Chlororespiration

The term 'chlororespiration' is used to describe the activity of a putative respiratory electron transler chain within the thylakoid membrane of chloroplasts and was originally proposed by Bennouon in 1982 to explain effects on the redox state of the plastoquinone pool in green algae in the absence of photosynthetic plastoquinone electrontransfer. In his original model, Bennoun suggested that the pool could be reduced through the action of a NAD(P) H dehydrogenase and could be oxidized by oxygen at an oxidase. At the same time an electrochemical gradient would be generated across the membrane. This review describes the current status of the chlororespiration model in light of the recent discoveries of novel respiratory components chloroplast thylakoid membrane.     

Chloroplasts as source and target of cellular redox regulation: a discussion on chloroplast redox signals in the context of plant physiology

During the evolution of plants, chloroplasts have lost the exclusive genetic control over redox regulation and antioxidant gene expression. Together with many other genes, all genes encoding antioxidant enzymes and enzymes involved in the biosynthesis of low molecular weight antioxidants were transferred to the nucleus. On the other hand, photosynthesis bears a high risk for photo-oxidative damage. Concomitantly, an intricate network for mutual regulation by anthero- and retrograde signals has emerged to co-ordinate the activities of the different genetic and metabolic compartments. A major focus of recent research in chloroplast regulation addressed the mechanisms of redox sensing and signal transmission, the identification of regulatory targets, and the understanding of adaptation mechanisms. In addition to redox signals communicated through signalling cascades also used in pathogen and wounding responses, specific chloroplast signals control nuclear gene expression. Signalling pathways are triggered by the redox state of the plastoquinone pool, the thioredoxin system, and the acceptor availability at photosystem I, in addition to control by oxolipins, tetrapyrroles, carbohydrates, and abscisic acid. The signalling function is discussed in the context of regulatory circuitries that control the expression of antioxidant enzymes and redox modulators, demonstrating the principal role of chloroplasts as the source and target of redox regulation.     

Thiol regulation of the thylakoid electron transport chain--a missing link in the regulation of photosynthesis?

Avoidance of over-reduction of the chloroplast ferredoxin pool is of paramount importance for plants in avoiding oxidative stress. The redox state of this pool can be controlled through regulation of the thylakoid electron transport chain. A model is presented for regulation of this chain via a thiol reduction mechanism, possibly involving a thioredoxin. It is shown in isolated thylakoids that electron transport is inhibited by the thiol reducing agent dithiothreitol. The kinetics of this reduction are rapid and readily reversible. The midpoint redox potential is -365 mV at pH 7.7, with a pH dependency of about -90 mV/pH. At physiological pH values, this places the potential of the species titrated between that of ferredoxin and NADPH and thus in the right potential range to be regulating the redox poise of the ferredoxin pool. This is also close to the potential of NADPH-malate dehydrogenase, an enzyme known to be regulated by thioredoxin. Regulation of electron transport by thioredoxin provides a mechanistic link between the regulation of photosynthesis and gene expression by sugars and the redox regulation of gene expression mediated through the plastoquinone pool.     

Photosystem II protein phosphorylation follows four distinctly different regulatory patterns induced by environmental cues

Differential redox regulation of thylakoid phosphoproteins was studied in winter rye plants in vivo. The redox state of chloroplasts was modulated by growing plants under different light/temperature conditions and by transient shifts to different light/temperature regimes. Phosphorylation of PSII reaction centre proteins D1 and D2, the chlorophyll a binding protein CP43, the major chlorophyll a/b binding proteins Lhcb1 and Lhcb2 (LHCII) and the minor light‐harvesting antenna protein CP29 seem to belong to four distinct regulatory groups. Phosphorylation of D1 and D2 was directly dependent on the reduction state of the plastoquinone pool. CP43 protein phosphorylation generally followed the same pattern, but often remained phosphorylated even in darkness. Phosphorylation of CP29 occurred upon strong reduction of the plastoquinone pool, and was further enhanced by low temperatures. In vitro studies further demonstrated that CP29 phosphorylation is independent of the redox state of both the cytochrome b₆/f complex and the thiol compounds. Complete phosphorylation of Lhcb1 and 2 proteins, on the contrary, required only modest reduction of the plastoquinone pool, and was subject to inhibition upon increase in the thiol redox state of the stroma. Furthermore, the reversible phosphorylation of Lhcb1 and 2 proteins appeared to be an extremely dynamic process, being rapidly modulated by short‐term fluctuations in chloroplast redox conditions.     

Temperature-induced greening of<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis>. The role of the cellular energy balance and zeaxanthin-dependent nonphotochemical quenching

When cells of the green alga Chlorella vulgaris Beij. are transferred from growth at 5 degrees C and an irradiance of 150 micromol photons m(-2) s(-1) to 27 degrees C and the same irradiance, they undergo what is normally considered a high-light to low-light phenotypic change. This involves a 3-fold increase in cellular chlorophyll content with a concomitant increase in light-harvesting complex polypeptide levels. This process appears to occur in response to the cellular capacity to utilize the products of photosynthesis, with the redox state of the plastoquinone pool sensing the cellular energy balance. The phenotypic adjustment can be enhanced or blocked using chemical inhibitors that modulate the redox state of the plastoquinone pool. The functional changes in the photosynthetic apparatus that occurred during the high-light to low-light acclimation were examined with special consideration paid to the paradox that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated cells, with non-functional photosystem II (PSII), accumulate light-harvesting polypeptides. At the structural and basic functional levels, the light-harvesting complex of the cells treated with DCMU was indistinguishable from that of the untreated, control cells. To examine how PSII was protected in the DCMU-treated cells, we measured the content of xanthophyll-cycle pigments. It appeared that a zeaxanthin-dependent nonphotochemical quenching process was involved in PSII protection during greening in the presence of DCMU. Metabolic inhibitors of mitochondrial respiration were used to examine how the change in cellular energy balance regulates the greening process. Apparently, the mitochondrion acts to supply energy to the chloroplast during greening, and inhibition of mitochondrial respiration diminishes chlorophyll accumulation apparently through an increase in the redox state of the plastoquinone pool.     

The relationship between changes in the redox state of plastoquinone and control of excitation energy distribution in photosynthesis

The hypothesis that excitation energy distribution between PS I and PS II is controlled by the redox state of the plastoquinone pool between the two photosystems was investigated using the green alga Chlorella vulgaris. Changes in the redox state of the pool were monitored by measurement of the area above the fluorescence induction curve on exposure to high-intensity light. In agreement with the hypothesis, exposure of state I adapted cells to light preferentially absorbed by PS II led to a reduction of the plastoquinone pool whilst exposure of State II adapted cells to light preferentially absorbed by PS I resulted in its oxidation. However, the limits within which these fluctuations occurred were much narrower than anticipated. The reasons for this are discussed in terms of the possible involvement of changes in the redox state of more specialised molecules associated with the main plastoquinone pool and the postulated role of plastoquinone as an electron shuttle between the two photosystems.     

Protein kinases and phosphatases involved in the acclimation of the photosynthetic apparatus to a changing light environment

Photosynthetic organisms are subjected to frequent changes in light quality and quantity and need to respond accordingly. These acclimatory processes are mediated to a large extent through thylakoid protein phosphorylation. Recently, two major thylakoid protein kinases have been identified and characterized. The Stt7/STN7 kinase is mainly involved in the phosphorylation of the LHCII antenna proteins and is required for state transitions. It is firmly associated with the cytochrome b₆f complex, and its activity is regulated by the redox state of the plastoquinone pool. The other kinase, Stl1/STN8, is responsible for the phosphorylation of the PSII core proteins. Using a reverse genetics approach, we have recently identified the chloroplast PPH1/TAP38 and PBPC protein phosphatases, which counteract the activity of STN7 and STN8 kinases, respectively. They belong to the PP2C-type phosphatase family and are conserved in land plants and algae. The picture that emerges from these studies is that of a complex regulatory network of chloroplast protein kinases and phosphatases that is involved in light acclimation, in maintenance of the plastoquinone redox poise under fluctuating light and in the adjustment to metabolic needs.     

Coregulation of light-harvesting complex II phosphorylation and lhcb mRNA accumulation in winter rye

Winter rye plants grown under contrasting environmental conditions or just transiently shifted to varying light and temperature conditions, were studied to elucidate the chloroplast signal involved in regulation of photosynthesis genes in the nucleus. Photosystem II excitation pressure, reflecting the plastoquinone redox state, and the phosphorylation level of thylakoid light-harvesting proteins (LHCII and CP29) were monitored together with changes occurring in the accumulation of lhcb, rbcS and psbA mRNAs. Short-term shifts of plants to changed conditions, from 1 h to 2 d, were postulated to reveal signals crucial for the initiation of the acclimation process. Comparison of these results with those obtained from plants acclimated during several weeks' growth at contrasting temperature and in different light regimes, allow us to make the following conclusions: (1) LHCII protein phosphoylation is a sensitive tool to monitor redox changes in chloroplasts; (2) LHCII protein phosphorylation and lhcb mRNA accumulation occur under similar conditions and are possibly coregulated via an activation state of the same kinase (the LHCII kinase); (3) Maximal accumulation of lhcb mRNA during the diurnal light phase seems to require an active LHCII kinase whereas inactivation of the kinase is accompanied by dampening of the circadian oscillation in the amount of lhcb mRNA; (4) Excitation pressure of photosystem II (reduction state of the plastoquinone pool) is not directly involved in the regulation of lhcb mRNA accumulation. Instead (5) the redox status of the electron acceptors of photosystem I in the stromal compartment seems to be highly regulated and crucial for the regulation of lhcb gene expression in the nucleus.     

Voltammetric measurement of the plastoquinone redox state in isolated thylakoids

The oxidation of plastoquinol by the cytochrome bf complex is commonly believed to be the rate limiting step in photosynthetic electron transport. When input of electrons from PS II exceeds electron flow through the cytochrome bf complex the plastoquinone pool becomes reduced. A voltammetric technique previously used to measure the redox state of the ubiquinone pool in plant mitochondria, was modified to measure the redox state of the plastoquinone pool in thylakoids. The presence or absence of a proton gradient strongly influenced the relationship between the redox state of the plastoquinone pool and other photosynthetic parameters. A linear relationship between the rate of electron transport and the reduction of plastoquinone was found. The slope of this relationship was greater in coupled than in uncoupled thylakoids, indicating that under coupled conditions the plastoquinone pool is more reduced at any given rate of electron flow. A complex relationship was found between QA reduction, calculated as 1 – q_p, and the redox state of the plastoquinone pool. The extent of Q_A reduction was similar in coupled and uncoupled thylakoids, but at any given level of Q_A reduction, PQ was always more reduced in coupled thylakoids. These results suggest that the presence of a proton gradient changes the equilibrium constant between Q_A and PQ.     

Thylakoid protein phosphorylation,state 1-state 2 transitions,and photosystem stoichiometry adjustment: redox control at multiple levels of gene expression

In photosynthesis in chloroplasts, control of thylakoid protein phosphorylation by redox state of inter-photosystem electron carriers makes distribution of absorbed excitation energy between the two photosystems self-regulating. During operation of this regulatory mechanism, reduction of plastoquinone activates a thylakoid protein kinase which phosphorylates the light-harvesting complex LHC II, causing a change in protein recognition that results in redistribution of energy to photosystem I at the expense of photosystem II, thus tending to oxidise the reduced plastoquinone pool. These events correspond to the transition from light-state 1 to light-state 2. The reverse transition (to light-state 1) is initiated by transient oxidation of plastoquinone, inactivation of the LHC II kinase, and return of dephosphorylated LHC II from photosystem I to photosystem II, supplying excitation energy to photosystem II and thereby reducing plastoquinone. State 1-state 2 transitions therefore operate by means of redox control of reversible, post-translational modification of pre-existing proteins. A balance in the rates of light utilization by photosystem I and photosystem II can also be achieved, on longer time-scales and between wider limits, by adjustment of the relative quantities, or stoichiometry, of photosystem I and photosystem II. Recent evidence suggests that adjustment of photosystem stoichiometry is also a response to perturbation of the redox state of inter-photosystem electron carriers, and involves specific redox control of de novo protein synthesis, assembly, and breakdown. It is therefore suggested that the same redox sensor initiates these different adaptations by control of gene expression at different levels, according to the time-scale and amplitude of the response. This integrated feedback control may serve to maintain redox homeostasis, and, as a result, quantum yield. Evidence for the components required by such systems is discussed.     

Investigating the production of foreign membrane proteins in tobacco chloroplasts: expression of an algal plastid terminal oxidase

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5'UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast.     

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.