Dynamic modulation of transthylakoid electric potential by chloroplast ATP synthases.

The light-induced transthylakoid membrane potential (ΔΨm) can function as a driving force to help catalyzing the formation of ATP molecules, proving a tight connection between ΔΨm and the ATP synthase. Naturally, a question can be raised on the effects of altered functioning of ATP synthases on regulating ΔΨm, which is attractive in the area of photosynthetic research. Lots of findings, when making efforts of solving this difficulty, can offer an in-depth understanding into the mechanism behind. However, the functional network on modulating ΔΨm is highly interdependent. It is difficult to comprehend the consequences of altered activity of ATP synthases on adjusting ΔΨm because parameters that have influences on ΔΨm would themselves be affected by ΔΨm. In this work, a computer model was applied to check the kinetic changes in polarization/depolarization across the thylakoid membrane (TM) regulated by the modified action of ATP synthases. The computing data revealed that under the extreme condition by numerically "switching off" the action of the ATP synthase, the complete inactivation of ATP synthase would markedly impede proton translocation at the cytb6f complex. Concurrently, the KEA3 (CLCe) porter, actively pumping protons into the stroma, further contributes to achieving a sustained low level of ΔΨm. Besides, the quantitative consequences on every particular component of ΔΨm adjusted by the modified functioning of ATP synthases were also explored. By employing the model, we bring evidence from the theoretical perspective that the ATP synthase is a key factor in forming a transmembrane proton loop thereby maintaining a propriate steady-state ΔΨm to meet variable environmental conditions.

Dissipation of the proton electrochemical gradient in chloroplasts promotes the oxidation of ATP synthase by thioredoxin-like proteins.

Chloroplast FoF1-ATP synthase (CFoCF1) uses an electrochemical gradient of protons across the thylakoid membrane (ΔµH+) as an energy source in the ATP synthesis reaction. CFoCF1 activity is regulated by the redox state of a Cys pair on its central axis, that is, the γ subunit (CF1-γ). When the ΔµH+ is formed by the photosynthetic electron transfer chain under light conditions, CF1-γ is reduced by thioredoxin (Trx), and the entire CFoCF1 enzyme is activated. The redox regulation of CFoCF1 is a key mechanism underlying the control of ATP synthesis under light conditions. In contrast, the oxidative deactivation process involving CFoCF1 has not been clarified. In the present study, we analyzed the oxidation of CF1-γ by two physiological oxidants in the chloroplast, namely the proteins Trx-like 2 and atypical Cys-His-rich Trx. Using the thylakoid membrane containing the reduced form of CFoCF1, we were able to assess the CF1-γ oxidation ability of these Trx-like proteins. Our kinetic analysis indicated that these proteins oxidized CF1-γ with a higher efficiency than that achieved by a chemical oxidant and typical chloroplast Trxs. Additionally, the CF1-γ oxidation rate due to Trx-like proteins and the affinity between them were changed markedly when ΔµH+ formation across the thylakoid membrane was manipulated artificially. Collectively, these results indicate that the formation status of the ΔµH+ controls the redox regulation of CFoCF1 to prevent energetic disadvantages in plants.

On the Edge of Dispensability, the Chloroplast ndh Genes.

The polypeptides encoded by the chloroplast ndh genes and some nuclear genes form the thylakoid NADH dehydrogenase (Ndh) complex, homologous to the mitochondrial complex I. Except for Charophyceae (algae related to higher plants) and a few Prasinophyceae, all eukaryotic algae lack ndh genes. Among vascular plants, the ndh genes are absent in epiphytic and in some species scattered among different genera, families, and orders. The recent identification of many plants lacking plastid ndh genes allows comparison on phylogenetic trees and functional investigations of the ndh genes. The ndh genes protect Angiosperms under various terrestrial stresses, maintaining efficient photosynthesis. On the edge of dispensability, ndh genes provide a test for the natural selection of photosynthesis-related genes in evolution. Variable evolutionary environments place Angiosperms without ndh genes at risk of extinction and, probably, most extant ones may have lost ndh genes recently. Therefore, they are evolutionary endpoints in phylogenetic trees. The low number of sequenced plastid DNA and the long lifespan of some Gymnosperms lacking ndh genes challenge models about the role of ndh genes protecting against stress and promoting leaf senescence. Additional DNA sequencing in Gymnosperms and investigations into the molecular mechanisms of their response to stress will provide a unified model of the evolutionary and functional consequences of the lack of ndh genes.

Binding and/or hydrolysis of purine-based nucleotides is not required for IM30 ring formation.

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.

Membrane Chaperoning of a Thylakoid Protease Whose Structural Stability Is Modified by the Protonmotive Force.

Protein folding is a complex cellular process often assisted by chaperones, but it can also be facilitated by interactions with lipids. Disulfide bond formation is a common mechanism to stabilize a protein. This can help maintain functionality amid changes in the biochemical milieu, including those relating to energy-transducing membranes. Plastidic Type I Signal Peptidase 1 (Plsp1) is an integral thylakoid membrane signal peptidase that requires an intramolecular disulfide bond for in vitro activity. We have investigated the interplay between disulfide bond formation, lipids, and pH in the folding and activity of Plsp1. By combining biochemical approaches with a genetic complementation assay using Arabidopsis thaliana plants, we provide evidence that interactions with lipids in the thylakoid membrane have reconstitutive chaperoning activity toward Plsp1. Further, the disulfide bridge appears to prevent an inhibitory conformational change resulting from proton motive force-mimicking pH conditions. Broader implications related to the folding of proteins in energy-transducing membranes are discussed.

Decreased photosystem II activity facilitates acclimation to fluctuating light in the understory plant Paris polyphylla.

In forests, understory plants are usually exposed to sunflecks on timescales of seconds or minutes. However, it is unclear how understory plants acclimate to fluctuating light. In this study, we compared chlorophyll fluorescence, PSI redox state and the electrochromic shift signal under fluctuating light between an understory plant Paris polyphylla (Liliaceae) and a light-demanding plant Bletilla striata (Orchidaceae). Within the first seconds after transition from low to high light, PSI was highly oxidized in P. polyphylla but was highly reduced in B. striata, although both species could not generate a sufficient trans-thylakoid proton gradient (ΔpH). Furthermore, the outflow of electrons from PSI to O2 was not significant in P. polyphylla, as indicated by the P700 redox kinetics upon dark-to-light transition. Therefore, the different responses of PSI to fluctuating light between P. polyphylla and B. striata could not be explained by ΔpH formation or alternative electron transport. In contrast, upon a sudden transition from low to high light, electron flow from PSII was much lower in P. polyphylla than in B. striata, suggesting that the rapid oxidation of PSI in P. polyphylla was largely attributed to the lower PSII activity. We propose, for the first time, that down-regulation of PSII activity is an important strategy used by some understory angiosperms to cope with sunflecks.

Chilling stress and hydrogen peroxide accumulation in Chrysanthemum morifolium and Spathiphyllum lanceifolium. Involvement of chlororespiration.

Plants of Chrysanthemum morifolium (sun species) and Spathiphyllum lanceifolium (shade species) were used to study the effects of chilling stems under high illumination. The stress conditions resulted in a greater accumulation of H2O2 in C. morifolium than in S. lanceifolium, and in the down-regulation of photosynthetic linear electron transport in both species. However, only a slight decrease in the maximal quantum yield of PSII was observed under unfavorable conditions in both species, suggesting that mechanisms exist in the chloroplasts that dissipate excess excitation energy and prevent damage to the photosynthetic apparatus. Additionally, changes were observed in the PGR5 polypeptide involved in cyclic electron flow around PSI and in chlororespiratory enzymes (plastidial NDH complex and PTOX). The level of PGR5 increased significantly only in chilled plants of C. morifolium, whereas the levels of the PTOX and NDH-H polypeptide of the plastidial NDH complex and the NDH activity increased significantly only in chilled plants of S. lanceifolium. These findings suggest that the cyclic electron flow involving PGR5 is more active in C. morifolium, while in S. lanceifolium, other mechanisms involving chlororespiratory enzymes are stimulated in response to chilling and high light, resulting in less H2O2 being accumulated in leaves.

Identification of Two Conserved Residues Involved in Copper Release from Chloroplast PIB-1-ATPases.

Copper is an essential transition metal for living organisms. In the plant model Arabidopsis thaliana, half of the copper content is localized in the chloroplast, and as a cofactor of plastocyanin, copper is essential for photosynthesis. Within the chloroplast, copper delivery to plastocyanin involves two transporters of the PIB-1-ATPases subfamily: HMA6 at the chloroplast envelope and HMA8 in the thylakoid membranes. Both proteins are high affinity copper transporters but share distinct enzymatic properties. In the present work, the comparison of 140 sequences of PIB-1-ATPases revealed a conserved region unusually rich in histidine and cysteine residues in the TMA-L1 region of eukaryotic chloroplast copper ATPases. To evaluate the role of these residues, we mutated them in HMA6 and HMA8. Mutants of interest were selected from phenotypic tests in yeast and produced in Lactococcus lactis for further biochemical characterizations using phosphorylation assays from ATP and Pi Combining functional and structural data, we highlight the importance of the cysteine and the first histidine of the CX3HX2H motif in the process of copper release from HMA6 and HMA8 and propose a copper pathway through the membrane domain of these transporters. Finally, our work suggests a more general role of the histidine residue in the transport of copper by PIB-1-ATPases.

A Novel Redoxin in the Thylakoid Membrane Regulates the Titer of Photosystem I.

In photosynthetic organisms like cyanobacteria and plants, the main engines of oxygenic photosynthesis are the pigment-protein complexes photosystem I (PSI) and photosystem II (PSII) located in the thylakoid membrane. In the cyanobacterium Synechocystis sp. PCC 6803, the slr1796 gene encodes a single cysteine thioredoxin-like protein, orthologs of which are found in multiple cyanobacterial strains as well as chloroplasts of higher plants. Targeted inactivation of slr1796 in Synechocystis 6803 resulted in compromised photoautotrophic growth. The mutant displayed decreased chlorophyll a content. These changes correlated with a decrease in the PSI titer of the mutant cells, whereas the PSII content was unaffected. In the mutant, the transcript levels of genes for PSI structural and accessory proteins remained unaffected, whereas the levels of PSI structural proteins were severely diminished, indicating that Slr1796 acts at a posttranscriptional level. Biochemical analysis indicated that Slr1796 is an integral thylakoid membrane protein. We conclude that Slr1796 is a novel regulatory factor that modulates PSI titer.

CARBONIC ANHYDRASE ACTIVITY OF INTEGRAL-FUNCTIONAL COMPLEXES OF THYLAKOID MEMBRANES OF SPINACH CHLOROPLASTS.

Isolated thylakoid membranes were disrupted by treatment with nonionic detergents digitonin or dodecyl maltoside. Solubilized polypeptide complexes were separated by native gel charge shift electrophoresis. The position of ATP-synthase complex and its isolated catalytic part (CF1) within gel was determined using the color reaction for ATPase activity. Due to the presence of cytochromes, the red band in unstained gels corresponded to the cytochrome b6f complex. Localization of the cytochrome b6f complex, ATP synthase and coupling CF1 in the native gel was confirmed by their subunit composition determined after SDS-electrophoretic analysis. Carbonic anhydrase (CA) activity in polypeptide zones of PS II, cytochrome b6f complex, and ATP-synthase CF1 was identified in native gels using indicator bromothymol blue. CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1 Thus, it was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase.

Ca(2+)-regulated cyclic electron flow supplies ATP for nitrogen starvation-induced lipid biosynthesis in green alga.

We previously showed that both the linear photosynthetic electron transportation rate and the respiration rate dropped significantly during N starvation-induced neutral lipid accumulation in an oil-producing microalga, Chlorella sorokiniana, and proposed a possible role for cyclic electron flow (CEF) in ATP supply. In this study, we further exploited this hypothesis in both Chlorella sorokiniana C3 and the model green alga Chlamydomonas. We found that both the rate of CEF around photosystem I and the activity of thylakoid membrane-located ATP synthetase increased significantly during N starvation to drive ATP production. Furthermore, we demonstrated that the Chlamydomonas mutant pgrl1, which is deficient in PGRL1-mediated CEF, accumulated less neutral lipids and had reduced rates of CEF under N starvation. Further analysis revealed that Ca(2+) signaling regulates N starvation-induced neutral lipid biosynthesis in Chlamydomonas by increasing calmodulin activity and boosting the expression of the calcium sensor protein that regulates Pgrl1-mediated CEF. Thus, Ca(2+)-regulated CEF supplies ATP for N starvation-induced lipid biosynthesis in green alga. The increased CEF may re-equilibrate the ATP/NADPH balance and recycle excess light energy in photosystems to prevent photooxidative damage, suggesting Ca(2+)-regulated CEF also played a key role in protecting and sustaining photosystems.

Violaxanthin de-epoxidase disulphides and their role in activity and thermal stability.

Violaxanthin de-epoxidase (VDE) catalyses the conversion of violaxanthin to zeaxanthin at the lumen side of the thylakoids during exposure to intense light. VDE consists of a cysteine-rich N-terminal domain, a lipocalin-like domain and a negatively charged C-terminal domain. That the cysteines are important for the activity of VDE is well known, but in what way is less understood. In this study, wild-type spinach VDE was expressed in E. coli as inclusion bodies, refolded and purified to give a highly active and homogenous preparation. The metal content (Fe, Cu, Ni, Mn, Co and Zn) was lower than 1 mol% excluding a metal-binding function of the cysteines. To investigate which of the 13 cysteines that could be important for the function of VDE, we constructed mutants where the cysteines were replaced by serines, one by one. For 12 out of 13 mutants the activity dropped by more than 99.9%. A quantification of free cysteines showed that only the most N-terminal of these cysteines was in reduced form in the native VDE. A disulphide pattern in VDE of C9-C27, C14-C21, C33-C50, C37-C46, C65-C72 and C118-C284 was obtained after digestion of VDE with thermolysin followed by mass spectroscopy analysis of reduced versus non-reduced samples. The residual activity found for the mutants showed a variation that was consistent with the results obtained from mass spectroscopy. Reduction of the disulphides resulted in loss of a rigid structure and a decrease in thermal stability of 15 °C.

2-cysteine peroxiredoxins and thylakoid ascorbate peroxidase create a water-water cycle that is essential to protect the photosynthetic apparatus under high light stress conditions.

Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H2O2)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H2O2, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H2O2- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX.

A novel chloroplast localized Rab GTPase protein CPRabA5e is involved in stress, development, thylakoid biogenesis and vesicle transport in Arabidopsis.

A novel Rab GTPase protein in Arabidopsis thaliana, CPRabA5e (CP = chloroplast localized) is located in chloroplasts and has a role in transport. Transient expression of CPRabA5e:EGFP fusion protein in tobacco (Nicotiana tabacum) leaves, and immunoblotting using Arabidopsis showed localization of CPRabA5e in chloroplasts (stroma and thylakoids). Ypt31/32 in the yeast Saccharomyces cerevisiae are involved in regulating vesicle transport, and CPRabA5e a close homolog of Ypt31/32, restores the growth of the ypt31Δ ypt32(ts) mutant at 37 °C in yeast complementation. Knockout mutants of CPRabA5e displayed delayed seed germination and growth arrest during oxidative stress. Ultrastructural studies revealed that after preincubation at 4 °C mutant chloroplasts contained larger plastoglobules, lower grana, and more vesicles close to the envelopes compared to wild type, and vesicle formation being enhanced under oxidative stress. This indicated altered thylakoid development and organization of the mutants. A yeast-two-hybrid screen with CPRabA5e as bait revealed 13 interacting partner proteins, mainly located in thylakoids and plastoglobules. These proteins are known or predicted to be involved in development, stress responses, and photosynthesis related processes, consistent with the stress phenotypes observed. The results observed suggest a role of CPRabA5e in transport to and from thylakoids, similar to cytosolic Rab proteins involved in vesicle transport.

Enhanced stability of thylakoid membrane proteins and antioxidant competence contribute to drought stress resistance in the tasg1 wheat stay-green mutant.

A wheat stay-green mutant, tasg1, was previously generated via mutation breeding of HS2, a common wheat cultivar (Triticum aestivum L.). Compared with wild-type (WT) plants, tasg1 exhibited delayed senescence indicated by the slower degradation of chlorophyll. In this study, the stability of proteins in thylakoid membranes was evaluated in tasg1 under drought stress compared with WT plants in the field as well as in seedlings in the laboratory. Drought stress was imposed by controlling irrigation and sheltering the plants from rain in the field, and by polyethylene glycol (PEG)-6000 in the laboratory. The results indicated that tasg1 plants could maintain higher Hill activity, actual efficiency (ΦPSII), maximal photochemical efficiency of PSII (Fv/Fm), and Ca(2+)-ATPase and Mg(2+)-ATPase activities than the WT plants under drought stress. Furthermore, the abundance of some polypeptides in thylakoid membranes of tasg1 was greater than that in the WT under drought stress. Expression levels of TaLhcb4 and TaLhcb6 were higher in tasg1 compared with the WT. Under drought stress, the accumulation of superoxide radical (O2·(-)) and hydrogen peroxide (H2O2) was lower in tasg1 compared with the WT not only at the senescence stage but also at the seedling stages. These results suggest greater functional stability of thylakoid membrane proteins in tasg1 compared with the WT, and the higher antioxidant competence of tasg1 may play an important role in the enhanced drought tolerance of tasg1.

S-sulfocysteine synthase function in sensing chloroplast redox status.

The minor chloroplastic O-acetylserine(thiol)lyase isoform encoded by the CS26 gene in Arabidopsis thaliana has been described as an S-sulfocysteine synthase enzyme that plays an important role in chloroplast function. This enzyme is located in the thylakoid lumen, and its S-sulfocysteine activity is essential for the proper photosynthetic performance of the chloroplast under long-day growth conditions. Based on the present knowledge of this enzyme, we suggest that S-sulfocysteine synthase functions as a protein sensor to detect the accumulation of thiosulfate as a result of the inadequate detoxification of reactive oxygen species generated under conditions of excess light to produce the S-sulfocysteine molecule that triggers protection mechanisms of the photosynthetic apparatus.

Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii.

BACKGROUND: Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO(2) fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO(2) conditions. RESULTS/CONCLUSIONS: In the present work we demonstrate that after transfer to low CO(2), Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO(2) conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO(2) conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO(2) conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO(2) the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. SIGNIFICANCE: This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

Photosynthetic adaptation to length of day is dependent on S-sulfocysteine synthase activity in the thylakoid lumen.

Arabidopsis (Arabidopsis thaliana) chloroplasts contain two O-acetyl-serine(thiol)lyase (OASTL) homologs, OAS-B, which is an authentic OASTL, and CS26, which has S-sulfocysteine synthase activity. In contrast with OAS-B, the loss of CS26 function resulted in dramatic phenotypic changes, which were dependent on the light treatment. We have performed a detailed characterization of the photosynthetic and chlorophyll fluorescence parameters in cs26 plants compared with those of wild-type plants under short-day growth conditions (SD) and long-day growth conditions (LD). Under LD, the photosynthetic characterization, which was based on substomatal CO(2) concentrations and CO(2) concentration in the chloroplast curves, revealed significant reductions in most of the photosynthetic parameters for cs26, which were unchanged under SD. These parameters included net CO(2) assimilation rate, mesophyll conductance, and mitochondrial respiration at darkness. The analysis also showed that cs26 under LD required more absorbed quanta per driven electron flux and fixed CO(2). The nonphotochemical quenching values suggested that in cs26 plants, the excess electrons that are not used in photochemical reactions may form reactive oxygen species. A photoinhibitory effect was confirmed by the background fluorescence signal values under LD and SD, which were higher in young leaves compared with mature ones under SD. To hypothesize the role of CS26 in relation to the photosynthetic machinery, we addressed its location inside of the chloroplast. The activity determination and localization analyses that were performed using immunoblotting indicated the presence of an active CS26 enzyme exclusively in the thylakoid lumen. This finding was reinforced by the observation of marked alterations in many lumenal proteins in the cs26 mutant compared with the wild type.

Reactive oxygen species affect ATP hydrolysis by targeting a highly conserved amino acid cluster in the thylakoid ATP synthase γ subunit.

The vast majority of organisms produce ATP by a membrane-bound rotating protein complex, termed F-ATP synthase. In chloroplasts, the corresponding enzyme generates ATP by using a transmembrane proton gradient generated during photosynthesis, a process releasing high amounts of molecular oxygen as a natural byproduct. Due to its chemical properties, oxygen can be reduced incompletely which generates several highly reactive oxygen species (ROS) that are able to oxidize a broad range of biomolecules. In extension to previous studies it could be shown that ROS dramatically decreased ATP synthesis in situ and affected the CF1 portion in vitro. A conserved cluster of three methionines and a cysteine on the chloroplast γ subunit could be identified by mass spectrometry to be oxidized by ROS. Analysis of amino acid substitutions in a hybrid F1 assembly system indicated that these residues were exclusive catalytic targets for hydrogen peroxide and singlet oxygen, although it could be deduced that additional unknown amino acid targets might be involved in the latter reaction. The cluster was tightly integrated in catalytic turnover since mutants varied in MgATPase rates, stimulation by sulfite and chloroplast-specific γ subunit redox-modulation. Some partial disruptions of the cluster by mutagenesis were dominant over others regarding their effects on catalysis and response to ROS.

The hetero-hexameric nature of a chloroplast AAA+ FtsH protease contributes to its thermodynamic stability.

FtsH is an evolutionary conserved membrane-bound metalloprotease complex. While in most prokaryotes FtsH is encoded by a single gene, multiple FtsH genes are found in eukaryotes. Genetic and biochemical data suggest that the Arabidopsis chloroplast FtsH is a hetero-hexamer. This raises the question why photosynthetic organisms require a heteromeric complex, whereas in most bacteria a homomeric one is sufficient. To gain structural information of the possible complexes, the Arabidopsis FtsH2 (type B) and FtsH5 (type A) were modeled. An in silico study with mixed models of FtsH2/5 suggests that heteromeric hexamer structure with ratio of 4:2 is more likely to exists. Specifically, calculation of the buried surface area at the interfaces between neighboring subunits revealed that a hetero-complex should be thermodynamically more stable than a homo-hexamer, due to the presence of additional hydrophobic and hydrophilic interactions. To biochemically assess this model, we generated Arabidopsis transgenic plants, expressing epitope-tagged FtsH2 and immuno-purified the protein. Mass-spectrometry analysis showed that FtsH2 is associated with FtsH1, FtsH5 and FtsH8. Interestingly, we found that 'type B' subunits (FtsH2 and FtsH8) were 2-3 fold more abundant than 'type A' (FtsH1 and FtsH5). The biochemical data corroborate the in silico model and suggest that the thylakoid FtsH hexamer is composed of two 'type A' and four 'type B' subunits.