Decay of cytochromes and appearance of a cyanide-insensitive electron transfer pathway in Euglena gracilis grown in anoxia

The effect of anoxia over a 3-week period on the respiratory ability of Euglena gracilis (strains Z and ZC) was studied. Low temperature absorption spectra indicated that comparable alterations of the cytochromes occurred in both cell types studies, leading to the disappearance of cytochrome oxidase and cytochrome c558 in the aplastic strain ZC and in the wild type strain Z. Both types of cells maintained their ability to consume O₂ when they were transferred from the nitrogen atmosphere to air. Resistance of respiration to cyanide and azide, and sensitivity to propyl gallate increased during anoxia, indicating the decreasing role of cytochrome oxidase in this O₂ consumption. Quantitative changes in the O₂ consumption capacity were followed during the course of anoxia; this capacity decreased during the 5 first days of anoxia and then increased to recover its initial value during the second week. This recovery of a high O₂, consumption capacity was linked to the appearance of a cyanide- and azide-resistant, propyl gallate-sensitive O₂ consumption pathway. This paper raises the question of the physiological significance of this electron transfer pathway induced by anoxia.     

Photosystem I light-harvesting proteins regulate photosynthetic electron transfer and hydrogen production

Linear electron flow (LEF) and cyclic electron flow (CEF) compete for light-driven electrons transferred from the acceptor side of photosystem I (PSI). Under anoxic conditions, such highly reducing electrons also could be used for hydrogen (H₂) production via electron transfer between ferredoxin and hydrogenase in the green alga Chlamydomonas reinhardtii. Partitioning between LEF and CEF is regulated through PROTON-GRADIENT REGULATION5 (PGR5). There is evidence that partitioning of electrons also could be mediated via PSI remodeling processes. This plasticity is linked to the dynamics of PSI-associated light-harvesting proteins (LHCAs) LHCA2 and LHCA9. These two unique light-harvesting proteins are distinct from all other LHCAs because they are loosely bound at the PSAL pole. Here, we investigated photosynthetic electron transfer and H₂ production in single, double, and triple mutants deficient in PGR5, LHCA2, and LHCA9. Our data indicate that lhca2 and lhca9 mutants are efficient in photosynthetic electron transfer, that LHCA2 impacts the pgr5 phenotype, and that pgr5/lhca2 is a potent H₂ photo-producer. In addition, pgr5/lhca2 and pgr5/lhca9 mutants displayed substantially different H₂ photo-production kinetics. This indicates that the absence of LHCA2 or LHCA9 impacts H₂ photo-production independently, despite both being attached at the PSAL pole, pointing to distinct regulatory capacities.

Alteration of the light-harvesting composition of photosystem I impacts photosynthetic electron transfer and hydrogen production.     

Isolation and characterization of mutants corresponding to the MENA,MENB, MENC and MENE enzymatic steps of 5′‐monohydroxyphylloquinone biosynthesis in Chlamydomonas reinhardtii

Phylloquinone (PhQ), or vitamin K₁, is an essential electron carrier (A₁) in photosystem I (PSI). In the green alga Chlamydomonas reinhardtii, which is a model organism for the study of photosynthesis, a detailed characterization of the pathway is missing with only one mutant deficient for MEND having been analyzed. We took advantage of the fact that a double reduction of plastoquinone occurs in anoxia in the A₁ site in the mend mutant, interrupting photosynthetic electron transfer, to isolate four new phylloquinone‐deficient mutants impaired in MENA, MENB, MENC (PHYLLO) and MENE. Compared with the wild type and complemented strains for MENB and MENE, the four men mutants grow slowly in low light and are sensitive to high light. When grown in low light they show a reduced photosynthetic electron transfer due to a specific decrease of PSI. Upon exposure to high light for a few hours, PSI becomes almost completely inactive, which leads in turn to lack of phototrophic growth. Loss of PhQ also fully prevents reactivation of photosynthesis after dark anoxia acclimation. In silico analyses allowed us to propose a PhQ biosynthesis pathway in Chlamydomonas that involves 11 enzymatic steps from chorismate located in the chloroplast and in the peroxisome.     

Cyclic electron flow around photosystem I in unicellular green algae

Cyclic electron flow around PSI, or cyclic photophosphorylation, is the photosynthetic process which recycles the reducing equivalents produced by photosystem I in the stroma towards the plastoquinone pool. Through the activity of cytochrome b ₆ f, which also transfers protons across the membrane, it promotes the synthesis of ATP. The literature dealing with cyclic electron flow in unicellular algae is far less abundant than it is for plants. However, in the chloroplast of algae such as Chlorella or Chlamydomonas, an efficient carbohydrate catabolism renders the redox poise much more reducing than in plant chloroplasts. It is therefore worthwhile highlighting the specific properties of unicellular algae because cyclic electron flow is highly dependent upon the accumulation of these stromal reducing equivalents. Such an increase of reducing power in the stroma stimulates the reduction of plastoquinones, which is the limiting step of cyclic electron flow. In anaerobic conditions in the dark, this reaction can lead to a fully reduced plastoquinone pool and induce state transitions, the migration of 80% of light harvesting complexes II and 20% of cytochrome b ₆ f complex from the PSII-enriched grana to the PSI-enriched lamella. These ultrastructural changes have been proposed to further enhance cyclic electron flow by increasing PSI antenna size, and forming PSI-cyt b ₆ f supercomplexes. These hypotheses are discussed in light of recently published data.     

Picosecond spectroscopy of the charge separation in reaction centers of Chloroflexus aurantiacus with selective excitation of the primary electron donor

Absorbance changes in the picosecond region were studied in isolated reaction centers of the green photosynthetic bacterium Chloroflexus aurantiacus upon selective excitation of the primary electron donor, P, at 870 nm. The results indicate that the first observed state is an excited state of P (P1) which appears to transfer an electron to a bacteriochlorophyll a molecule absorbing at 812 nm (B₁) in 10 ± 2 ps as indicated by a bleaching at this wavelength. This reaction is followed by a rapid electron transfer (3 ± 1 ps) from B₁⁻ to bacteriopheophytin a, so that the fraction of reaction centers in the state P⁺B₁⁻ remains small during the experiment. An apparent bleaching at 925 nm is ascribed to stimulated emission from excited P, which emission disappears upon formation of P⁺. The difference between these time constants for electron transfer and those observed for the same reactions in reaction centers of the purple photosynthetic bacterium Rhodopseudomonas (Rhodobacter) sphaeroides is discussed in terms of the energy difference between P1 and P⁺B₁⁻, which appears to be larger for C. aurantiacus.     

Autotrophic and Mixotrophic Hydrogen Photoproduction in Sulfur-Deprived Chlamydomonas Cells

In Chlamydomonas reinhardtii cells, H₂ photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H₂ production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H₂ photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H₂ production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H₂ photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H₂ production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H₂ production was measured. H₂ production was enhanced in autotrophic conditions by removing O₂ using N₂ bubbling, thereby showing that substantial H₂ production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H₂ photoproduction.     

Unfolding pathway and intermolecular interactions of the cytochrome subunit in the bacterial photosynthetic reaction center

Precise folding of photosynthetic proteins and organization of multicomponent assemblies to form functional entities are fundamental to efficient photosynthetic electron transfer. The bacteriochlorophyll b-producing purple bacterium Blastochloris viridis possesses a simplified photosynthetic apparatus. The light-harvesting (LH) antenna complex surrounds the photosynthetic reaction center (RC) to form the RC-LH1 complex. A non-membranous tetraheme cytochrome (4Hcyt) subunit is anchored at the periplasmic surface of the RC, functioning as the electron donor to transfer electrons from mobile electron carriers to the RC. Here, we use atomic force microscopy (AFM) and single-molecule force spectroscopy (SMFS) to probe the long-range organization of the photosynthetic apparatus from Blc. viridis and the unfolding pathway of the 4Hcyt subunit in its native supramolecular assembly with its functional partners. AFM images reveal that the RC-LH1 complexes are densely organized in the photosynthetic membranes, with restricted lateral protein diffusion. Unfolding of the 4Hcyt subunit represents a multi-step process and the unfolding forces of the 4Hcyt α-helices are approximately 121 picoNewtons. Pulling of 4Hcyt could also result in the unfolding of the RC L subunit that binds with the N-terminus of 4Hcyt, suggesting strong interactions between RC subunits. This study provides new insights into the protein folding and interactions of photosynthetic multicomponent complexes, which are essential for their structural and functional integrity to conduct photosynthetic electron flow.     

Auxiliary electron transport pathways in chloroplasts of microalgae

Microalgae are photosynthetic organisms which cover an extraordinary phylogenic diversity and have colonized extremely diverse habitats. Adaptation to contrasted environments in terms of light and nutrient’s availabilities has been possible through a high flexibility of the photosynthetic machinery. Indeed, optimal functioning of photosynthesis in changing environments requires a fine tuning between the conversion of light energy by photosystems and its use by metabolic reaction, a particularly important parameter being the balance between phosphorylating (ATP) and reducing (NADPH) power supplies. In addition to the main route of electrons operating during oxygenic photosynthesis, called linear electron flow or Z scheme, auxiliary routes of electron transfer in interaction with the main pathway have been described. These reactions which include non-photochemical reduction of intersystem electron carriers, cyclic electron flow around PSI, oxidation by molecular O₂ of the PQ pool or of the PSI electron acceptors, participate in the flexibility of photosynthesis by avoiding over-reduction of electron carriers and modulating the NADPH/ATP ratio depending on the metabolic demand. Forward or reverse genetic approaches performed in model organisms such as Arabidopsis thaliana for higher plants, Chlamydomonas reinhardtii for green algae and Synechocystis for cyanobacteria allowed identifying molecular components involved in these auxiliary electron transport pathways, including Ndh-1, Ndh-2, PGR5, PGRL1, PTOX and flavodiiron proteins. In this article, we discuss the diversity of auxiliary routes of electron transport in microalgae, with particular focus in the presence of these components in the microalgal genomes recently sequenced. We discuss how these auxiliary mechanisms of electron transport may have contributed to the adaptation of microalgal photosynthesis to diverse and changing environments.     

Kinetic properties and physiological role of the plastoquinone terminal oxidase (PTOX) in a vascular plant

The physiological role of the plastid terminal oxidase (PTOX) involved in plastoquinol oxidation in chloroplasts has been investigated in vivo in tomato leaves. Enzyme activity was assessed by non-invasive methods based on the analysis of the kinetics of chlorophyll fluorescence changes. In the dark, the maximum PTOX rate was smaller than 1 electron per second per PSII. This value was further decreased upon light acclimation, and became almost negligible upon inhibition of the photosynthetic performances by reducing the CO₂ availability. In contrast, prolonged exposure to high light resulted in an increase of the overall PTOX activity, which was paralleled by an increased protein accumulation. Under all the conditions tested the enzyme activity always remained about two orders of magnitude lower than that of electron flux through the linear photosynthetic electron pathway. Therefore, PTOX cannot have a role of a safety valve for photogenerated electrons, while it could be involved in acclimation to high light. Moreover, by playing a major role in the control of the stromal redox poise, PTOX is also capable of modulating the balance between linear and cyclic electron flow around PSI during the deactivation phase of carbon assimilation that follows a light to dark transition.     

Hydrogen photo-evolution upon S deprivation stepwise: an illustration of microalgal photosynthetic and metabolic flexibility and a step stone for future biotechnological methods of renewable H2 production

The metabolic flexibility of some photosynthetic microalgae enables them to survive periods of anaerobiosis in the light by developing a particular photofermentative metabolism. The latter entails compounds of the photosynthetic electron transfer chain and an oxygen-sensitive hydrogenase in order to reoxidize reducing equivalents and to generate ATP for maintaining basal metabolic function. This pathway results in the photo-evolution of hydrogen gas by the algae. A decade ago, Melis and coworkers managed to reproduce such a condition in a laboratory context by depletion of sulfur in the algal culture media, making the photo-evolution by the algae sustainable for several days (Melis et al. in Plant Physiol 122:127–136, 2000). This observation boosted research in algal H₂ evolution. A feature, which due to its transient nature was long time considered as a curiosity of algal photosynthesis suddenly became a phenomenon with biotechnological potential. Although the Melis procedure has not been developed into a biotechnological process of renewable H₂ generation so far, it has been a useful tool for studying microalgal metabolic and photosynthetic flexibility and a possible step stone for future H₂ production procedures. Ten years later most of the critical steps and limitations of H₂ production by this protocol have been studied from different angles particularly with the model organism Chlamydomonas reinhardtii, by introducing various changes in culture conditions and making use of mutants issued from different screens or by reverse genomic approaches. A synthesis of these observations with the most important conclusions driven from recent studies will be presented in this review.     

Carbon limitation enhances CO2 concentrating mechanism but reduces trichome size in Arthrospira platensis (cyanobacterium)

Arthrospira species grow well under highly enriched inorganic carbon concentrations, but little is known on the effects of inorganic carbon (Ci) limitation on its physiological performance. When Arthrospira platensis D-0083 was grown in a modified medium without NaHCO₃ under ambient air of 380 ppm CO₂, its trichomes became disassembled while the growth and photosynthetic rates were severely reduced. Phycocyanin and allophycocyanin contents decreased but the carotenoid content increased under the Ci limitation. Compared with the cells grown in Zarrouk medium, the trichomes grown under the Ci limitation increased their photosynthetic apparent affinity for Ci by about 14 times but photochemical quenching capacity was reduced. It appeared that A. platensis increased its CO₂ concentrating mechanism by inducing HCO₃ ^? transporters and reducing the trichome size which increased filamentous surface to volume ratio.     

The impact of cell-specific absorption properties on the correlation of electron transport rates measured by chlorophyll fluorescence and photosynthetic oxygen production in planktonic algae

Photosynthesis–irradiance (P–E)-curves describe the photosynthetic performance of autotrophic organisms. From these P–E-curves the photosynthetic parameters α-slope, P_max, and E_k can be deduced which are often used to characterize and to compare different organisms or organisms in acclimation to different environmental conditions. Particularly, for in situ-measurements of P–E curves of phytoplankton the analysis of variable chlorophyll fluorescence proved its potential as a sensitive and rapid method. By using Chlorella vulgaris (Trebouxiophyceae), Nannochloropsis salina (Eustigmatophyceae), Skeletonema costatum and Cyclotella meneghiniana (Bacillariophyceae), the present study investigated the influence of cellular bio-optical properties on the correlation of the photosynthetic parameters derived from fluorescence-based P–E-curves with photosynthetic parameters obtained from the measurement of oxygen evolution. It is demonstrated that small planktonic algae show a wide range of cellular absorptivity which was subject to species-specifity, growth stage and environmental conditions, e.g. nutrient limitation. This variability in bio-optical properties resulted in a great deviation of relative electron transport rates (rETRs) from oxygen-based photosynthesis rates. Thus, the photosynthetic parameters α-slope and P_max derived from rETRs strongly depend on the specific cellular absorptivity and cannot be used to compare the photosynthetic performance of cells with different optical properties. However, it was shown that E_k is independent of cellular absorptivity and could be used to compare samples with unknown optical properties.     

Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aerobic conditions

Assimilation of atmospheric CO₂ by photosynthetic organisms such as plants, cyanobacteria and green algae, requires the production of ATP and NADPH in a ratio of 3:2. The oxygenic photosynthetic chain can function following two different modes: the linear electron flow which produces reducing power and ATP, and the cyclic electron flow which only produces ATP. Some regulation between the linear and cyclic flows is required for adjusting the stoichiometric production of high-energy bonds and reducing power. Here we explore, in the green alga Chlamydomonas reinhardtii, the onset of the cyclic electron flow during a continuous illumination under aerobic conditions. In mutants devoid of Rubisco or ATPase, where the reducing power cannot be used for carbon fixation, we observed a stimulation of the cyclic electron flow. The present data show that the cyclic electron flow can operate under aerobic conditions and support a simple competition model where the excess reducing power is recycled to match the demand for ATP.     

‘Birth defects’ of photosystem II make it highly susceptible to photodamage during chloroplast biogenesis

High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is susceptible to photodamage (also known as photoinactivation) in strong light, resulting in severe loss of water oxidation capacity and destruction of the water‐oxidizing complex (WOC). The repair of damaged PSIIs comes at a high energy cost and requires de novo biosynthesis of damaged PSII subunits, reassembly of the WOC inorganic cofactors and membrane remodeling. Employing membrane‐inlet mass spectrometry and O₂‐polarography under flashing light conditions, we demonstrate that newly synthesized PSII complexes are far more susceptible to photodamage than are mature PSII complexes. We examined these ‘PSII birth defects’ in barley seedlings and plastids (etiochloroplasts and chloroplasts) isolated at various times during de‐etiolation as chloroplast development begins and matures in synchronization with thylakoid membrane biogenesis and grana membrane formation. We show that the degree of PSII photodamage decreases simultaneously with biogenesis of the PSII turnover efficiency measured by O₂‐polarography, and with grana membrane stacking, as determined by electron microscopy. Our data from fluorescence, Q_B‐inhibitor binding, and thermoluminescence studies indicate that the decline of the high‐light susceptibility of PSII to photodamage is coincident with appearance of electron transfer capability Q_A^? → Q_B during de‐etiolation. This rate depends in turn on the downstream clearing of electrons upon buildup of the complete linear electron transfer chain and the formation of stacked grana membranes capable of longer‐range energy transfer.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F_V/F_M, whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F_V/F_M of ∼0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.     

The Regulation of Photosynthetic Electron Transport during Nutrient Deprivation in Chlamydomonas reinhardtii

The light-saturated rate of photosynthetic O₂ evolution in Chlamydomonas reinhardtii declined by approximately 75% on a per-cell basis after 4 d of P starvation or 1 d of S starvation. Quantitation of the partial reactions of photosynthetic electron transport demonstrated that the light-saturated rate of photosystem (PS) I activity was unaffected by P or S limitation, whereas light-saturated PSII activity was reduced by more than 50%. This decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). In addition to a decline in the light-saturated rate of O₂ evolution, there was reduced efficiency of excitation energy transfer to the reaction centers of PSII (because of dissipation of absorbed light energy as heat and because of a transition to state 2). These findings establish a common suite of alterations in photosynthetic electron transport that results in decreased linear electron flow when C. reinhardtii is limited for either P or S. It was interesting that the decline in the maximum quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers were regulated specifically during S-limited growth by the SacI gene product, which was previously shown to be critical for the acclimation of C. reinhardtii to S limitation (J.P. Davies, F.H. Yildiz, and A.R. Grossman [1996] EMBO J 15: 2150–2159).     

Respiration in energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus: I. Relation of the respiratory and photosynthetic electron transport

The thermophilic cyanobacterium Mastigocladus laminosus was grown at different CO₂ concentrations and temperatures. Respiratory and photosynthetic electron transport in isolated membranes were measured and their activities were compared. Cells grown at low CO₂ concentration showed respiratory electron transport, whereas Photosystem-II-dependent transport was optimal in cells grown at high CO₂ concentrations. The respiratory electron transport from NADH and succinate were KCN-sensitive, whereas NADPH-dependent O₂ uptake was not. It could be shown that NADH and succinate donate electrons in the photosynthetic electron pathway via Photosystem I. In cytochrome-c-553-depleted membranes added cytochrome c-553 could stimulate photosynthetic and respiratory electron transport. A common electron transport pathway between the quinone and cytochrome c is postulated.     

Developmental acclimation of the thylakoid proteome to light intensity in Arabidopsis

Photosynthetic acclimation, the ability to adjust the composition of the thylakoid membrane to optimise the efficiency of electron transfer to the prevailing light conditions, is crucial to plant fitness in the field. While much is known about photosynthetic acclimation in Arabidopsis, to date there has been no study that combines both quantitative label-free proteomics and photosynthetic analysis by gas exchange, chlorophyll fluorescence and P700 absorption spectroscopy. Using these methods we investigated how the levels of 402 thylakoid proteins, including many regulatory proteins not previously quantified, varied upon long-term (weeks) acclimation of Arabidopsis to low (LL), moderate (ML) and high (HL) growth light intensity and correlated these with key photosynthetic parameters. We show that changes in the relative abundance of cytb₆f, ATP synthase, FNR2, TIC62 and PGR6 positively correlate with changes in estimated PSII electron transfer rate and CO₂ assimilation. Improved photosynthetic capacity in HL grown plants is paralleled by increased cyclic electron transport, which positively correlated with NDH, PGRL1, FNR1, FNR2 and TIC62, although not PGR5 abundance. The photoprotective acclimation strategy was also contrasting, with LL plants favouring slowly reversible non-photochemical quenching (qI), which positively correlated with LCNP, while HL plants favoured rapidly reversible quenching (qE), which positively correlated with PSBS. The long-term adjustment of thylakoid membrane grana diameter positively correlated with LHCII levels, while grana stacking negatively correlated with CURT1 and RIQ protein abundance. The data provide insights into how Arabidopsis tunes photosynthetic electron transfer and its regulation during developmental acclimation to light intensity.