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International audience; During photosynthesis, electron transport is necessary for carbon assimilation and must be regulated to minimize free radical damage. There is a longstanding controversy over the role of a critical enzyme in this process (ferredoxin:NADP(H) oxidoreductase, or FNR), and in particular its location within chloroplasts. Here we use immunogold labelling to prove that FNR previously assigned as soluble is in fact membrane associated. We combined this technique with a genetic approach in the model plant Arabidopsis to show that the distribution of this enzyme between different membrane regions depends on its interaction with specific tether proteins. We further demonstrate a correlation between the interaction of FNR with different proteins and the activity of alternative photosynthetic electron transport pathways. This supports a role for FNR location in regulating photosynthetic electron flow during the transition from dark to light.

Biogenesis of photosystem II (PSII), nature’s water-splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate from Thermosynechococcus elongatus at 2.94 A resolution. It contains three assembly factors (Psb27, Psb28 and Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace the bicarbonate ligand of non-haem iron with glutamate, a structural motif found in reaction centres of non-oxygenic photosynthetic bacteria. These results reveal mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water-splitting reaction. Photosystems need auxiliary proteins to assist their assembly. Cryo-electron microscopy of a cyanobacterial photosystem II assembly intermediate at 2.94 A reveals mechanisms protecting against photodamage during vulnerable stages of biogenesis.

Biogenesis of photosystem II (PSII), nature’s water splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate with 2.94 A resolution. It contains three assembly factors (Psb27, Psb28, Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace bicarbonate with glutamate as a ligand of the non-heme iron, a structural motif found in reaction centers of non-oxygenic photosynthetic bacteria. These results reveal novel mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water splitting reaction. One Sentence Highlight The high-resolution Cryo-EM structure of the photosystem II assembly intermediate PSII-I reveals how nature’s water splitting catalyst is assembled, protected and prepared for photoactivation by help of the three assembly factors Psb27, Psb28 and Psb34.

Pgr5 proteins play a major direct role in cyclic electron flow paths in plants and eukaryotic phytoplankton. The genomes of many cyanobacterial species code for Pgr5-like proteins but their function is still uncertain. Here, we present evidence that supports a link between the Synechocystis sp. PCC6803 Pgr5-like protein and the regulation of intracellular redox balance. The knockout strain, pgr5KO, did not display substantial phenotypic response under our experimental conditions, confirming results obtained in earlier studies. However, the overexpression strain, pgr5OE, accumulated 2.5-fold more chlorophyll than the wild type and displayed increased content of photosystems matching the chlorophyll increase. As a result, electron transfer rates through the photosynthetic apparatus of pgr5OE increased, as did the amount of energy stored as glycogen. While, under photoautotrophic conditions, this metabolic difference had only minor effects, under mixotrophic conditions, pgr5OE cultures collapsed. Interestingly, this specific phenotype of pgr5OE mutants displayed a tendency for reverting, and cultures which previously collapsed in the presence of glucose were now able to survive. DNA sequencing of a pgr5OE strain revealed a second site suppression mutation in slr1916, a putative esterase associated with redox regulation. The phenotype of the slr1916 knockout is very similar to that of the strain reported here and to that of the pmgA regulator knockout. These data demonstrate that, in Synechocystis 6803, there is strong selection against overexpression of the Pgr5-like protein. The pseudoreversion event in a gene involved in redox regulation suggests a connection of the Pgr5-like protein to this network.

Summary Heavy metal contamination is a serious environmental problem. Understanding the toxicity mechanisms may allow to lower concentration of metals in the metal‐based antimicrobial treatments of crops, and reduce metal content in soil and groundwater. Here, we investigate the interplay between metal efflux systems and the superoxide dismutase (SOD) in the purple bacterium Rubrivivax gelatinosus and other bacteria through analysis of the impact of metal accumulation. Exposure of the Cd2+‐efflux mutant ΔcadA to Cd2+ caused an increase in the amount and activity of the cytosolic Fe‐Sod SodB, thereby suggesting a role of SodB in the protection against Cd2+. In support of this conclusion, inactivation of sodB gene in the ΔcadA cells alleviated detoxification of superoxide and enhanced Cd2+ toxicity. Similar findings were described in the Cu+‐efflux mutant with Cu+. Induction of the Mn‐Sod or Fe‐Sod in response to metals in other bacteria, including Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Vibrio cholera and Bacillus subtilis, was also shown. Both excess Cd2+ or Cu+ and superoxide can damage [4Fe‐4S] clusters. The additive effect of metal and superoxide on the [4Fe‐4S] could therefore explain the hypersensitive phenotype in mutants lacking SOD and the efflux ATPase. These findings underscore that ROS defence system becomes decisive for bacterial survival under metal excess., Superoxide detoxification system became determinant for bacterial survival under metal stress in bacteria. Targeting the ROS defense system together with the metal efflux systems may allow lowering the concentration of metals in the metal‐based antimicrobial treatments in agriculture and farming.

In photosynthesis research, non-invasive in vivo spectroscopic analyses have been used as a practical tool for studying photosynthetic electron transport. Klas-NIR spectrophotometer has been recently developed by Klughammer and Schreiber (Photosynth Res 128:195-214, 2016) for in vivo measurements of redox changes of P700, plastocyanin (Pcy) and ferredoxin (Fd). Here we show examples using the Klas-NIR spectrophotometer for the evaluation of the redox states and quantities of these components in plant leaves and cyanobacterial suspensions. The redox poise under light of the electron transport components is different in leaves from higher plants compared with cyanobacteria. During a short illumination with an actinic light, P700, Pcy, and Fd are kept reduced in barley leaves but are oxidized in cyanobacteria. During far-red light illumination, P700 and Pcy are mostly oxidized in the leaves but are partially kept reduced in cyanobacteria. In the cyanobacterium, Thermosynechococcus elongatus, which has no Pcy but uses cytochrome c6 (cyt c6) as the electron donor to photosystem I, a cyt c6 signal was detected in vivo. To show the potential of Klas-NIR spectrophotometer for studying different developmental stages of a leaf, we performed measurements on fully mature and early senescing barley leaves. Pcy content in leaves decreased during senescence at an early stage. The Pcy loss was quantitatively analyzed using Klas-NIR spectrophotometer, giving absolute ratios of Pcy to PSI of 2.5 and 1.6 in younger and older leaves, respectively. For quantification of the signals in vivo, in vitro data (Sétif et al. in Photosynth Res142:307-319, 2019) obtained with Klas-NIR spectrophotometer were used.

At high O2 tensions, as encountered prehistorically, Chlamydomonas reinhardtii elevated levels of NPQ-related proteins and LHCSR3 had an important function in protecting PSI., Non-photochemical quenching (NPQ) helps dissipate surplus light energy, preventing formation of reactive oxygen species (ROS). In Chlamydomonas reinhardtii, the thylakoid membrane protein LHCSR3 is involved in pH-dependent (qE-type) NPQ, lacking in the npq4 mutant. Preventing PSII repair revealed that npq4 lost PSII activity faster than the wild type (WT) in elevated O2, while no difference between strains was observed in O2-depleted conditions. Low Fv/Fm values remained 1.5 h after moving cells out of high light, and this qH-type quenching was independent of LHCSR3 and not accompanied by losses of maximum PSII activity. Culturing cells in historic O2 atmospheres (30–35%) increased the qE of cells, due to increased LHCSR1 and PsbS levels, and LHCSR3 in the WT, showing that atmospheric O2 tensions regulate qE capacity. Colony growth of npq4 was severely restricted at elevated O2, and npq4 accumulated more reactive electrophile species (RES) than the WT, which could damage PSI. Levels of PsaA (PSI) were lower in npq4 grown at 35% O2, while PsbA (PSII) levels remained stable. We conclude that even at high O2 concentrations, the PSII repair cycle is sufficient to maintain net levels of PSII. However, LHCSR3 has an important function in protecting PSI against O2-mediated damage, such as via RES.

International audience; Flavodiiron proteins (FDPs) of photosynthetic organisms play a photoprotective role by reducing oxygen to water and thus avoiding the accumulation of excess electrons on the photosystem I (PSI) acceptor side under stress conditions. In Synechocystis sp. PCC 6803 grown under high CO 2 , both FDPs Flv1 and Flv3 are indispensable for oxygen reduction. We performed a detailed in vivo kinetic study of wild-type (WT) and flv1/3 strains of Synechocystis using light-induced NADPH fluorescence and near-infrared absorption of ironsulfur clusters from ferredoxin and the PSI acceptors (F A F B), collectively named FeS. These measurements were performed under conditions where the Calvin-Benson cycle is inactive or poorly activated. Under such conditions, the NADPH decay following a short illumination decays in parallel in both strains and exhibits a time lag which is correlated to the presence of reduced FeS. On the contrary, reduced FeS decays much faster in WT than in flv1/3 (13 vs 2 s-1). These data unambiguously show that reduced ferredoxin, or possibly reduced F A F B , is the direct electron donor to the Flv1/Flv3 heterodimer. Evidences for large reduction of (F A F B) and recombination reactions within PSI were also provided by near-infrared absorption. Mutants lacking either the NDH1-L complex, the homolog of complex I of respiration, or the Pgr5 protein show no difference with WT in the oxidation of reduced FeS following a short illumination. These observations question the participation of a significant cyclic electron flow in cyanobacteria during the first seconds of the induction phase of photosynthesis.

International audience; The liverwort Marchantia polymorpha contains two isoforms of the plastid terminal oxidase (PTOX), an enzyme that catalyzes the reduction of oxygen to water using plastoquinol as substrate. Phylogenetic analyses showed that one isoform, here called MpPTOXa, is closely related to isoforms occurring in plants and some algae, while the other isoform, here called MpPTOXb, is closely related to the two isoforms occurring in Chlamydomonas reinhardtii. Mutants of each isoform were created in Marchantia polymorpha using CRISPR/Cas9 technology. While no obvious phenotype was found for these mutants, chlorophyll fluorescence analyses demonstrated that the plastoquinone pool was in a higher reduction state in both mutants. This was visible at the level of fluorescence measured in dark-adapted material and by post illumination fluorescence rise. These results suggest that both isoforms have a redundant function. However, when P700 oxidation and re-reduction was studied, differences between these two isoforms were observed. Furthermore, the mutant affected in MpPTOXb showed a slight alteration in the pigment composition, a higher non-photochemical quenching and a slightly lower electron transport rate through photosystem II. These differences may be explained either by differences in the enzymatic activities or by different activities attributed to preferential involvement of the two PTOX isoforms to either linear or cyclic electron flow.

44 páginas, 6 figuras, 1 tabla. -- The definitive version is available at http://www.elsevier.com, The two Arabidopsis thaliana mutants, aba1 and max4, were previously identified as sharing a number of co-regulated genes with both the flu mutant and Arabidopsis cell suspension cultures exposed to high light (HL). On this basis, we investigated whether aba1 and max4 were generating high amounts of singlet oxygen (1O2) and activating 1O2-mediated cell death. Thylakoids of aba1 produced twice as much 1O2 as thylakoids of max4 and wild type (WT) plants when illuminated with strong red light. 1O2 was measured using the spin probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride. 77-K chlorophyll fluorescence emission spectra of thylakoids revealed lower aggregation of the light harvesting complex II in aba1. This was rationalized as a loss of connectivity between photosystem II (PSII) units and as the main cause for the high yield of 1O2 generation in aba1. Up-regulation of the 1O2 responsive gene AAA-ATPase was only observed with statistical significant in aba1 under HL. Two early jasmonate (JA)-responsive genes, JAZ1 and JAZ5, encoding for two repressor proteins involved in the negative feedback regulation of JA signalling, were not up-regulated to the WT plant levels. Chloroplast aggregation followed by chloroplast rupture and eventual cell death was observed by confocal imaging of the fluorescence emission of leaf cells of transgenic aba1 plants expressing the chimeric fusion protein SSU-GFP. Cell death was not associated with direct 1O2 cytotoxicity in aba1, but rather with a delayed stress response. In contrast, max4 did not show evidence of 1O2-mediated cell death. In conclusion, aba1 may serve as an alternative model to other 1O2-overproducing mutants of Arabidopsis for investigating 1O2-mediated cell death., This work was supported by MINECO (AGL2013-41363-R, ERDF) and Junta de Castilla y León (CSI002A10-2 and CSI083U16, ERDF) to J.B.A., ERC.KBBE.2012.1.1-01 (EcoSeed-311840) to Ó.L and A. K.-L., and MINECO (BIO2014-57107-R) and Junta de Castilla y León (SA239U13 and SA093U16) to Ó.L.

International audience; During daffodilflower development,chloroplasts differentiate into photosynthetically inactive chromoplasts, which have lostfunctional photosynthetic reaction centers. Chromoplasts exhibit a respiratory activity reducing oxygen to water and generating ATP. Immunoblots revealed the presence of the plastid terminal oxidase(PTOX), the NAD(P)H dehydrogenase (NDH) complex, the cytochrome b6fcomplex, ATP synthase and several isoforms of ferredoxin-NADP+oxidoreductase (FNR) and of ferredoxin (Fd). Fluorescence spectroscopy allowed the detection of chlorophyll a in the cytochrome b6fcomplex. Here we characterize the electron transport pathway of chromorespiration by using specific inhibitors forthe NDH complex, the cytochrome b6fcomplex, FNR and redox-inactive Fd in which the iron was replaced by gallium. Our data suggest anelectron flowvia twoseparatepathways, both reducing plastoquinone and using PTOX as oxidase. The first oxidizes NADPH via FNR, Fd,and cytochrome bh of the cytochrome b6fcomplex and does not result in the pumpingofprotons across the membrane. In the second,electron transport takes place via the NDH complex using preferentially NADH but also NADPH as electron donor. FNR and Fd are not involved in this pathway. The NDH-complex is responsible for the generation of the proton gradient. We propose a new model for chromorespiration which may also be relevant for the understanding of chlororespiration and for the characterization of the electron input from Fd to the cytochrome b6fcomplex during cyclic electron transport in chloroplasts.

International audience; Abscisic acid (ABA) is an important hormone for seed development and germination whose physiological action is modulated by its endogenous levels. Cleavage of carotenoid precursors by 9-cis epoxycarotenoid dioxygenase (NCED) and inactivation of ABA by ABA 8'-hydroxylase (CYP707A) are key regulatory metabolic steps. In Arabidopsis (Arabidopsis thaliana), both enzymes are encoded by multigene families, having distinctive expression patterns. To evaluate the genome-wide impact of ABA deficiency in developing seeds at the maturation stage when dormancy is induced, we used a nced2569 quadruple mutant in which ABA deficiency is mostly restricted to seeds, thus limiting the impact of maternal defects on seed physiology. ABA content was very low in nced2569 seeds, similar to the severe mutant aba2; unexpectedly, ABA glucose ester was detected in aba2 seeds, suggesting the existence of an alternative metabolic route. Hormone content in nced2569 seeds compared with nced259 and wild-type strongly suggested that specific expression of NCED6 in the endosperm is mainly responsible for ABA production. In accordance, transcriptome analyses revealed broad similarities in gene expression between nced2569 and either wild type or nced259 developing seeds. Gene ontology enrichments revealed a large spectrum of ABA activation targets involved in reserve storage and desiccation tolerance, and repression of photosynthesis and cell cycle. Proteome and metabolome profiles in dry nced2569 seeds, compared with wild-type and cyp707a1a2 seeds, also highlighted an inhibitory role of ABA on remobilisation of reserves, ROS production, and protein oxidation. Down-regulation of these oxidative processes by ABA may have an essential role in dormancy control.

A kinetic-LED-array-spectrophotometer (Klas) was recently developed for measuring in vivo redox changes of P700, plastocyanin (PCy), and ferredoxin (Fd) in the near-infrared (NIR). This spectrophotometer is used in the present work for in vitro light-induced measurements with various combinations of photosystem I (PSI) from tobacco and two different cyanobacteria, spinach plastocyanin, cyanobacterial cytochrome c6 (cyt. c6), and Fd. It is shown that cyt. c6 oxidation contributes to the NIR absorption changes. The reduction of (FAFB), the terminal electron acceptor of PSI, was also observed and the shape of the (FAFB) NIR difference spectrum is similar to that of Fd. The NIR difference spectra of the electron-transfer cofactors were compared between different organisms and to those previously measured in vivo, whereas the relative absorption coefficients of all cofactors were determined by using single PSI turnover conditions. Thus, the (840 nm minus 965 nm) extinction coefficients of the light-induced species (oxidized minus reduced for PC and cyt. c6, reduced minus oxidized for (FAFB), and Fd) were determined with values of 0.207 ± 0.004, – 0.033 ± 0.006, – 0.036 ± 0.008, and – 0.021 ± 0.005 for PCy, cyt. c6, (FAFB) (single reduction), and Fd, respectively, by taking a reference value of + 1 for P700+. The fact that the NIR P700 coefficient is larger than that of PCy and much larger than that of other contributing species, combined with the observed variability in the NIR P700 spectral shape, emphasizes that deconvolution of NIR signals into different components requires a very precise determination of the P700 spectrum.

International audience; Senescence is the last stage of leaf development preceding the death of the organ, and it is important for nutrient remobilization and for feeding sink tissues. There are many reports on leaf senescence but the mechanisms initiating leaf senescence are still poorly understood. Leaf senescence is affected by many environmental factors and seems to vary in different species and even varieties of plants, which makes it difficult to generalize the mechanism. Here, we give an overview on studies reporting about alterations in the composition of the photosynthetic electron transport chain in chloroplasts during senescence. We hypothesize that alternative electron flow and related generation of the proton motive force required for ATP synthesis become increasingly important during progression of senescence. We address the generation of reactive oxygen species (ROS) in chloroplasts in the initiation of senescence, retrograde signaling from the chloroplast to the nucleus and ROS-dependent signaling associated with leaf senescence. Finally, differences between natural senescence and dark-induced senescence are pointed out and a few ideas for increasing crop yields by increasing the chloroplast lifespan are presented. This article is protected by copyright. All rights reserved.

High irradiances may lead to photooxidative stress in plants, and non-photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under light-limiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NTRC). Whereas Arabidopsis thaliana plants lacking NTRC tolerate high light intensities, these plants display drastically elevated qE, have larger trans-thylakoid ΔpH and have 10-fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an ntrc-psbs double-knockout mutant, which is devoid of qE. This double mutant grows faster than the ntrc mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the ntrc-psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking ntrc alone. These data uncover a new role for NTRC in the control of photosynthetic yield.

The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2 •− in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2 •− and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2 •− production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

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International audience; Plants and algae must tightly coordinate photosynthetic electron transport and metabolic activities given that they often face fluctuating light and nutrient conditions. The exchange of metabolites and signaling molecules between organelles is thought to be central to this regulation but evidence for this is still fragmentary. Here we show that knocking out the peroxisome-located MALATE DEHYDROGENASE 2 (MDH2) of Chlamydomonas reinhardtii results in dramatic alterations not only in peroxisomal fatty acid breakdown but also in chloroplast starch metabolism and photosynthesis. mdh2 mutants accumulated 50% more storage lipid and twofold more starch than wild type during nitrogen deprivation. In parallel, mdh2 showed increased PSII yield and photosynthetic CO 2 fixation. Metabolite analyses revealed a >60% reduction in malate, together with increased levels of NADPH and H 2 O 2 in mdh2. Similar phenotypes were found upon high light exposure. Furthermore, based on the lack of starch accumulation in a knockout mutant of the H 2 O 2-producing peroxisomal ACYL-COA OXIDASE 2 and on the effects of H 2 O 2 supplementation, we propose that peroxisome-derived H 2 O 2 acts as a regulator of chloroplast metabolism. We conclude that peroxisomal MDH2 helps photoautotrophs cope with nitrogen scarcity and high light by transmitting the redox state of the peroxisome to the chloroplast by means of malate shuttle-and H 2 O 2-based redox signaling.

International audience; In this article a novel mechanism of retrograde signaling by chloroplasts during stress is described. This mechanism involves the DNA/RNA binding protein WHIRLY1 as a regulator of microRNA levels. By virtue of its dual localization in chloroplasts and the nucleus of the same cell, WHIRLY1 was proposed as an excellent candidate coordinator of chloroplast function and nuclear gene expression. Comparison of wild-type and transgenic plants with an RNAi-mediated knockdown of WHIRLY1 showed, that the transgenic plants were unable to cope with continuous high light conditions. They were impaired in production of several microRNAs mediating post-transcriptional responses during stress. The results support a central role of WHIRLY1 in retrograde signaling and also underpin a so far underestimated role of microRNAs in this process.

The hot ABA-deficiency suppressor2 (has2) mutation increases drought tolerance and the ABA sensitivity of stomata closure and seed germination. Here we report that the HAS2 locus encodes the MITOCHONDRIAL EDITING FACTOR11 (MEF11), also known as LOVASTATIN INSENSITIVE1. has2/mef11 mutants exhibited phenotypes very similar to the ABA-hypersensitive mutant, hai1-1 pp2ca-1 hab1-1 abi1-2, which is impaired in four genes encoding type 2C protein phosphatases (PP2C) that act as upstream negative regulators of the ABA signaling cascade. Like pp2c, mef11 plants were more resistant to progressive water stress and seed germination was more sensitive to paclobutrazol (a gibberellin biosynthesis inhibitor) as well as mannitol and NaCl, compared with the wild-type plants. Phenotypic alterations in mef11 were associated with the lack of editing of transcripts for the mitochondrial cytochrome c maturation FN2 (ccmFN2) gene, which encodes a cytochrome c-heme lyase subunit involved in cytochrome c biogenesis. Although the abundance of electron transfer chain complexes was not affected, their dysfunction could be deduced from increased respiration and altered production of hydrogen peroxide and nitric oxide in mef11 seeds. As minor defects in mitochondrial respiration affect ABA signaling, this suggests an essential role for ABA in mitochondrial retrograde regulation.

International audience; The production of reactive oxygen species (ROS) is an unavoidable part of photosynthesis. Stress that accompanies high light levels and low CO2 availability putatively includes enhanced ROS production in the so-called Mehler reaction. Such conditions are thought to encourage O2 to become an electron acceptor at photosystem I, producing the ROS superoxide anion radical (O2·-) and hydrogen peroxide (H2 O2 ). In contrast, here it is shown in Chlamydomonas reinhardtii that CO2 depletion under high light levels lowered cellular H2 O2 production, and that elevated CO2 levels increased H2 O2 production. Using various photosynthetic and mitochondrial mutants of C. reinhardtii, the chloroplast was identified as the main source of elevated H2 O2 production under high CO2 availability. High light levels under low CO2 availability induced photoprotective mechanisms called non-photochemical quenching, or NPQ, including state transitions (qT) and high energy state quenching (qE). The qE-deficient mutant npq4 produced more H2 O2 than wild-type cells under high light levels, although less so under high CO2 availability, whereas it demonstrated equal or greater enzymatic H2 O2 -degrading capacity. The qT-deficient mutant stt7-9 produced the same H2 O2 as wild-type cells under high CO2 availability. Physiological levels of H2 O2 were able to hinder qT and the induction of state 2, providing an explanation for why under high light levels and high CO2 availability wild-type cells behaved like stt7-9 cells stuck in state 1.

In this article a novel mechanism of retrograde signaling by chloroplasts during stress is described. This mechanism involves the DNA/RNA binding protein WHIRLY1 as a regulator of microRNA levels. By virtue of its dual localization in chloroplasts and the nucleus of the same cell, WHIRLY1 was proposed as an excellent candidate coordinator of chloroplast function and nuclear gene expression (Grabowski et al., 2008; Foyer et al., 2014). In this study the putative involvement of WHIRLY1 in stress dependent retrograde signaling was investigated by comparison of barley (Hordeum vulgare L., cv. Golden Promise) wild-type and transgenic plants with an RNAi-mediated knockdown of WHIRLY1. In contrast to the wild type, the transgenic plants were unable to cope with continuous high light conditions. They were impaired in production of several microRNAs mediating post-transcriptional responses during stress (Kruszka et al., 2012, Sunkar et al., 2012). The results support a central role of WHIRLY1 in retrograde signaling and underpin a so far underestimated role of microRNAs in this process.

Cyanobacteria are the most ancient organisms performing oxygenic photosynthesis, and they are the ancestors of plant plastids. All plastids contain the plastid terminal oxidase (PTOX), while only certain cyanobacteria contain PTOX. Many putative functions have been discussed for PTOX in higher plants including a photoprotective role during abiotic stresses like high light, salinity and extreme temperatures. Since PTOX oxidizes PQH 2 and reduces oxygen to water, it is thought to protect against photo-oxidative damage by removing excess electrons from the plastoquinone (PQ) pool. To investigate the role of PTOX we overexpressed rice PTOX fused to the maltose-binding protein (MBP-OsPTOX) in Synechocystis sp. PCC 6803, a model cyanobacterium that does not encode PTOX. The fusion was highly expressed and OsPTOX was active, as shown by chlorophyll fluorescence and P 700 absorption measurements. The presence of PTOX led to a highly oxidized state of the NAD(P)H/NAD(P) + pool, as detected by NAD(P)H fluorescence. Moreover, in the PTOX overexpressor the electron transport capacity of PSI relative to PSII was higher, indicating an alteration of the photosystem I (PSI) to photosystem II (PSII) stoichiometry. We suggest that PTOX controls the expression of responsive genes of the photosynthetic apparatus in a different way from the PQ/PQH 2 ratio. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’.

High light causes photosystem II to generate singlet oxygen (1O2), a reactive oxygen species (ROS) that can react with membrane lipids, releasing reactive electrophile species (RES), such as acrolein. To investigate how RES may contribute to light stress responses, Chlamydomonas reinhardtii was high light-treated in photoautotrophic and mixotrophic conditions and also in an oxygen-enriched atmosphere to elevate ROS production. The responses were compared to exogenous acrolein. Non-photochemical quenching (NPQ) was higher in photoautotrophic cells, as a consequence of a more de-epoxidized state of the xanthophyll cycle pool and more LHCSR3 protein, showing that photosynthesis was under more pressure than in mixotrophic cells. Photoautotrophic cells had lowered α-tocopherol and β-carotene contents and a higher level of protein carbonylation, indicators of elevated 1O2 production. Levels of glutathione, glutathione peroxidase (GPX5) and glutathione-S-transferase (GST1), important antioxidants against RES, were also increased in photoautotrophic cells. In parallel to the wild-type, the LHCSR3-deficient npq4 mutant was high light-treated, which in photoautotrophic conditions exhibited particular sensitivity under elevated oxygen, the treatment that induced the highest RES levels, including acrolein. The npq4 mutant had more GPX5 and GST1 alongside higher levels of carbonylated protein and a more oxidized glutathione redox state. In wild-type cells glutathione contents doubled after 4 h treatment, either with high light under elevated oxygen or with a non-critical dose (600 ppm) of acrolein. Exogenous acrolein also increased GST1 levels, but not GPX5. Overall, RES-associated oxidative damage and glutathione metabolism are prominently associated with light stress and potentially in signaling responses of C. reinhardtii.

International audience; Carnosic acid, a phenolic diterpene specific to the Lamiaceae family, is highly abundant in rosemary (Rosmarinus officinalis). Despite numerous industrial and medicinal/pharmaceutical applications of its antioxidative features, this compound in planta and its antioxidant mechanism have received little attention, except a few studies of rosemary plants under natural conditions. In vitro analyses, using high-performance liquid chromatography-ultraviolet and luminescence imaging, revealed that carnosic acid and its major oxidized derivative, carnosol, protect lipids from oxidation. Both compounds preserved linolenic acid and monogalactosyldiacylglycerol from singlet oxygen and from hydroxyl radical. When applied exogenously, they were both able to protect thylakoid membranes prepared from Arabidopsis (Arabidopsis thaliana) leaves against lipid peroxidation. Different levels of carnosic acid and carnosol in two contrasting rosemary varieties correlated with tolerance to lipid peroxidation. Upon reactive oxygen species (ROS) oxidation of lipids, carnosic acid was consumed and oxidized into various derivatives, including into carnosol, while carnosol resisted, suggesting that carnosic acid is a chemical quencher of ROS. The antioxidative function of carnosol relies on another mechanism, occurring directly in the lipid oxidation process. Under oxidative conditions that did not involve ROS generation, carnosol inhibited lipid peroxidation, contrary to carnosic acid. Using spin probes and electron paramagnetic resonance detection, we confirmed that carnosic acid, rather than carnosol, is a ROS quencher. Various oxidized derivatives of carnosic acid were detected in rosemary leaves in low light, indicating chronic oxidation of this compound, and accumulated in plants exposed to stress conditions, in parallel with a loss of carnosic acid, confirming that chemical quenching of ROS by carnosic acid takes place in planta.

Understanding of the molecular mechanisms of photosynthetic electron and proton transports and their regulation in plants and algae in response to changes in environmental conditions is an important issue for fundamental research on photosynthesis, and may extend even to practical applications by identifying important sites for improvement of photosynthesis. The significance and often centrality of regulatory mechanisms of photosynthetic electron transport is well established for processes in plant acclimation. In recent years, significant advancements have been achieved in understanding of regulatory processes such as dissipation of excess energy in the antenna systems, state transitions, cyclic electron flow, oxygen reduction by flavodiiron enzymes and many others.

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.

The constitutive expression of the bacterial carotene desaturase (CRTI) in Arabidopsis thaliana leads to increased susceptibility of leaves to light-induced damage. Changes in the photosynthetic electron transport chain rather than alterations of the carotenoid composition in the antenna were responsible for the increased photoinhibition. A much higher level of superoxide/hydrogen peroxide was generated in the light in thylakoid membranes from the CRTI expressing lines than in wild-type while the level of singlet oxygen generation remained unchanged. The increase in reactive oxygen species was related to the activity of plastid terminal oxidase (PTOX) since their generation was inhibited by the PTOX-inhibitor octyl gallate, and since the protein level of PTOX was increased in the CRTI-expressing lines. Furthermore, cyclic electron flow was suppressed in these lines. We propose that PTOX competes efficiently with cyclic electron flow for plastoquinol in the CRTI-expressing lines and that it plays a crucial role in the control of the reduction state of the plastoquinone pool.

International audience; Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

International audience; AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. Recombinant AIR12 from Arabidopsis accepted electrons from ascorbate or superoxide, and donated electrons to either monodehydroascorbate or oxygen. AIR12 was found associated in vivo to the plasma membrane. Though linked to the membrane by a glycophosphatidylinositol anchor, AIR12 is a hydrophilic and glycosylated protein predicted to be fully exposed to the apoplast. The expression pattern of AIR12 in Arabidopsis is developmentally regulated and correlated to sites of controlled cell separation (e.g. micropilar endosperm during germination, epidermal cells surrounding the emerging lateral root) and cells around wounds. Arabidopsis (Landsberg erecta-0) mutants with altered levels of AIR12 did not show any obvious phenotype. However, AIR12-overexpressing plants accumulated ROS (superoxide, hydrogen peroxide) and lipid peroxides in leaves, indicating that AIR12 may alter the redox state of the apoplast under particular conditions. On the other hand, AIR12-knock out plants displayed a strongly decreased susceptibility to Botrytis cinerea infection, which in turn induced AIR12 expression in susceptible wild type plants. Altogether, the results suggest that AIR12 plays a role in the regulation of the apoplastic redox state and in the response to necrotrophic pathogens. Possible relationships between these functions are discussed.

In photosynthetic organisms, excess light is a stress that induces production of reactive oxygen species inside the chloroplasts. As a response, the capacity of antioxidative defence mechanisms increases. However, when cells of Chlamydomonas reinhardtii were shifted from dark to high light, a reversible partial inactivation of catalase activity was observed, which correlated with a transient increase in the level of H2 O2 in the 10 μm range. This concentration range seems to be necessary to activate H2 O2 -dependent signalling pathways stimulating the expression of H2 O2 responsive genes, such as the heat shock protein HSP22C. Catalase knock-down mutants had lost the transient accumulation of H2 O2 , suggesting that a decrease in catalase activity was the key element for establishing a transient H2 O2 burst. Catalase was inactivated by a one-electron event consistent with the reduction of a single cysteine. We propose that under high light intensity, the redox state of the photosynthetic electron transport chain is sensed and transmitted to the cytosol to regulate the catalase activity. This allows a transient accumulation of H2 O2 , inducing a signalling event that is transmitted to the nucleus to modulate the expression of chloroplast-directed protection enzymes.

Plastid-localized NADPH-dependent thioredoxin reductase C (NTRC) is a unique NTR enzyme containing both reductase and thioredoxin domains in a single polypeptide. Arabidopsis thaliana NTRC knockout lines (ntrc) show retarded growth, especially under short-day (SD) photoperiods. This study identified chloroplast processes that accounted for growth reduction in SD-acclimated ntrc. The strongest reduction in ntrc growth occurred under photoperiods with nights longer than 14 h, whereas knockout of the NTRC gene did not alter the circadian-clock-controlled growth of Arabidopsis. Lack of NTRC modulated chloroplast reactive oxygen species (ROS) metabolism, but oxidative stress was not the primary cause of retarded growth of SD-acclimated ntrc. Scarcity of starch accumulation made ntrc leaves particularly vulnerable to photoperiods with long nights. Direct interaction of NTRC and ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was confirmed by yeast two-hybrid analysis. The ntrc line was not able to maximize starch synthesis during the light period, which was particularly detrimental under SD conditions. Acclimation of Arabidopsis to SD conditions also involved an inductive rise of ROS production in illuminated chloroplasts that was not counterbalanced by the activation of plastidial anti-oxidative systems. It is proposed that knockout of NTRC challenges redox regulation of starch synthesis, resulting in stunted growth of the mutant lines acclimated to the SD photoperiod.

WOS: 000381303300003, PubMed ID: 27255485, Photosynthesis is a high-rate redox metabolic process that is subjected to rapid changes in input parameters, particularly light. Rapid transients of photon capture, electron fluxes, and redox potentials during photosynthesis cause reactive oxygen species (ROS) to be released, including singlet oxygen, superoxide anion radicals, and hydrogen peroxide. Thus, the photosynthesizing chloroplast functions as a conditional source of important redox and ROS information, which is exploited to tune processes both inside the chloroplast and, following retrograde release or processing, in the cytosol and nucleus. Analyses of mutants and comparative transcriptome profiling have led to the identification of these processes and associated players and have allowed the specificity and generality of response patterns to be defined. The release of ROS and oxidation products, envelope permeabilization (for larger molecules), and metabolic interference with mitochondria and peroxisomes produce an intricate ROS and redox signature, which controls acclimation processes. This photosynthesis-related ROS and redox information feeds into various pathways (e.g. the mitogen-activated protein kinase and OXI1 signaling pathways) and controls processes such as gene expression and translation., German Science FoundationGerman Research Foundation (DFG) [DI346, SPP1710, 1935], This work was supported by the German Science Foundation (grant nos. DI346, SPP1710, and 1935 to K.-J.D.).

International audience; Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3'-phosphoadenosine 5'-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5'-3' exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response.

International audience; The plastid terminal oxidase PTOX is a plastohydroquinone:oxygen oxidoreductase that is important for carotenoid biosynthesis and plastid development. Its role in photosynthesis is controversially discussed. Under a number of abiotic stress conditions, the protein level of PTOX increases. PTOX is thought to act as a safety valve under high light protecting the photosynthetic apparatus against photodamage. However, transformants with high PTOX level were reported to suffer from photoinhibition. To analyze the effect of PTOX on the photosynthetic electron transport, tobacco expressing PTOX-1 from Chlamydomonas reinhardtii (Cr-PTOX1) was studied by chlorophyll fluorescence, thermoluminescence, P700 absorption kinetics and CO2 assimilation. Cr-PTOX1 was shown to compete very efficiently with the photosynthetic electron transport for PQH2 . High pressure liquid chromatography (HPLC) analysis confirmed that the PQ pool was highly oxidized in the transformant. Immunoblots showed that, in the wild-type, PTOX was associated with the thylakoid membrane only at a relatively alkaline pH value while it was detached from the membrane at neutral pH. We present a model proposing that PTOX associates with the membrane and oxidizes PQH2 only when the oxidation of PQH2 by the cytochrome b6 f complex is limiting forward electron transport due to a high proton gradient across the thylakoid membrane.

The plastid terminal oxidase (PTOX) is a non-heme diiron quinol oxidase that oxidizes plastoquinol and reduced O2 to H2O. PTOX was discovered in the so-called immutans mutant of A. thaliana showing a variegated phenotype (Wetzel et al., 1994; Carol et al., 1999). PTOX is localized in the non-appressed regions of the thylakoid membrane (Lennon et al., 2003) and is involved in carotenoid biosynthesis, plastid development, and chlororespiration. Reviews have focused on the role of PTOX in chlororespiration (Bennoun, 2002; Rumeau et al., 2007), in chloroplast biogenesis (Putarjunan et al., 2013) and in stress responses (McDonald et al., 2011; Sun and Wen, 2011). A recent review by Nawrocki et al. (2015) has addressed the role of PTOX in poising the chloroplast redox potential in darkness. However, its role and interplay with the photosynthetic electron flow remains unclear. Plants grown in moderate light under non-stress conditions have low PTOX concentrations (about 1 PTOX protein per 100 PSII; Lennon et al., 2003). By contrast, elevated PTOX levels have been found in plants exposed to abiotic stresses such as high temperatures, high light and drought (Quiles, 2006), salinity (Stepien and Johnson, 2009), low temperatures and high intensities of visible (Ivanov et al., 2012), and UV light (Laureau et al., 2013). PTOX has been proposed to act as a safety valve by protecting the plastoquinone pool from overreduction under abiotic stress. A highly reduced PQ pool hinders forward electron flow and triggers charge recombination in photosystem II (PSII) leading to the generation of triplet chlorophyll and highly toxic singlet oxygen. However, overexpression of PTOX in A. thaliana did not protect against light-induced photodamage (Rosso et al., 2006) and even enhanced photo-oxidative stress in tobacco expressing, in addition to its endogenous enzyme, either PTOX from A. thaliana (Heyno et al., 2009) or PTOX1 from C. reinhardtii (Ahmad et al., 2012). Different to higher plants C. reinhardtii possesses two isoforms, PTOX1 and PTOX2. PTOX1 is most likely responsible for regenerating PQ for phytoene desaturation and shows a lower rate of plastoquinol oxidation during photosynthesis than PTOX2 (Houille-Vernes et al., 2011). Using purified PTOX, Yu and coworkers have recently shown that depending on the quinol concentration PTOX can act as an anti-oxidant or pro-oxidant (Feilke et al., 2014; Yu et al., 2014). PTOX activity was found to be pH insensitive between pH 6.0–8.5 when as substrate decylPQH2 dissolved in methanol was used (Yu et al., 2014). During the catalysis, peroxide intermediates are formed at the diiron center. Depending on the lifetime of these intermediates, reactive oxygen species (ROS) can be generated as a side reaction. Isolated PTOX generates superoxide radicals at both high, but physiologically relevant, quinol concentrations at pH 8.0 and substrate limiting concentrations at pH 6.0–6.5 (Feilke et al., 2014; Yu et al., 2014). When substrate is limited, the second quinol may not arrive in time leading to superoxide formation directly at the catalytic center. Alternatively, since at pH 8.0 the semiquinone is more stable than at pH 6.0, it is conceivable that the high pH stabilized semiquinone acts as a ROS generator. PTOX in overexpressors has also been found to generate superoxide in the light (Heyno et al., 2009). By oxidizing plastoquinol PTOX reduces the number of electrons available for photosynthetic electron flow. It is generally accepted that PTOX has low activity compared to photosynthetic electron flow. The maximum rate of PTOX was reported to be 5 e− s−1 PSII−1 for PTOX2 in C. reinhardtii and 0.3 e− s−1 PSII−1 in tomato while the maximal rate of photosynthesis is approximately 150 e− s−1 PSII−1 (Nawrocki et al., 2015). However in plants exposed to stress, PTOX activity can account for 30% of the PSII activity (Stepien and Johnson, 2009). The in vitro enzyme activity of PTOX is high when substrate concentrations are saturating (up to 19.01 ± 1.1 μmol O2 mg protein−1 min−1; Yu et al., 2014). This corresponds to a turnover rate of 320 e− s−1 PTOX−1 at 35°C, the optimum temperature for PTOX from rice. The discrepancy between the reported PTOX activities in planta and the Vmax measured with the purified protein points to a mechanism that allows the regulation of PTOX activity depending on the reduction state of the electron transport chain. Since PTOX can compete with linear and cyclic electron flow (Feilke et al., 2015) and consequently lowers NADPH, ATP production and CO2 fixation and potentially generates ROS, its activity must be tightly controlled. High activity is beneficial for the plant to protect the photosynthetic apparatus against photoinhibition when the electron transport chain is in a highly reduced state as it is the case under abiotic stress when the stomata are closed due to water stress or when CO2 fixation is limited by unfavorable temperatures. However, high PTOX activity is detrimental to high photosynthetic activity when light and CO2 are not limiting. These observations have led us to postulate the following hypothesis (Figure ​(Figure1)1) that explains the discrepancies in the literature about the safety valve function of PTOX. When stromal pH is alkaline (in high light), PTOX may become associated with the membrane giving it access to its substrate, lipophilic plastoquinol, leading to efficient oxidation of the quinol and reduction of O2 to H2O. By contrast when stroma pH becomes less alkaline (under non-saturating light conditions) PTOX may be soluble. Soluble PTOX cannot access its substrate plastoquinol that is located in the thylakoid membrane and the enzyme is effectively inactive. Activity of carotenoid biosynthesis enzymes may be regulated in a similar manner. Phytoene desaturase, which catalyzes the reaction of lipophilic phytoene to ζ-carotene, is found in the stroma both as a tetrameric membrane-bound form which has access to substrate and a soluble multi-oligomeric form in the stroma that does not (Gemmecker et al., 2015). Another example of an enzyme known to associate with the membrane in a pH-dependent manner is the violaxanthin de-epoxidase (Hager and Holocher, 1994). This enzyme associates with the thylakoid membrane when the luminal pH decreases. Figure 1 Hypothetical model of the regulation of PTOX activity by the proton gradient in higher plants. Under non-saturating light conditions linear electron transport between PSII and PSI takes place and a moderate proton gradient is established across the thylakoid ... The model of pH-dependent regulation of PTOX activity by membrane association allows us to rationalize how PTOX could act as a safety valve under conditions of stress such as drought, high light and extreme temperatures when the stomata are closed and the CO2 assimilation rate is low and the stromal pH is alkaline. Its dissociation from the membrane at less alkaline pH would hinder its competition with the photosynthetic electron chain for its substrate plastoquinol. Chlororespiration in the dark requires membrane associated PTOX. In our model, this can only take place when a proton gradient is created in the dark by hydrolysis of ATP that is either present in the chloroplast or delivered to the chloroplast from mitochondria. Additionally, when the plastoquinone pool is highly reduced, PTOX can generate superoxide, a potential signaling mechanism that causes the expression levels of responsive genes to change allowing the plant to acclimate to changes in its environment.

Cadmium (Cd) is highly toxic to plants causing growth reduction and chlorosis. It binds thiols and competes with essential transition metals. It affects major biochemical processes such as photosynthesis and the redox balance, but the connection between cadmium effects at the biochemical level and its deleterious effect on growth has seldom been established. In this study, two Cd hypersensitive mutants, cad1-3 impaired in phytochelatin synthase (PCS1), and nramp3nramp4 impaired in release of vacuolar metal stores, have been compared. The analysis combines genetics with measurements of photosynthetic and antioxidant functions. Loss of AtNRAMP3 and AtNRAMP4 function or of PCS1 function leads to comparable Cd sensitivity. Root Cd hypersensitivities conferred by cad1-3 and nramp3nramp4 are cumulative. The two mutants contrast in their tolerance to oxidative stress. In nramp3nramp4, the photosynthetic apparatus is severely affected by Cd, whereas it is much less affected in cad1-3. In agreement with chloroplast being a prime target for Cd toxicity in nramp3nramp4, the Cd hypersensitivity of this mutant is alleviated in the dark. The Cd hypersensitivity of nramp3nramp4 mutant highlights the critical role of vacuolar metal stores to supply essential metals to plastids and maintain photosynthetic function under Cd and oxidative stresses.

The redox potential of Q A in Photosystem II (PSII) from Thermosynechococcus elongatus was titrated monitoring chlorophyll fluorescence. A high potential form ( E m = +60 ± 25 mV) was found in the absence of Mn 4 Ca, the active site for water oxidation. The low potential form ( E m = −60 ± 48 mV), which is difficult to measure in conventional titration experiments, could be “locked in” by cross-linking the active enzyme. This indicates that the presence of Mn 4 Ca is relayed to the quinone site by significant structural changes in the protein. The presence of high and low potential forms agrees with what has been seen in plants, algae from our lab and in T. elongatus (Shibamoto et al., Biochemistry 48 (2009) 10682–10684). In the latter work, the potentials of Q A were shifted to lower potentials compared to other measurements. The redox potential of Q A in Mn-depleted PSII from spinach was titrated in the presence of redox mediators and the midpoint potential was shifted by 80 mV towards a more negative value compared to titrations without mediators. The lower values of the midpoint potential of the ( Q A / Q A - ) redox couple in the literature could be due to a perturbation due to a specific mediator.

Hydrogen peroxide (H(2)O(2)) is recognized as an important signalling molecule. There are two important aspects to this function: H(2)O(2) production and its diffusion to its sites of action. The production of H(2)O(2) by photosynthetic electron transport and its ability to diffuse through the chloroplast envelope membranes has been investigated using spin trapping electron paramagnetic resonance spectroscopy and H(2)O(2)-sensitive fluorescence dyes. It was found that, even at low light intensity, a portion of H(2)O(2) produced inside the chloroplasts can leave the chloroplasts thus escaping the effective antioxidant systems located inside the chloroplast. The production of H(2)O(2) by chloroplasts and the appearance of H(2)O(2) outside chloroplasts increased with increasing light intensity and time of illumination. The amount of H(2)O(2) that can be detected outside the chloroplasts has been shown to be up to 5% of the total H(2)O(2) produced inside the chloroplasts at high light intensities. The fact that H(2)O(2) produced by chloroplasts can be detected outside these organelles is an important finding in terms of understanding how chloroplastic H(2)O(2) can serve as a signal molecule.

The function of cytochrome b559 (cyt b559) in photosystem II (PSII) was studied in a tobacco mutant in which the conserved phenylalanine at position 26 in the beta-subunit was changed to serine. Young leaves of the mutant showed no significant difference in chloroplast ultra structure or in the amount and activity of PSII, while in mature leaves the size of the grana stacks and the amount of PSII were significantly reduced. Mature leaves of the mutant showed a higher susceptibility to photoinhibition and a higher production of singlet oxygen, as shown by spin trapping electron paramagnetic resonance (EPR) spectroscopy. Oxygen consumption and superoxide production were studied in thylakoid membranes in which the Mn cluster was removed to ensure that all the cyt b559 was present in its low potential form. In thylakoid membranes, from wild-type plants, the larger fraction of superoxide production was 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive. This type of superoxide formation was absent in thylakoid membranes from the mutant. The physiological importance of the plastoquinol oxidation by cyt b559 for photosynthesis is discussed.

Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical ((OH)-O-center dot)-mediated cell wall loosening during plant seed germination and seedling growth. We used electron paramagnetic resonance spectroscopy to show that (OH)-O-center dot is generated in the cell wall during radicle elongation and weakening of the endosperm of cress (Lepidium sativum; Brassicaceae) seeds. Endosperm weakening precedes radicle emergence, as demonstrated by direct biomechanical measurements. By H-3 fingerprinting, we showed that wall polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic (OH)-O-center dot in radicles and endosperm caps: the production and action of (OH)-O-center dot increased during endosperm weakening and radicle elongation and were inhibited by the germination-inhibiting hormone abscisic acid. Both effects were reversed by gibberellin. Distinct and tissue-specific target sites of (OH)-O-center dot attack on polysaccharides were evident. In vivo (OH)-O-center dot attack on cell wall polysaccharides were evident not only in germinating seeds but also in elongating maize (Zea mays; Poaceae) seedling coleoptiles. We conclude that plant cell wall loosening by (OH)-O-center dot is a controlled action of this type of reactive oxygen species.

A specific signaling role for H(2)O(2) in Chlamydomonas reinhardtii was demonstrated by the definition of a promoter that specifically responded to this ROS. Expression of a nuclear-encoded reporter gene driven by this promoter was shown to depend not only on the level of exogenously added H(2)O(2) but also on light. In the dark, the induction of the reporter gene by H(2)O(2) was much lower than in the light. This lower induction was correlated with an accelerated disappearance of H(2)O(2) from the culture medium in the dark. Due to a light-induced reduction in catalase activity, H(2)O(2) levels in the light remained higher. Photosynthetic electron transport mediated the light-controlled down-regulation of the catalase activity since it was prevented by 3-(3'4'-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. In the presence of light and DCMU, expression of the reporter gene was low while the addition of aminotriazole, a catalase inhibitor, led to a higher induction of the reporter gene by H(2)O(2) in the dark. The role of photosynthetic electron transport and thioredoxin in this regulation was investigated by using mutants deficient in photosynthetic electron flow and by studying the correlation between NADP-malate dehydrogenase and catalase activities. It is proposed that, contrary to expectations, a controlled down-regulation of catalase activity occurs upon a shift of cells from dark to light. This down-regulation apparently is necessary to maintain a certain level of H(2)O(2) required to activate H(2)O(2)-dependent signaling pathways.

* Cadmium (Cd(2+)) is an environmental pollutant that causes increased reactive oxygen species (ROS) production. To determine the site of ROS production, the effect of Cd(2+) on ROS production was studied in isolated soybean (Glycine max) plasma membranes, potato (Solanum tuberosum) tuber mitochondria and roots of intact seedlings of soybean or cucumber (Cucumis sativus). * The effects of Cd(2+) on the kinetics of superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and hydroxyl radical ((*OH) generation were followed using absorption, fluorescence and spin-trapping electron paramagnetic resonance spectroscopy. * In isolated plasma membranes, Cd(2+) inhibited O2*- production. This inhibition was reversed by calcium (Ca(2+)) and magnesium (Mg(2+)). In isolated mitochondria, Cd(2+) increased and H(2)O(2) production. In intact roots, Cd(2+) stimulated H(2)O(2) production whereas it inhibited O2*- and (*)OH production in a Ca(2+)-reversible manner. * Cd(2+) can be used to distinguish between ROS originating from mitochondria and from the plasma membrane. This is achieved by measuring different ROS individually. The immediate (or= 1 h) consequence of exposure to Cd(2+) in vivo is stimulation of ROS production in the mitochondrial electron transfer chain and inhibition of NADPH oxidase activity in the plasma membrane.

A reporter system for the assay of reactive oxygen species (ROS) was developed in Chlamydomonas reinhardtii, a plant model organism well suited for the application of inhibitors and generators of various types of ROS. This system employs various HSP70A promoter segments fused to a Renilla reniformis luciferase gene as a reporter. Transformants with the complete HSP70A promoter were inducible by both hydrogen peroxide and singlet oxygen. Constructs that lacked upstream heat-shock elements (HSEs) were inducible by hydrogen peroxide, indicating that this induction does not require such HSEs. Rather, downstream elements located between positions -81 to -149 with respect to the translation start site appear to be involved. In contrast, upstream sequences are essential for the response to singlet oxygen. Thus, activation by singlet oxygen appears to require promoter elements that are different from those used by hydrogen peroxide. ROS generated endogenously by treatment of the alga with metronidazole, protoporphyrin IX, dinoterb or high light intensities were detected by this reporter system, and distinguished as production of hydrogen peroxide (metronidazole) and singlet oxygen (protoporphyrin IX, dinoterb, high light). This system thus makes it possible to test whether, under varying environmental conditions including the application of abiotic stress, hydrogen peroxide or singlet oxygen or both are produced.

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

The molecular defense mechanisms against photooxidative stress in photosynthetic organisms are essential to protect cells from damaging effects of high light illumination and photoinhibition but also to protect against effects by endogenous and exogenous photosensitizers. Here, we analyzed the genetic response of Chlamydomonas reinhardtii to the model type I photosensitizer neutral red (NR) and the type II photosensitizer rose bengal (RB) using DNA-microarrays. Many oxidative and general stress response genes, which were also induced by other oxidative stress conditions, were strongly induced by NR. Only one gene was upregulated by RB, the glutathione (GSH) peroxidase homologous gene Gpxh , which was also induced by NR. In addition NR exposure resulted in the reduced expression of most nuclear photosynthetic genes and subunits of the light harvesting complex (LHC) indicating an effect on the photosynthetic activity. This is supported by a stimulation of singlet oxygen generation in NR-treated thylakoids. Thus, in C. reinhardtii the Gpxh expression is most probably induced by the formation of singlet oxygen in both the NR and RB-treated cells via the activation of a very sensitive and specific sensor, whereas general oxidative stress response mechanisms seem to be involved in the response of most other genes to the type I photooxidative stress.

The connection between the mode of toxic action and the genetic response caused by the type I photosensitizer and photosynthesis inhibitor neutral red (NR) and the type II photosensitizer rose bengal (RB) was investigated in the green alga Chlamydomonas reinhardtii. For both photosensitizers, a light intensity-dependent increase in toxicity and expression of the glutathione peroxidase homologous gene (Gpxh) was found. The toxicity of RB was reduced by the singlet oxygen (1O2) quenchers 1,4-diazabicyclo[2.2.2]octane and L-histidine, and the RB-induced Gpxh expression was stimulated in deuterium oxide-supplemented growth medium. These observations clearly indicate the involvement of 1O2 in both toxicity and the genetic response caused by RB. NR up-regulated the expression of typical oxidative and general stress response genes, probably by a type I mechanism, and also strongly induced the Gpxh expression. The stimulating effect of deuterium oxide in the growth medium suggested the involvement of 1O2 also in the NR-induced response. Indeed, an increased 1O2 formation was detected with EPR-spin trapping in NR-treated spinach thylakoids. However, none of the 102 quenchers could reduce the light-dependent toxicity of NR in C. reinhardtii, indicating that NR has a different mode of toxic action than RB.

Alb3 homologs Oxa1 and YidC have been shown to be required for the integration of newly synthesized proteins into membranes. Here, we show that although Alb3.1p is not required for integration of the plastid-encoded photosystem II core subunit D1 into the thylakoid membrane of Chlamydomonas reinhardtii, the insertion of D1 into functional photosystem II complexes is retarded in the Alb3.1 deletion mutant ac29. Alb3.1p is associated with D1 upon its insertion into the membrane, indicating that Alb3.1p is essential for the efficient assembly of photosystem II. Furthermore, levels of nucleus-encoded light-harvesting proteins are vastly reduced in ac29; however, the remaining antenna systems are still connected to photosystem II reaction centers. Thus, Alb3.1p has a dual function and is required for the accumulation of both nucleus- and plastid-encoded protein subunits in photosynthetic complexes of C. reinhardtii.

Myriophyllum spicatum (Haloragaceae) is a highly competitive freshwater macrophyte that produces and releases algicidal and cyanobactericidal polyphenols. Among them, β-1,2,3-tri-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-d-glucose (tellimagrandin II) is the major active substance and is an effective inhibitor of microalgal exoenzymes. However, this mode of action does not fully explain the strong allelopathic activity observed in bioassays. Lipophilic extracts of M. spicatum inhibit photosynthetic oxygen evolution of intact cyanobacteria and other photoautotrophs. Fractionation of the extract provided evidence for tellimagrandin II as the active compound. Separate measurements of photosystem I and II activity with spinach (Spinacia oleracea) thylakoid membranes indicated that the site of inhibition is located at photosystem II (PSII). In thermoluminescence measurements with thylakoid membranes and PSII-enriched membrane fragments M. spicatum extracts shifted the maximum temperature of the B-band (S2QB −recombination) to higher temperatures. Purified tellimagrandin II in concentrations as low as 3 μm caused a comparable shift of the B-band. This demonstrates that the target site of this inhibitor is different from the QB-binding site, a common target of commercial herbicides like 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Measurements with electron paramagnetic resonance spectroscopy suggest a higher redox midpoint potential for the non-heme iron, located between the primary and the secondary quinone electron acceptors, QA and QB. Thus, tellimagrandin II has at least two modes of action, inhibition of exoenzymes and inhibition of PSII. Multiple target sites are a common characteristic of many potent allelochemicals.