE m(-2) s(-1) and incubated under 150 microE m(-2) s(-1) during sulfur deprivation. In contrast, the potential PSII activity decreased in cultures pre-grown under 100 microE m(-2) s(-1) and incubated under 420 microE m(-2) s(-1). This indicates that cultures grown under higher light experience irreversible inhibition of PSII in addition to reversible down regulation. High dissolved O(2) content during the oxygen evolving stage of sulfur deprivation has a negative regulatory role on PSII activity. To increase hydrogen production by C. reinhardtii pre-grown under 100 microE m(-2) s(-1), cultures were incubated under elevated PFD and decreased oxygen pressure during the oxygen evolving stage. These cultures reproducibly reached anaerobic stage, accumulated significant quantities of starch and produced significant quantities of H(2). It was found that elevation of pH from 7.4 to 7.7 during the oxygen producing stage of sulfur deprivation led to a significant increase of accumulated starch. Thus, control of pH during sulfur deprivation is a possible way to further optimize hydrogen production by photoautotrophic cultures.