Photobiological hydrogen production and artificial photosynthesis for clean energy: from bio to nanotechnologies.

Global energy demand is increasing rapidly and due to intensive consumption of different forms of fuels, there are increasing concerns over the reduction in readily available conventional energy resources. Because of the deleterious atmospheric effects of fossil fuels and the uncertainties of future energy supplies, there is a surge of interest to find environmentally friendly alternative energy sources. Hydrogen (H2) has attracted worldwide attention as a secondary energy carrier, since it is the lightest carbon-neutral fuel rich in energy per unit mass and easy to store. Several methods and technologies have been developed for H2 production, but none of them are able to replace the traditional combustion fuel used in automobiles so far. Extensively modified and renovated methods and technologies are required to introduce H2 as an alternative efficient, clean, and cost-effective future fuel. Among several emerging renewable energy technologies, photobiological H2 production by oxygenic photosynthetic microbes such as green algae and cyanobacteria or by artificial photosynthesis has attracted significant interest. In this short review, we summarize the recent progress and challenges in H2-based energy production by means of biological and artificial photosynthesis routes.

Abnormal regulation of photosynthetic electron transport in a chloroplast ycf9 inactivation mutant.

The ycf9 (orf62) gene of the plastid genome encodes a 6.6-kDa protein (ORF62) of thylakoid membranes. To elucidate the role of the ORF62 protein, the coding region of the gene was disrupted with an aadA cassette, yielding mutant plants that were nearly (more than 95%) homoplasmic for ycf9 inactivation. The ycf9 mutant had no altered phenotype under standard growth conditions, but its growth rate was severely reduced under suboptimal irradiances. On the other hand, it was less susceptible to photodamage than the wild type. ycf9 inactivation resulted in a clear reduction in protein amounts of CP26, the NAD(P)H dehydrogenase complex, and the plastid terminal oxidase. Furthermore, depletion of ORF62 led to a faster flow of electrons to photosystem I without a change in the maximum electron transfer capacity of photosystem II. Despite the reduction of CP26 in the mutant thylakoids, no differences in PSII oxygen evolution rates were evident even at low light intensities. On the other hand, the ycf9 mutant presented deficiencies in the capacity for PSII-independent electron transport (ferredoxin-dependent cyclic electron transport and NAD(P)H dehydrogenase-mediated plastoquinone reduction). Altogether, it is shown that depletion of ORF62 leads to anomalies in the photosynthetic electron transfer chain and in the regulation of electron partitioning among the different routes of electron transport.

The Kautsky curve is a built-in barcode.

We identify objects from their visually observable morphological features. Automatic methods for identifying living objects are often needed in new technology, and these methods try to utilize shapes. When it comes to identifying plant species automatically, machine vision is difficult to implement because the shapes of different plants overlap and vary greatly because of different viewing angles in field conditions. In the present study we show that chlorophyll a fluorescence, emitted by plant leaves, carries information that can be used for the identification of plant species. Transient changes in fluorescence intensity when a light is turned on were parameterized and then subjected to a variety of pattern recognition procedures. A Self-Organizing Map constructed from the fluorescence signals was found to group the signals according to the phylogenetic origins of the plants. We then used three different methods of pattern recognition, of which the Bayesian Minimum Distance classifier is a parametric technique, whereas the Multilayer Perceptron neural network and k-Nearest Neighbor techniques are nonparametric. Of these techniques, the neural network turned out to be the most powerful one for identifying individual species or groups of species from their fluorescence transients. The excellent recognition accuracy, generally over 95%, allows us to speculate that the method can be further developed into an application in precision agriculture as a means of automatically identifying plant species in the field.

Exposure of Synechocystis 6803 cells to series of single turnover flashes increases the psbA transcript level by activating transcription and down-regulating psbA mRNA degradation.

Exposure of Synechocystis sp. PCC 6803 cells to series of single turnover flashes increases specifically the level of psbA and psbD2 messages, encoding the D1 and D2 proteins of photosystem II, as compared to light exposed cells. This increase is due to maintenance the transcription rate as high as in growth light and to the down-regulation of transcript degradation as in darkness. Inhibition of the plastoquinone pool reduction by DCMU or its oxidation by DBMIB does not diminish the transcription of the psbA gene under growth conditions. However, the degradation rate of psbA transcript, as well as of other transcripts encoding proteins of thylakoid complexes, is down-regulated in all conditions leading to the oxidation of the plastoquinone pool. We conclude that single turnover flashes are sensed as 'light' by transcription machinery of the cells irrespective of the plastoquinone pool reduction state and as 'dark' by the transcript degradation system.

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.

Increase in the quantum yield of photoinhibition contributes to copper toxicity in vivo

The effect of copper on photoinhibition of photosystem II in vivo was studied in bean (Phaseolus vulgaris L. cv Dufrix). The plants were grown hydroponically in the presence of various concentrations of Cu2+ ranging from the optimum 0.3 &mgr;m (control) to 15 &mgr;m. The copper concentration of leaves varied according to the nutrient medium from a control value of 13 mg kg-1 dry weight to 76 mg kg-1 dry weight. Leaf samples were illuminated in the presence and absence of lincomycin at different light intensities (500-1500 &mgr;mol photons m-2 s-1). Lincomycin prevents the concurrent repair of photoinhibitory damage by blocking chloroplast protein synthesis. The photoinhibitory decrease in the light-saturated rate of O2 evolution measured from thylakoids isolated from treated leaves correlated well with the decrease in the ratio of variable to maximum fluorescence measured from the leaf discs; therefore, the fluorescence ratio was used as a routine measurement of photoinhibition in vivo. Excess copper was found to affect the equilibrium between photoinhibition and repair, resulting in a decrease in the steady-state concentration of active photosystem II centers of illuminated leaves. This shift in equilibrium apparently resulted from an increase in the quantum yield of photoinhibition (PhiPI) induced by excess copper. The kinetic pattern of photoinhibition and the independence of PhiPI on photon flux density were not affected by excess copper. An increase in PhiPI may contribute substantially to Cu2+ toxicity in certain plant species.

Mutagenesis of the D-E loop of photosystem II reaction centre protein D1. Function and assembly of photosystem II.

The sequence connecting alpha-helices D and E of the D1 protein in photosystem II (PSII) is longer than that found in the corresponding loop of the L subunit in the rhodobacterial reaction centre. This sequence was mutated in order to determine its role in oxygenic photosynthesis. Site-specific mutants, including point mutations and deletions of different size, of the PEST-like region and the putative cleavage area in the D-E loop of the D protein were constructed in Synechocystis sp. PCC 6803. The effects of mutations on the functional and structural properties of PSII and turnover of the D1 protein were examined. Our results demonstrate that deletion of either the PEST-like sequence (deltaR225-F239) or the putative cleavage region (deltaG240-V249, deltaR225-V249) of the D1 protein resulted in severe perturbations on the function of the QB electron acceptor of PSII. However, PSII centres of the mutant with deleted PEST region remained functional enough to support autotrophic growth whereas deletions of the putative cleavage region prevented autotrophic growth. Although enhanced degradation rates of the mutant D1 proteins under low-light growth conditions demonstrate that neither the PEST-like sequence nor the putative cleavage region are required for D1 proteolysis, it became clear that the extension in the D-E loop of the D1 protein is essential for proper PSII assembly and photoautotrophic growth. Moreover, modifications of the D-E loop resulted in transcriptional activation of the psbA gene, indicating that neither light intensity, as such, nor the activity of the electron transfer chain are the only determinants in regulation of psbA gene transcription.

Degradation pattern of photosystem II reaction center protein D1 in intact leaves. The major photoinhibition-induced cleavage site in D1 polypeptide is located amino terminally of the DE loop.

Photoinhibition-induced degradation of the D1 protein of the photosystem II reaction center was studied in intact pumpkin (Cucurbita pepo L.) leaves. Photoinhibition was observed to cause the cleavage of the D1 protein at two distinct sites. The main cleavage generated an 18-kD N-terminal and a 20-kD C-terminal degradation fragment of the D1 protein. this cleavage site was mapped to be located clearly N terminally of the DE loop. The other, less-frequent cleavage occurred at the DE loop and produced the well-documented 23-kD, N-terminal D1 degradation product. Furthermore, the 23-kD, N-terminal D1 fragment appears to be phosphorylated and can be detected only under severe photoinhibition in vivo. Comparison of the D1 degradation pattern after in vivo photoinhibition to that after in vitro acceptor-side and donor-side photoinhibition, performed with isolated photosystem II core particles, gives indirect evidence in support of donor-side photoinhibition in intact leaves.

The rate constant of photoinhibition, measured in lincomycin-treated leaves, is directly proportional to light intensity.

Pumpkin leaves grown under high light (500-700 micromol of photons m-2.s-1) were illuminated under photon flux densities ranging from 6.5 to 1500 micromol.m-2.s-1 in the presence of lincomycin, an inhibitor of chloroplast protein synthesis. The illumination at all light intensities caused photoinhibition, measured as a decrease in the ratio of variable to maximum fluorescence. Loss of photosystem II (PSII) electron transfer activity correlated with the decrease in the fluorescence ratio. The rate constant of photoinhibition, determined from first-order fits, was directly proportional to photon flux density at all light intensities studied. The fluorescence ratio did not decrease if the leaves were illuminated in low light in the absence of lincomycin or incubated in darkness in the presence of lincomycin. The constancy of the quantum yield of photoinhibition under different photon flux densities strongly suggests that photoinhibition in vivo occurs by one dominant mechanism under all light intensities. This mechanism probably is not the acceptor side mechanism characterized in the anaerobic case in vitro. Furthermore, there was an excellent correlation between the loss of PSII activity and the loss of the D1 protein from thylakoid membranes under low light. At low light, photoinhibition occurs so slowly that inactive PSII centers with the D1 protein waiting to be degraded do not accumulate. The kinetic agreement between D1 protein degradation and the inactivation of PSII indicates that the turnover of the D1 protein depends on photoinhibition under both low and high light.

A Mutation in the D-de Loop of D1 Modifies the Stability of the S2QA- and S2QB- States in Photosystem II.

Photosystem II electron transfer, charge stabilization, and photoinhibition were studied in three site-specific mutants of the D1 polypeptide of Synechocystis PCC 6803: E243K, E229D, and CA1 (deletion of three glutamates 242-244 and a substitution, glutamine-241 to histidine). The phenotypes of the E229D and E243K mutants were similar to that of the control strain (AR) in all of the studied aspects. The characteristics of CA1 were very different. Formate, which inhibits the QA- to QB- reaction, was severalfold less effective in CA1 than in AR. The S2QA- and S2QB- states were stabilized in CA1. It was previously shown that the electron transfer between QA- and QB was modified in CA1 (P Maenpaa, T. Kallio, P. Mulo, G. Salih, E.-M. Aro, E. Tyystjarvi, C. Jansson [1993] Plant Mol Biol 22: 1-12). A change in the redox potential of the QA/QA- couple, which renders the reoxidation of QA- by back or forward reactions more difficult, could explain the phenotype of CA1. Although the rates of photoinhibition measured as inhibition of oxygen evolution, Chl fluorescence quenching, and decrease of thermoluminescence B and Q bands were similar in AR and CA1, the CA1 strain more quickly reached a state from which the cells were unable to recover their activity. The results described in this paper suggest that a modification in the structure of the D-de loop of D1 could influence the properties of the couple QA/QA- in D2 and the mechanism of recovery from photoinhibition.

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.

Mathematical modelling of photoinhibition and Photosystem II repair cycle. I. Photoinhibition and D1 protein degradation in vitro and in the absence of chloroplast protein synthesis in vivo.

The kinetics of photoinhibition of Photosystem II and D1 protein degradation were studied by applying mathematical modelling to new and published data. The word 'photoinhibition' refers here only to such inhibition of PS II activity that requires chloroplast protein synthesis for recovery. It is shown that acceptor-side photoinhibition in vitro as well as in vivo photoinhibition in higher plants and cyanobacteria in the presence of prokaryotic translation inhibitors follow first-order kinetics. Degradation of damaged D1 protein also fits in a first-order reaction equation with respect to the concentration of photoinhibited PS II centres. It is shown that photoprotective lowering of the ratio of variable to maximum fluorescence can be distinguished from the lowering of this ratio associated with photoinhibition.

Site-specific mutations in the D1 polypeptide affect the susceptibility of Synechocystis 6803 cells to photoinhibition.

Photoinhibition of photosystem II in the cyanobacterium Synechocystis 6803 was followed after site-specific mutagenesis of the D1 polypeptide. Mutations were created in the stromal/cytosolic loop connecting helices D and E. Two mutations E243K and CA1, a deletion of the three glutamates 242-244 and a substitution Q241H, were made in the putative cleavage area of the D1 polypeptide. A third mutation E229D was made in the PEST-like sequence. Mutants and control cells were illuminated and FV/FM was recorded. Compared to the control, the mutants were less photoinhibited. Fluorescence relaxation after a single flash was delayed in CA1. Restoration of FV/FM after photoinhibition in the mutants was totally dependent on protein synthesis while control cells were able to recover partially also when protein synthesis was inhibited. In addition, the protein synthesis-dependent recovery of CA1 was slowed down. Our results indicate a correlation between the mutated amino acids and photoinhibition of photosystem II.

Slow degradation of the d1 protein is related to the susceptibility of low-light-grown pumpkin plants to photoinhibition.

Photoinhibition of photosystem II (PSII) electron transport and subsequent degradation of the D1 protein were studied in pumpkin (Cucurbita pepo L.) leaves developed under high (1000 mumol m(-2) s(-1)) and low (80 mumol m(-2) s(-1)) photon flux densities. The low-light leaves were more susceptible to high light. This difference was greatly diminished when illumination was performed in the presence of chloramphenicol, indicating that a poor capacity to repair photodamaged PSII centers is decisive in the susceptibility of low-light leaves to photoinhibition. In fact, the first phases of the repair cycle, degradation and removal of photodamaged D1 protein from the reaction center complex, occurred slowly in low-light leaves, whereas in high-light leaves the degradation of the D1 protein more readily followed photoinhibition of PSII electron transport. A modified form of the D1 protein, with slightly slower electrophoretic mobility than the original D1, accumulated in the appressed thylakoid membranes of low-light leaves during illumination and was subsequently degraded only slowly.

ATP and light regulate D1 protein modification and degradation. Role of D1* in photoinhibition.

We have recently shown that during in vivo photoinhibition the D1 protein is degraded via a modified form, designated D1*. Depending on light conditions, the amount of D1* varies in leaves between 0 and 50% of total D1 content. By isolating thylakoids from leaves acclimated to different light levels, and performing photoinhibition experiments on these thylakoids, the following results on D1 protein degradation were obtained: (i) the protease involved in D1 degradation requires activation by light; (ii) neither acceptor nor donor side photoinhibition of PSII induces formation of D1* in vitro; (iii) in isolated thylakoids, the transformation of D1 to D1* can be induced in low light in the presence of ATP, which suggests that D1* is a phosphorylated form of the D1 protein; (iv) D1*, induced either in vivo or in vitro, is much less susceptible to degradation during illumination of isolated thylakoids than the original D1 protein. We suggest that the modification to D1* is a means to prevent disassembly of photodamaged photosystem II complex in appressed membranes.

Small light-harvesting antenna does not protect from photoinhibition.

High-light-induced decrease in photosystem II (PSII) electron transfer activity was studied in high- and low-light-grown pumpkin (Cucurbita pepo L.) plants in vivo and in vitro. The PSII light-harvesting antenna of the low-light leaves was estimated to be twice as big as that of the high-light leaves. The low-light leaves were more susceptible to photoinhibition in vivo. However, thylakoids isolated from these two plant materials were equally sensitive to photoinhibition when illuminated in the absence of external electron acceptors. Only the intensity of the photoinhibitory light and the chlorophyll concentration of the sample, not the size of the light-harvesting antenna, determined the rate of PSII photoinhibition in vitro. Because excitation of the reaction center and not only the antenna chlorophylls is a prerequisite for photoinhibition of PSII activity, independence of photoinhibition on antenna size provides support for the hypothesis (Schatz EH, Brock H, Holzwarth AR [1988] Biophys J 54: 397-405) that the excitations of the antenna chlorophylls are in equilibrium with the excitations of the reaction centers. Better tolerance of the high-light leaves in vivo was due to a more active repair process and more powerful protective mechanisms, including photosynthesis. Apparently, some protective mechanism of the high-light-grown plants is at least partially active at low temperature. The protective mechanisms do not appear to function in vitro.

D1 protein degradation during photoinhibition of intact leaves. A modification of the D1 protein precedes degradation.

Illumination of intact pumpkin leaves with high light led to severe photoinhibition of photosystem II with no net degradation of the D1 protein. Instead, however, a modified form of D1 protein with slightly slower electrophoretic mobility was induced with corresponding loss in the original form of the D1 protein. When the leaves were illuminated in the presence of chloramphenicol the modified form was degraded, which led to a decrease in the total amount of the D1 protein. Subfractionation of the thylakoid membranes further supported the conclusion that the novel form of the D1 protein was not a precursor but a high-light modified form that was subsequently degraded.

Temperature-dependent changes in Photosystem II heterogeneity of attached leaves under high light.

Attached leaves of pumpkin (Cucurbita pepo L. cv. Jattiläismeloni) were exposed to high light intensity at room temperature (ca 23°C) and at 1°C. Fluorescence parameters and electron transport activities measured from isolated thylakoids indicated faster photoinhibition of PSII at low temperature. Separation of the α and ß components of the complementary area above the fluorescence induction curve of dichlorophenyl-dimethylurea-poisoned thylakoids revealed that at low temperature only the α-centers declined during exposure to high light intensity while the content of functional ß-centers remained constant. Freeze-fracture electron microscopy showed no decrease in the density of particles on the appressed exoplasmic fracture face, indicating that the photoinhibited α-centers remained in the appressed membranes at 1°C. Because of the function of the repair and protective mechanisms of PSII, strong light induced less photoinhibition at room temperature, but more complicated changes occurred in the α/ß-heterogeneity of PSII. During the first 30 min at high light intensity the decrease in α-centers was almost as large as at 1°C, but in contrast to the situation at low temperature the decrease in α-centers was compensated for by a significant increase in PSIIß-centers. Changes in the density and size of freeze-fracture particles suggest that this increase in ß-centers was due to migration of phosphorylated light-harvesting complex from appressed to non-appressed thylakoid membranes while the PSII core remained in the appressed membranes. This situation, however, was only transient and was followed by a rapid decrease in the functionalß-centers.

Temperature-dependent changes in Photosystem II heterogeneity support a cycle of Photosystem II during photoinhibition.

High light treatments were given to attached leaves of pumpkin (Cucurbita pepo L.) at room temperature and at 1°C where the diffusion- and enzyme-dependent repair processes of Photosystem II are at a minimum. After treatments, electron transfer activities and fluorescence induction were measured from thylakoids isolated from the treated leaves. When the photoinhibition treatment was given at 1°C, the Photosystem II electron transfer assays that were designed to require electron transfer to the plastoquinone pool showed greater inhibition than electron transfer from H2O to paraphenyl-benzoquinone, which measures all PS II centers. When the light treatment was given at room temperature, electron transfer from H2O to paraphenyl-benzoquinone was inhibited more than whole-chain electron transfer. Variable fluorescence measured in the presence of ferricyanide decreased only during room-temperature treatments. These results suggest that reaction centers of one pool of Photosystem II, non-QB-PS II, replace photoinhibited reaction centers at room temperature, while no replacement occurs at 1°C. A simulation of photoinhibition at 1°C supports this conclusion.

A microcomputer program and fast analog to digital converter card for the analysis of fluorescence induction transients.

A microcomputer program and an analog to digital conversion card were developed for the analysis of fluorescence induction curves. The program and the analog to digital conversion card are compatible with all commercially available fluorometers. Most of the current analysis methods for fluorescence induction curves are implemented in the program, including analysis of OIDPSMT-kinetics, dissection of fluorescence quenching into two components, measurement of the slope of the fluorescence curve, complementary area analysis and analysis of energy 'spillover' from PS II to PS I. The program can also do basic statistical calculations from the measured parameters. The architecture of the program is open, allowing the user to add new methods to the main body of the program. Split time-scale is used in data capture and analysis. A new procedure facilitates accurate determination of F0.