Towards clarifying what distinguishes cyanobacteria able to resurrect after desiccation from those that cannot: The photosynthetic aspect.

Organisms inhabiting biological soil crusts (BSCs) are able to cope with extreme environmental conditions including daily hydration/dehydration cycles, high irradiance and extreme temperatures. The photosynthetic machinery, potentially the main source of damaging reactive oxygen species during cessation of CO(2) fixation in desiccating cells, must be protected to avoid sustained photodamage. We compared certain photosynthetic parameters and the response to excess light of BCS-inhabiting, desiccation-tolerant cyanobacteria Leptolyngbya ohadii and Nostoc reinholdii with those observed in the "model" organisms Nostoc sp. PCC 7120, able to resurrect after mild desiccation, and Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803 that are unable to recover from dehydration. Desiccation-tolerant strains exhibited a transient decline in the photosynthetic rate at light intensities corresponding to the inflection point in the PI curve relating the O(2) evolution rate to light intensity. They also exhibited a faster and larger loss of variable fluorescence and profoundly faster Q(A)(-) re-oxidation rates after exposure to high illumination. Finally, a smaller difference was found in the temperature of maximal thermoluminescence signal in the absence or presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) than observed in "model" cyanobacteria. These parameters indicate specific functional differences of photosystem II (PSII) between desiccation tolerant and sensitive cyanobacteria. We propose that exposure to excess irradiation activates a non-radiative electron recombination route inside PSII that minimizes formation of damaging singlet oxygen in the desiccation-tolerant cyanobacteria and thereby reduces photodamage.

An easily reversible structural change underlies mechanisms enabling desert crust cyanobacteria to survive desiccation.

Biological desert sand crusts are the foundation of desert ecosystems, stabilizing the sands and allowing colonization by higher order organisms. The first colonizers of the desert sands are cyanobacteria. Facing the harsh conditions of the desert, these organisms must withstand frequent desiccation-hydration cycles, combined with high light intensities. Here, we characterize structural and functional modifications to the photosynthetic apparatus that enable a cyanobacterium, Leptolyngbya sp., to thrive under these conditions. Using multiple in vivo spectroscopic and imaging techniques, we identified two complementary mechanisms for dissipating absorbed energy in the desiccated state. The first mechanism involves the reorganization of the phycobilisome antenna system, increasing excitonic coupling between antenna components. This provides better energy dissipation in the antenna rather than directed exciton transfer to the reaction center. The second mechanism is driven by constriction of the thylakoid lumen which limits diffusion of plastocyanin to P700. The accumulation of P700(+) not only prevents light-induced charge separation but also efficiently quenches excitation energy. These protection mechanisms employ existing components of the photosynthetic apparatus, forming two distinct functional modes. Small changes in the structure of the thylakoid membranes are sufficient for quenching of all absorbed energy in the desiccated state, protecting the photosynthetic apparatus from photoinhibitory damage. These changes can be easily reversed upon rehydration, returning the system to its high photosynthetic quantum efficiency.

The mechanisms whereby the green alga Chlorella ohadii, isolated from desert soil crust, exhibits unparalleled photodamage resistance.

Excess illumination damages the photosynthetic apparatus with severe implications with regard to plant productivity. Unlike model organisms, the growth of Chlorella ohadii, isolated from desert soil crust, remains unchanged and photosynthetic O2 evolution increases, even when exposed to irradiation twice that of maximal sunlight. Spectroscopic, biochemical and molecular approaches were applied to uncover the mechanisms involved. D1 protein in photosystem II (PSII) is barely degraded, even when exposed to antibiotics that prevent its replenishment. Measurements of various PSII parameters indicate that this complex functions differently from that in model organisms and suggest that C. ohadii activates a nonradiative electron recombination route which minimizes singlet oxygen formation and the resulting photoinhibition. The light-harvesting antenna is very small and carotene composition is hardly affected by excess illumination. Instead of succumbing to photodamage, C. ohadii activates additional means to dissipate excess light energy. It undergoes major structural, compositional and physiological changes, leading to a large rise in photosynthetic rate, lipids and carbohydrate content and inorganic carbon cycling. The ability of C. ohadii to avoid photodamage relies on a modified function of PSII and the dissipation of excess reductants downstream of the photosynthetic reaction centers. The biotechnological potential as a gene source for crop plant improvement is self-evident.

Cross-talk between photomixotrophic growth and CO(2) -concentrating mechanism in Synechocystis sp. strain PCC 6803.

Simultaneous catabolic and anabolic glucose metabolism occurs in the same compartment during photomixotrophic growth of the model cyanobacterium Synechocystis sp. PCC 6803. The presence of glucose is stressful to the cells; it is reflected in the high frequency of suppression mutations in glucose-sensitive mutants. We show that glucose affects many cellular processes. It stimulates respiration and the rate of photosynthesis and quantum yield in low- but not high-CO(2) -grown cells. Fluorescence and thermoluminescence parameters of photosystem II are also affected but the results did not lend support to sustained glucose driven over reduction in the light. Glucose-sensitive mutants such as ΔpmgA (impaired in photomixotrophic growth) and Δhik31 (lacking histidine kinase 31) are far more susceptible under high than low air level of CO(2) . A glycine to tryptophan mutation in position 354 in NdhF3, involved in the high-affinity CO(2) uptake, rescued ΔpmgA. A rise in the apparent photosynthetic affinity to external inorganic carbon is observed in high-CO(2) -grown wild-type cells after the addition of glucose, but not in mutant ΔpmgA. This is attributed to upregulation of certain low-CO(2) -induced genes, involved in inorganic carbon uptake, in the wild type but not in ΔpmgA. These data uncovered a new level of interaction between CO(2) fixation (and the CO(2) -concentrating mechanism) and photomixotrophic growth in cyanobacteria.

Photoinactivation of photosystem II: is there more than one way to skin a cat?

We briefly review the main mechanisms proposed for photodamage to photosystem II (PSII), at the donor and acceptor sides, and then discuss the mechanism whereby filamentous cyanobacteria inhabiting biological sand crusts such as Microcoleus sp. are able to avoid serious damage to their photosynthetic machinery. We show that the decline in fluorescence following exposure to excess light does not reflect a reduction in PSII activity but rather the activation of a non-radiative charge recombination in PSII. Furthermore, we show that the difference in the thermoluminescent peak temperature intensities in these organisms, in the presence and absence of inhibitors such as dichlorophenyl-dimethylurea (DCMU), is smaller than observed in model organisms suggesting that the redox gap between Q(A)⁻ and P680+ is smaller. On the basis of these data, we propose that this could enable an alternative, pheophytin-independent recombination, thereby minimizing the damaging ¹O2 production associated with radiative recombination.

Light, redox state, thylakoid-protein phosphorylation and signaling gene expression.

Two recent publications concerning the chloroplast membrane-protein phosphorylation and state transition might lead to further progress in the elucidation of the mechanism and role of this process. A thylakoid-bound protein TSP9 is released to the chloroplast matrix upon redox-dependent stepwise phosphorylation of three threonine sites and might signal redox-dependent gene transcription. The state-transition process and phosphorylation of the light-harvesting complex II require the activity of a novel protein kinase Stt7.

Changes in the photosynthetic reaction centre II in the diatom Phaeodactylum tricornutum result in non-photochemical fluorescence quenching.

Diatoms are an important group of primary producers in the aquatic environment. They are able to acclimate to fast changes in the light intensity by various mechanisms including a rise in non-photochemical fluorescence quenching (NPQ). The latter has been attributed to the xanthophyll cycle (XC) following activation of diadinoxanthin de-epoxidase by the acidification of the thylakoid lumen. To examine whether fluorescence quenching in the diatom Phaeodactylum tricornutum depends on the DeltapH generated by the photosynthetic electron transport, we arrested the latter by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). This treatment hardly affected the NPQ or XC, even when methylviologen was present. Dissipation of the DeltapH by 2,4-dinitrophenol inhibited the XC but did not alter NPQ. Similar results, i.e. inhibition of the XC but normal fluorescence quenching, were observed when the experiments were performed at 3 degrees C. Measurements of thermoluminescence showed that excess light treatment caused a marked decline in the signals obtained as a result of recombination of Q(B) (-) with the S(3) state of the Mn cluster; this was also observed in cells treated with DCMU (recombination of Q(A) (-) with S(2)). Light treatment also diminished the Q(A) (-) re-oxidation signals. The data suggest that changes in PSII core centre itself due to exposure to excess light conditions play an important part in the acclimation of P. tricornutum to the changing light conditions.

Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria.

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).

Structural basis for the thermostability of ferredoxin from the cyanobacterium Mastigocladus laminosus.

Plant-type ferredoxins (Fds) carry a single [2Fe-2S] cluster and serve as electron acceptors of photosystem I (PSI). The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus displays optimal activity at 65 degrees C. In order to reveal the molecular factors that confer thermostability, the crystal structure of M.laminosus Fd (mFd) was determined to 1.25 A resolution and subsequently analyzed in comparison with four similar plant-type mesophilic ferredoxins. The topologies of the plant-type ferredoxins are similar, yet two structural determinants were identified that may account for differences in thermostability, a salt bridge network in the C-terminal region, and the flexible L1,2 loop that increases hydrophobic accessible surface area. These conclusions were verified by three mutations, i.e. substitution of L1,2 into a rigid beta-turn ((Delta)L1,2) and two point mutations (E90S and E96S) that disrupt the salt bridge network at the C-terminal region. All three mutants have shown reduced electron transfer (ET) capabilities and [2Fe-2S] stability at high temperatures in comparison to the wild-type mFd. The results have also provided new insights into the involvement of the L1,2 loop in the Fd interactions with its electron donor, the PSI complex.

Redox regulation of thylakoid protein phosphorylation.

The photosystem II of chloroplast thylakoid membranes contains several proteins phosphorylated by redox-activated protein kinases. The mechanism of the reversible activation of the light-harvesting antenna complex II (LHCII) kinase(s) is one of the best understood and related to the regulation of energy transfer to photosystem II or I, thereby optimizing their relative excitation (state transition). The deactivated LHCII protein kinase(s) is associated with cytochrome b(6)f and dissociates from the complex upon activation. Activation of the LHCII protein kinase occurs via dynamic conformational changes in the cytochrome b(6)f complex taking place during plastoquinol oxidation. Deactivation of the kinase involves its reassociation with an oxidized cytochrome complex. A fine-tuning redox-dependent regulatory loop inhibits the activation of the kinase via reduction of protein disulfide groups, possibly involving the thioredoxin complex. Phosphorylation of LHCII is further modulated by light-induced conformational changes of the LHCII substrate. The reversible phosphorylation of LHCII and other thylakoid phosphoproteins, catalyzed by respective kinases and phosphatases, is under strict regulation in response to environmental changes.

Photoinhibition - a historical perspective.

Photoinhibition is a state of physiological stress that occurs in all oxygen evolving photosynthetic organisms exposed to light. The primary damage occurs within the reaction center of Photosystem II (PS II). While irreversible photoinduced damage to PS II occurs at all light intensities, the efficiency of photosynthetic electron transfer decreases markedly only when the rate of damage exceeds the rate of its repair, which requires de novo PS II protein synthesis. Photoinhibition has been studied for over a century using a large variety of biochemical, biophysical and genetic methodologies. The discovery of the light induced turnover of a protein, encoded by the plastid psbA gene (the D1 protein), later identified as one of the photochemical reaction center II proteins, has led to the elucidation of the underlying mechanism of photoinhibition and to a deeper understanding of the PS II 'life cycle.'

Cold-acclimation protects photosystem II against freezing damage in the halotolerant alga Dunaliella salina.

Cold-acclimation (CA) of the halotolerant alga Dunaliella was inhibited by light and by high salt. CA was associated with enhanced resistance to freezing in saline growth solutions, as manifested by protection of photosynthetic oxygen evolution and by reduced permeabilisation of the plasma membrane. Oxygen evolution activity in isolated chloroplasts was not affected by freezing, but was inhibited by high salt and the inhibition could be reversed or protected by glycerol. The activity of chloroplasts from cold-acclimated cells was more resistant to salt than of non-acclimated cells. Electron transport measurements in chloroplasts indicated that high salt inhibited PS-II, but not PS-I electron transport. High salt also inhibited PS-II thermoluminescence (TL) activity in chloroplasts. Similar inhibition of PS-II TL was observed by freezing intact cells in saline solutions. Chloroplasts from cold-acclimated cells had enhanced resistance to inhibition of PS-II electron transport and of PS-II TL by high salt. These results suggest that inhibition of oxygen evolution upon freezing Dunaliella cells may result from inactivation of PS-II due to massive influx of salt and loss of glycerol. The enhanced freeze-resistance of cold-acclimated cells to inhibition of oxygen evolution can be accounted for partly by protection of PS-II against high salt.

Light-induced changes within photosystem II protects Microcoleus sp. in biological desert sand crusts against excess light.

The filamentous cyanobacterium Microcoleus vaginatus, a major primary producer in desert biological sand crusts, is exposed to frequent hydration (by early morning dew) followed by desiccation during potentially damaging excess light conditions. Nevertheless, its photosynthetic machinery is hardly affected by high light, unlike "model" organisms whereby light-induced oxidative stress leads to photoinactivation of the oxygen-evolving photosystem II (PSII). Field experiments showed a dramatic decline in the fluorescence yield with rising light intensity in both drying and artificially maintained wet plots. Laboratory experiments showed that, contrary to "model" organisms, photosynthesis persists in Microcoleus sp. even at light intensities 2-3 times higher than required to saturate oxygen evolution. This is despite an extensive loss (85-90%) of variable fluorescence and thermoluminescence, representing radiative PSII charge recombination that promotes the generation of damaging singlet oxygen. Light induced loss of variable fluorescence is not inhibited by the electron transfer inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), nor the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), thus indicating that reduction of plastoquinone or O(2), or lumen acidification essential for non-photochemical quenching (NPQ) are not involved. The rate of Q(A) (-) re-oxidation in the presence of DCMU is enhanced with time and intensity of illumination. The difference in temperatures required for maximal thermoluminescence emissions from S(2)/Q(A) (-) (Q band, 22 degrees C) and S(2,3)/Q(B) (-) (B band, 25 degrees C) charge recombinations is considerably smaller in Microcoleus as compared to "model" photosynthetic organisms, thus indicating a significant alteration of the S(2)/Q(A) (-) redox potential. We propose that enhancement of non-radiative charge recombination with rising light intensity may reduce harmful radiative recombination events thereby lowering (1)O(2) generation and oxidative photodamage under excess illumination. This effective photo-protective mechanism was apparently lost during the evolution from the ancestor cyanobacteria to the higher plant chloroplast.

Thylakoid membrane remodeling during state transitions in Arabidopsis.

Adaptability of oxygenic photosynthetic organisms to fluctuations in light spectral composition and intensity is conferred by state transitions, short-term regulatory processes that enable the photosynthetic apparatus to rapidly adjust to variations in light quality. In green algae and higher plants, these processes are accompanied by reversible structural rearrangements in the thylakoid membranes. We studied these structural changes in the thylakoid membranes of Arabidopsis thaliana chloroplasts using atomic force microscopy, scanning and transmission electron microscopy, and confocal imaging. Based on our results and on the recently determined three-dimensional structure of higher-plant thylakoids trapped in one of the two major light-adapted states, we propose a model for the transitions in membrane architecture. The model suggests that reorganization of the membranes involves fission and fusion events that occur at the interface between the appressed (granal) and nonappressed (stroma lamellar) domains of the thylakoid membranes. Vertical and lateral displacements of the grana layers presumably follow these localized events, eventually leading to macroscopic rearrangements of the entire membrane network.

Impact of PsbTc on forward and back electron flow, assembly, and phosphorylation patterns of photosystem II in tobacco.

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn(4)Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in DeltapsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electron-accepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in DeltapsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in DeltapsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.

Thylakoid membrane perforations and connectivity enable intracellular traffic in cyanobacteria.

Cyanobacteria, the progenitors of plant and algal chloroplasts, enabled aerobic life on earth by introducing oxygenic photosynthesis. In most cyanobacteria, the photosynthetic membranes are arranged in multiple, seemingly disconnected, concentric shells. In such an arrangement, it is unclear how intracellular trafficking proceeds and how different layers of the photosynthetic membranes communicate with each other to maintain photosynthetic homeostasis. Using electron microscope tomography, we show that the photosynthetic membranes of two distantly related cyanobacterial species contain multiple perforations. These perforations, which are filled with particles of different sizes including ribosomes, glycogen granules and lipid bodies, allow for traffic throughout the cell. In addition, different layers of the photosynthetic membranes are joined together by internal bridges formed by branching and fusion of the membranes. The result is a highly connected network, similar to that of higher-plant chloroplasts, allowing water-soluble and lipid-soluble molecules to diffuse through the entire membrane network. Notably, we observed intracellular membrane-bounded vesicles, which were frequently fused to the photosynthetic membranes and may play a role in transport to these membranes.

Deletion of PsbM in tobacco alters the QB site properties and the electron flow within photosystem II.

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.

CHRD, a plant member of the evolutionarily conserved YjgF family, influences photosynthesis and chromoplastogenesis.

Studies on the carotenoid-overaccumulating structures in chromoplasts have led to the characterization of proteins termed plastid lipid-associated proteins (PAPs), involved in the sequestration of hydrophobic compounds. Here we characterize the PAP CHRD, which, based on sequence homology, belongs to a highly conserved group of proteins, YER057c/YjgF/UK114, involved in the regulation of basic and vital cellular processes in bacteria, yeast and animals. Two nuclear genes were characterized in tomato plants: one (LeChrDc) is constitutively expressed in various tissues and the other (LeChrDi) is induced by stress in leaves and is upregulated by developmental cues in floral tissues. Using RNAi and antisense approaches, we show their involvement in biologically significant processes such as photosynthesis. The quantum yield of photosynthetic electron flow in transgenic tomato leaves with suppressed LeChrDi/c expression was 30-50% of their control, non-transgenic counterparts and was ascribed to lower PSI activity. Transgenic flowers with suppressed LeChrDi/c also accumulated up to 30% less carotenoids per unit protein as compared to control plants, indicating an interrelationship between PAPs and floral-specific carotenoid accumulation in chromoplasts. We suggest that CHRD's role in the angiosperm reproductive unit may be a rather recent evolutionary development; its original function may have been to protect the plant under stress conditions by preserving plastid functionality.

Arabidopsis seed development and germination is associated with temporally distinct metabolic switches.

While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.