State transitions at the crossroad of thylakoid signalling pathways

In order to maintain optimal photosynthetic activity under a changing light environment, plants and algae need to balance the absorbed light excitation energy between photosystem I and photosystem II through processes called state transitions. Variable light conditions lead to changes in the redox state of the plastoquinone pool which are sensed by a protein kinase closely associated with the cytochrome b ₆ f complex. Preferential excitation of photosystem II leads to the activation of the kinase which phosphorylates the light-harvesting system (LHCII), a process which is subsequently followed by the release of LHCII from photosystem II and its migration to photosystem I. The process is reversible as dephosphorylation of LHCII on preferential excitation of photosystem I is followed by the return of LHCII to photosystem II. State transitions involve a considerable remodelling of the thylakoid membranes, and in the case of Chlamydomonas, they allow the cells to switch between linear and cyclic electron flow. In this alga, a major function of state transitions is to adjust the ATP level to cellular demands. Recent studies have identified the thylakoid protein kinase Stt7/STN7 as a key component of the signalling pathways of state transitions and long-term acclimation of the photosynthetic apparatus. In this article, we present a review on recent developments in the area of state transitions.     

Interaction between starch breakdown, acetate assimilation, and photosynthetic cyclic electron flow in Chlamydomonas reinhardtii

Spectroscopic studies on photosynthetic electron transfer generally are based upon the monitoring of dark to light changes in the electron transfer chain. These studies, which focus on the light reactions of photosynthesis, also indirectly provide information on the redox or metabolic state of the chloroplast in the dark. Here, using the unicellular microalga Chlamydomonas reinhardtii, we study the impact of heterotrophic/mixotrophic acetate feeding on chloroplast carbon metabolism by using the spectrophotometric detection of P700(+), the photooxidized primary electron donor of photosystem I. We show that, when photosynthetic linear and cyclic electron flows are blocked (DCMU inhibiting PSII and methylviologen accepting electrons from PSI), the post-illumination reduction kinetics of P700(+) directly reflect the dark metabolic production of reductants (mainly NAD(P)H) in the stroma of chloroplasts. Such results can be correlated to other metabolic studies: in the absence of acetate, for example, the P700(+) reduction rate matches the rate of starch breakdown reported previously, confirming the chloroplast localization of the upstream steps of the glycolytic pathway in Chlamydomonas. Furthermore, the question of the interplay between photosynthetic and non-photosynthetic carbon metabolism can be addressed. We show that cyclic electron flow around photosystem I is twice as fast in a starchless mutant fed with acetate than it is in the WT, and we relate how changes in the flux of electrons from carbohydrate metabolism modulate the redox poise of the plastoquinone pool in the dark through chlororespiration.     

Repressible chloroplast gene expression in Chlamydomonas: A new tool for the study of the photosynthetic apparatus

A repressible/inducible chloroplast gene expression system has been used to conditionally inhibit chloroplast protein synthesis in the unicellular alga Chlamydomonas reinhardtii. This system allows one to follow the fate of photosystem II and photosystem I and their antennae upon cessation of chloroplast translation. The main results are that the levels of the PSI core proteins decrease at a slower rate than those of PSII. Amongst the light-harvesting complexes, the decrease of CP26 proceeds at the same rate as for the PSII core proteins whereas it is significantly slower for CP29, and for the antenna complexes of PSI this rate is comprised between that of CP26 and CP29. In marked contrast, the components of trimeric LHCII, the major PSII antenna, persist for several days upon inhibition of chloroplast translation. This system offers new possibilities for investigating the biosynthesis and turnover of individual photosynthetic complexes in the thylakoid membranes. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

The light sensitivity of ATP synthase mutants of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii mutants defective in the chloroplast ATP synthase are highly sensitive to light. The ac46 mutant is affected in the MDH1 gene, required for production or stability of the monocistronic atpH mRNA encoding CF(O)-III. In this and other ATP synthase mutants, we show that short-term exposure to moderate light intensities-a few minutes-induces an inhibition of electron transfer after the primary quinone acceptor of photosystem II (PSII), whereas longer exposure-several hours-leads to a progressive loss of PSII cores. An extensive swelling of thylakoids accompanies the initial inhibition of electron flow. Thylakoids deflate as PSII cores are lost. The slow process of PSII degradation involves the participation of ClpP, a chloroplast-encoded peptidase that is part of a major stromal protease Clp. In the light of the above findings, we discuss the photosensitivity of ATP synthase mutants with respect to the regular photoinhibition process that affects photosynthetic competent strains at much higher light intensities.     

The phosphorylation status of the chloroplast protein kinase STN7 of Arabidopsis affects its turnover

The chloroplast serine-threonine protein kinase STN7 of Arabidopsis (Arabidopsis thaliana) is required for the phosphorylation of the light-harvesting system of photosystem II and for state transitions, a process that allows the photosynthetic machinery to balance the light excitation energy between photosystem II and photosystem I and thereby to optimize the photosynthetic yield. Because the STN7 protein kinase of Arabidopsis is known to be phosphorylated at four serine-threonine residues, we have changed these residues by site-directed mutagenesis to alanine (STN7-4A) or aspartic acid (STN7-4D) to assess the role of these phosphorylation events. The corresponding mutants were still able to phosphorylate the light-harvesting system of photosystem II and to perform state transitions. Moreover, we noticed a marked decrease in the level of the STN7 kinase in the wild-type strain under prolonged state 1 conditions that no longer occurs in the STN7-4D mutant. The results suggest a possible role of phosphorylation of the STN7 kinase in regulating its turnover.     

Photosystem II antenna complexes CP26 and CP29 are essential for nonphotochemical quenching in Chlamydomonas reinhardtii

Photosystems must balance between light harvesting to fuel the photosynthetic process for CO₂ fixation and mitigating the risk of photodamage due to absorption of light energy in excess. Eukaryotic photosynthetic organisms evolved an array of pigment-binding proteins called light harvesting complexes constituting the external antenna system in the photosystems, where both light harvesting and activation of photoprotective mechanisms occur. In this work, the balancing role of CP29 and CP26 photosystem II antenna subunits was investigated in Chlamydomonas reinhardtii using CRISPR-Cas9 technology to obtain single and double mutants depleted of monomeric antennas. Absence of CP26 and CP29 impaired both photosynthetic efficiency and photoprotection: Excitation energy transfer from external antenna to reaction centre was reduced, and state transitions were completely impaired. Moreover, differently from higher plants, photosystem II monomeric antenna proteins resulted to be essential for photoprotective thermal dissipation of excitation energy by nonphotochemical quenching.     

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.     

Photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas

When there is an imbalance between the light energy absorbed by a photosynthetic organism and that which can be utilized in photosynthesis, photo-oxidative stress can damage pigments, proteins, lipids, and nucleic acids. In this work we compared the wild type and a xanthophyll-deficient mutant of Chlamydomonas reinhardtii in their response to high amounts of light. Wild-type Chlamydomonas cells were able to acclimate to high amounts of light following transfer from low light conditions. In contrast, the npq1 lor1 double mutant, which lacks protective xanthophylls (zeaxanthin and lutein) in the chloroplast, progressively lost viability and photosynthetic capacity along with destruction of thylakoid membrane protein-pigment complexes and accumulation of reactive oxygen species and membrane lipid peroxides. Loss of viability was partially rescued by lowered oxygen tension, suggesting that the high sensitivity of the mutant to light stress is caused by the production of reactive oxygen species in the chloroplast. Cell death was not prevented by the addition of an organic carbon source to the growth medium, demonstrating that the photo-oxidative damage can target other essential chloroplast processes besides photosynthesis. From the differential sensitivity of the mutant to exogenously added pro-oxidants, we infer that the reactive oxygen species produced during light stress in npq1 lor1 may be singlet oxygen and/or superoxide but not hydrogen peroxide. The bleaching phenotype of npq1 lor1 was not due to enhanced photodamage to photosystem II but rather to a less localized phenomenon of accumulation of photo-oxidation products in chloroplast membranes.     

Role of singlet oxygen in chloroplast to nucleus retrograde signaling in Chlamydomonas reinhardtii

High light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II and causes photooxidative stress. In Chlamydomonas reinhardtii, singlet oxygen also induces the expression of the nuclear-encoded glutathione peroxidase homologous gene GPXH. We provide evidence that singlet oxygen stimulates GPXH expression by activating a signaling mechanism outside the thylakoid membrane. Singlet oxygen from photosystem II could be detected with specific probes in the aqueous phase of isolated thylakoid suspensions and the cytoplasm of high light stressed cells. This indicates that singlet oxygen can stimulate a response farther from its production site than generally believed.     

Functional Organization of the Chlorophyll-Containing Complexes of Chlamydomonas reinhardi: A Study of Their Formation and Interconnection with Reaction Centers in the Greening Process of the y-1 Mutant

The stepwise synthesis and assembly of photosynthetic membrane components in the y-1 mutant of Chlamydomonas reinhardi have been previously demonstrated (Ohad 1975 In Membrane Biogenesis, Mitochondria, Chloroplasts and Bacteria, Plenum, pp 279-350). This experimental system was used here in order to investigate the process of formation and interconnection of the energy collecting chlorophylls with the reaction centers of both photosystems I and II. The following measurements were carried out: photosynthetic electron flow at various light intensities, including parts or the entire electron transfer chain; analysis of the kinetics of fluorescence emission at room temperature and fluorescence emission spectra at 77 K, and electrophoretic separation of membrane polypeptides and chlorophyll protein complexes. Based on the data obtained it is concluded that: (a) each photosystem (PSI and PSII) contains, in addition to the reaction center, an interconnecting antenna and a main or light harvesting antenna complex; (b) the formation of the light harvesting complex, interconnecting antenna, and reaction centers for each photosystem can occur independently. (c) the interconnecting antennae link the light harvesting complexes with the respective reaction centers. In their absence, energy transfer between the light harvesting chlorophylls and the reaction centers is inefficient. The formation of the interconnecting antennae and efficient assembly of photosystem components occur simultaneously with the de novo synthesis of chlorophyll and at least three polypeptides, one translated in the cytoplasm and two translated in the chloroplast. The synthesis of these polypeptides was found to be light dependent.     

Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes

Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO(2) availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes.     

Involvement of state transitions in the switch between linear and cyclic electron flow in Chlamydomonas reinhardtii

The energetic metabolism of photosynthetic organisms is profoundly influenced by state transitions and cyclic electron flow around photosystem I. The former involve a reversible redistribution of the light-harvesting antenna between photosystem I and photosystem II and optimize light energy utilization in photosynthesis whereas the latter process modulates the photosynthetic yield. We have used the wild-type and three mutant strains of the green alga Chlamydomonas reinhardtii—locked in state I (stt7), lacking the photosystem II outer antennae (bf4) or accumulating low amounts of cytochrome b₆f complex (A-AUU)—and measured electron flow though the cytochrome b₆f complex, oxygen evolution rates and fluorescence emission during state transitions. The results demonstrate that the transition from state 1 to state 2 induces a switch from linear to cyclic electron flow in this alga and reveal a strict cause–effect relationship between the redistribution of antenna complexes during state transitions and the onset of cyclic electron flow.     

Multilevel regulation of non‐photochemical quenching and state transitions by chloroplast NADPH‐dependent thioredoxin reductase

In natural growth habitats, plants face constant, unpredictable changes in light conditions. To avoid damage to the photosynthetic apparatus on thylakoid membranes in chloroplasts, and to avoid wasteful reactions, it is crucial to maintain a redox balance both within the components of photosynthetic electron transfer chain and between the light reactions and stromal carbon metabolism under fluctuating light conditions. This requires coordinated function of the photoprotective and regulatory mechanisms, such as non‐photochemical quenching (NPQ) and reversible redistribution of excitation energy between photosystem II (PSII) and photosystem I (PSI). In this paper, we show that the NADPH‐dependent chloroplast thioredoxin system (NTRC) is involved in the control of the activation of these mechanisms. In plants with altered NTRC content, the strict correlation between lumenal pH and NPQ is partially lost. We propose that NTRC contributes to downregulation of a slow‐relaxing constituent of NPQ, whose induction is independent of lumenal acidification. Additionally, overexpression of NTRC enhances the ability to adjust the excitation balance between PSII and PSI, and improves the ability to oxidize the electron transfer chain during changes in light conditions. Thiol regulation allows coupling of the electron transfer chain to the stromal redox state during these changes.     

Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes

Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO₂ availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes.     

Chloroplast site-directed mutagenesis of photosystem I in Chlamydomonas: electron transfer reactions and light sensitivity

The photosystem I (PSI) complex is a multisubunit protein-pigment complex embedded in the thylakoid membrane which acts as a light-driven plastocyanin/cytochrome c(6)-ferredoxin oxido-reductase. The use of chloroplast transformation and site-directed mutagenesis coupled with the biochemical and biophysical analysis of mutants of the green alga Chlamydomonas reinhardtii with specific amino acid changes in several subunits of PSI has provided new insights into the structure-function relationship of this important photosynthetic complex. In particular, this molecular-genetic analysis has identified key residues of the reaction center polypeptides of PSI which are the ligands of some of the redox cofactors and it has also provided important insights into the orientation of the terminal electron acceptors of this complex. Finally this analysis has also shown that mutations affecting the donor side of PSI are limiting for overall electron transfer under high light and that electron trapping within the terminal electron acceptors of PSI is highly deleterious to the cells.     

Regulation of electron-transport pathways in cells of Chlamydomonas reinhardtii under stress conditions

The kinetics of interactions between electron-transport pathways in the thylakoid membrane was examined. A mathematical model was proposed to describe the kinetics of redox transitions in photosystem II, proton concentration changes in the chloroplast stroma, and the plastoquinone pool reduction due to photosynthetic and chlororespiratory pathways. A kinetic mechanism is considered that redirects electron flows between photosynthetic and chlororespiratory pathways in response to the increased NADPH content under mineral deficiency. According to the simulation model, the electron transport flows via different routes are switched over in a stepwise manner. The results of numerical simulations are qualitatively consistent with experimental data for Chlamydomonas reinhardtii cells subjected to mineral deprivation.     

Membrane protein damage and repair: removal and replacement of inactivated 32-kilodalton polypeptides in chloroplast membranes

Incubation of Chlamydomonas reinhardii cells at light levels that are several times more intense than those at which the cells were grown results in a loss of photosystem II function (termed photoinhibition). The loss of activity corresponded to the disappearance from the chloroplast membranes of a lysine-deficient, herbicide-binding protein of 32,000 daltons which is thought to be the apoprotein of the secondary quinone electron acceptor of photosystem II (the QB protein). In vivo recovery from the damage only occurred following de novo synthesis (replacement) of the chloroplast-encoded QB protein. We believe that the turnover of this protein is a normal consequence of its enzymatic function in vivo and is a physiological process that is necessary to maintain the photosynthetic integrity of the thylakoid membrane. Photoinhibition occurs when the rate of inactivation and subsequent removal exceeds the rate of resynthesis of the QB protein.     

Isolation of state transition mutants of Chlamydomonas reinhardtii by fluorescence video imaging

Oxygenic photosynthetic organisms adapt to varying light conditions by changing the distribution of light energy between Photosystem II (PS II) and photosystem I (PS I) during so-called state transitions. To identify the genes involved in this process, we have exploited a simple chlorophyll fluorescence video-imaging technique to screen a library of nuclear mutants of Chlamydomonas reinhardtii for colonies grown on agar plates that are disturbed in their ability to regulate light energy distribution between PS I and PS II. Subsequent modulated fluorescence measurements at room temperature and 77 K fluorescence emission spectra confirmed that 5 mutants (0.025% of total number screened) were defective in state transitions. [³²P]orthophosphate phosphorylation experiments in vivo revealed that in one of these mutants, designated stm1, the level of LHC II polypeptide phosphorylation was drastically reduced compared with wild type. Despite WT levels of PS I and PS II, stm1 grew photoautotrophically at reduced rates, compared with WT especially under low light conditions, which is consistent with an important physiological role for state transitions. Our results highlight the feasibility of video imaging in tandem with mutagenesis as a means of identifying the genes involved in controlling state transitions in eukaryotic photosynthetic organisms.