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Plastid terminal oxidase (PTOX) accepts electrons from plastoquinol to reduce molecular oxygen to water. We introduced the gene encoding Chlamydomonas reinhardtii (Cr)PTOX2 into the Arabidopsis (Arabidopsis thaliana) wild-type (WT) and proton gradient regulation5 (pgr5) mutant defective in cyclic electron transport around photosystem I (PSI). The accumulation of CrPTOX2 only mildly affected photosynthetic electron transport in the WT background during steady-state photosynthesis but partly complemented the induction of nonphotochemical quenching (NPQ) in the pgr5 background. During the induction of photosynthesis by actinic light (AL) of 130 µmol photons m−2 s−1, the high level of PSII yield (Y(II)) was induced immediately after the onset of AL in WT plants accumulating CrPTOX2. NPQ was more rapidly induced in the transgenic plants than in WT plants. P700 was also oxidized immediately after the onset of AL. Although CrPTOX2 does not directly induce a proton concentration gradient (ΔpH) across the thylakoid membrane, the coupled reaction of PSII generated ΔpH to induce NPQ and the downregulation of the cytochrome b6f complex. Rapid induction of Y(II) and NPQ was also observed in the pgr5 plants accumulating CrPTOX2. In contrast to the WT background, P700 was not oxidized in the pgr5 background. Although the thylakoid lumen was acidified by CrPTOX2, PGR5 was essential for oxidizing P700. In addition to acidification of the thylakoid lumen to downregulate the cytochrome b6f complex (donor-side regulation), PGR5 may be required for draining electrons from PSI by transferring them to the plastoquinone pool. We propose a reevaluation of the contribution of this acceptor-side regulation by PGR5 in the photoprotection of PSI.

We report here a detailed analysis of the proteome adjustments that accompany chromoplast differentiation from chloroplasts during bell-pepper fruit ripening. While the two photosystems are disassembled and their constituents degraded, the cytochrome b6f complex, the ATPase complex as well as Calvin cycle enzymes are maintained at high levels up to fully mature chromoplasts. This is also true for ferredoxin (Fd) and Fd-dependent NADP reductase, suggesting that ferredoxin retains a central role in the chromoplasts redox metabolism. There is a significant increase in the amount of enzymes of the typical metabolism of heterotrophic plastids such as the oxidative pentose phosphate pathway (OPPP), amino acid and fatty acid biosynthesis. Enzymes of chlorophyll catabolism and carotenoid biosynthesis increase in abundance, supporting the pigment reorganization that goes together with chromoplast differentiation. The majority of plastid encoded proteins declines but constituents of the plastid ribosome and AccD increase in abundance. Furthermore, the amount of plastid terminal oxidase (PTOX) remains unchanged despite a significant increase in phytoene desaturase (PDS) levels, suggesting that the electrons from phytoene desaturation may be consumed by another oxidase. This may be a particularity of non-climacteric fruits such as bell pepper, that lack a respiratory burst at the onset of fruit ripening.

The green alga Chlorella ohadii was isolated from a desert biological soil crust, one of the harshest environments on Earth. When grown under optimal laboratory settings it shows the fastest growth rate ever reported for a photosynthetic eukaryote and a complete resistance to photodamage even under unnaturally high light intensities. Here we examined the energy distribution along the photosynthetic pathway under four light and carbon regimes. This was performed using various methodologies such as membrane inlet mass spectrometer with stable O2 isotopes, variable fluorescence, electrochromic shift and fluorescence assessment of NADPH level, as well as the use of specific inhibitors. We show that the preceding illumination and CO2 level during growth strongly affect the energy dissipation strategies employed by the cell. For example, plastid terminal oxidase (PTOX) plays an important role in energy dissipation, particularly in high light- and low-CO2-grown cells. Of particular note is the reliance on PSII cyclic electron flow as an effective and flexible dissipation mechanism in all conditions tested. The energy management observed here may be unique to C. ohadii, as it is the only known organism to cope with such conditions. However, the strategies demonstrated may provide an insight into the processes necessary for photosynthesis under high-light conditions.

Disulfide-based regulation links the activity of numerous chloroplast proteins with photosynthesis-derived redox signals. The plastid terminal oxidase (PTOX) is a thylakoid-bound plastoquinol oxidase that has been implicated in multiple roles in the light and in the dark, which could require different levels of PTOX activity. Here we show that Arabidopsis PTOX contains a conserved C-terminus domain (CTD) with cysteines that evolved progressively following the colonization of the land by plants. Furthermore, the CTD contains a regulatory disulfide that is in the oxidized state in the dark and is rapidly reduced, within 5 min, in low light intensity (1-5 µE m-2 sec-1 ). The reduced PTOX form in the light was reoxidized within 15 min after transition to the dark. Mutation of the cysteines in the CTD prevented the formation of the oxidized form. This resulted in higher levels of reduced plastoquinone when measured at transition to the onset of low light. This is consistent with the reduced state of PTOX exhibiting diminished PTOX oxidase activity under conditions of limiting PQH2 substrate. Our findings suggest that AtPTOX-CTD evolved to provide light-dependent regulation of PTOX activity for the adaptation of plants to terrestrial conditions.

The element arsenic (As) is a non-essential metalloid that is found naturally in all soils and at high concentrations it is toxic to plant cells. As (V) can act as a chemical analogue of phosphate, it can disrupt phosphate-related energy metabolism and lipid structure. in this study, the contribution of mitochondrial alternative oxidase (AOX) and chloroplastic plastid terminal oxidase (PTOX) to As (V) stress tolerance was investigated. Our data indicate that As (V) stress (100, 200 and 300 mu M) induces AOX gene expression by 3.3- to 10.5-fold depending on AOX gene, but not PTOX expression in wild-type A. thaliana plants. To further elucidate the role of AOX in As (V) stress tolerance, we utilized aox1a mutants and observed that aox1a mutants had decreased growth and higher oxidative stress damage under stress conditions, while there were no differences under control conditions. Moreover, acclimation of aox1a plants to new cellular redox environment was investigated by measuring the activities of reactive oxygen species (ROS)-scavenging enzymes. Induction of mitochondrial MnSOD activity at 300 mu M As (V) was higher in aox1a plants (70%), when compared to wild type (43%). However, total ascorbate peroxidase and dehydroascorbate reductase activities were lower in aox1a plants when compared to wild type, which might explain higher oxidative damage observed in this genotype. on the other hand, NADPH oxidase activity, which is involved in ROS signaling, was lower in aox1a plants under normal conditions but a higher induction was observed with As (V) stress. Overall, our data indicate that AOX1a is involved in adaptation to As (V)-induced oxidative stress., Korean Acad Sci & Technol, Assoc Acad & Sco Sci Asia

Plastid terminal oxidase (PTOX) is a chloroplast enzyme that catalyzes oxidation of plastoquinol (PQH2) and reduction of molecular oxygen to water. Its function has been associated with carotenoid biosynthesis, chlororespiration and environmental stress responses in plants. In the majority of plant species, a single gene encodes the protein and little is known about events of PTOX gene duplication and their implication to plant metabolism. Previously, two putative PTOX (PTOX1 and 2) genes were identified in Glycine max, but the evolutionary origin and the specific function of each gene was not explored. Phylogenetic analyses revealed that this gene duplication occurred apparently during speciation involving the Glycine genus ancestor, an event absent in all other available plant leguminous genomes. Gene expression evaluated by RT-qPCR and RNA-seq data revealed that both PTOX genes are ubiquitously expressed in G. max tissues, but their mRNA levels varied during development and stress conditions. In development, PTOX1 was predominant in young tissues, while PTOX2 was more expressed in aged tissues. Under stress conditions, the PTOX transcripts varied according to stress severity, i.e., PTOX1 mRNA was prevalent under mild or moderate stresses while PTOX2 was predominant in drastic stresses. Despite the high identity between proteins (97%), molecular docking revealed that PTOX1 has higher affinity to substrate plastoquinol than PTOX2. Overall, our results indicate a functional relevance of this gene duplication in G. max metabolism, whereas PTOX1 could be associated with chloroplast effectiveness and PTOX2 to senescence and/or apoptosis.

During photosynthesis, organisms respond to their energy demand and ensure the supply of energy and redox equivalents that sustain metabolism. Hence, the photosynthetic apparatus can, and in fact should, be treated as an integrated supply-demand system. Any imbalance in the energy produced and consumed can lead to adverse reactions, such as the production of reactive oxygen species (ROS). Reaction centres of both photosystems are known sites of ROS production. Here, we investigate in particular the central role of Photosystem I (PSI) in this tightly regulated system. Using a computational approach we have expanded a previously published mechanistic model of C3 photosynthesis by including ROS producing and scavenging reactions around PSI. These include two water to water reactions mediated by Plastid terminal oxidase (PTOX) and Mehler and the ascorbate-glutathione (ASC-GSH) cycle, as a main non-enzymatic antioxidant. We have used this model to predict flux distributions through alternative electron pathways under various environmental stress conditions by systematically varying light intensity and enzymatic activity of key reactions. In particular, we studied the link between ROS formation and activation of pathways around PSI as potential scavenging mechanisms. This work shines light on the role of alternative electron pathways in photosynthetic acclimation and investigates the effect of environmental perturbations on PSI activity in the context of metabolic productivity.

The diatom Stauroneis sp. was previously identified as a promising source of fucoxanthin and omega-3 oils. Methyl jasmonate (MJ) supplementation is known to enhance metabolite yields in this species without impacting on growth or photosynthesis. Therefore, a label-free proteomics approach was undertaken to further evaluate the functional role of MJ on the diatom's physiology. Of the twenty cultivation regimes were screened, Uf/2 medium with green+white LED's induced the greatest metabolic response when exposed to 10 μM MJ treatment. These conditions significantly enhanced the pigment and total cellular lipids contents. The increase in fucoxanthin correlating with a 20% increase in Trolox reducing equivalent in the total antioxidant assay, indicating a non-enzymatic antioxidant role of fucoxanthin to mitigate the detrimental effects of a redox imbalance within chloroplasts. The proteomics identified 197 proteins up-regulated 48 h after MJ exposure including cell signalling cascades, photosynthetic processes, carbohydrate metabolism, lipid biosynthesis and chloroplast biogenesis. MJ strengthened the dark reactions of photosynthesis to support growth and metabolite fluxes. The MJ-induced ER stress protein triggered lipid body production, facilitating metabolite turnover and trafficking between cellular organelles. Plastid terminal oxidase and glutamate 1-semialdehyde 2,1-aminomutase may act as MJ-induced ROS responsive regulatory switch to support chloroplast biosynthesis. Significance statement Phytohormones represents a promising tool to enhance the high-value metabolite yields in plants and algae, however little is known of the role of methyl jasmonate in diatoms at a molecular level. A shotgun proteomics approach was undertaken to determine the influence of MJ on the diatom's cellular physiology in the marine diatom Stauroneis sp., revealing a signal transduction cascade leading to increased lipid and pigment content and identified promising targets for genetic engineering.

International audience; Photosynthesis has to work efficiently in contrasting environments such as in shade and full sun. Rapid changes in light intensity and over-reduction of the photosynthetic electron transport chain cause production of reactive oxygen species, which can potentially damage the photosynthetic apparatus. Thus, to avoid such damage, photosynthetic electron transport is regulated on many levels, including light absorption in antenna, electron transfer reactions in the reaction centers, and consumption of ATP and NADPH in different metabolic pathways. Many regulatory mechanisms involve the movement of protein-pigment complexes within the thylakoid membrane. Furthermore, a certain number of chloroplast proteins exist in different oligomerization states, which temporally associate to the thylakoid membrane and modulate their activity. This review starts by giving a short overview of the lipid composition of the chloroplast membranes, followed by describing supercomplex formation in cyclic electron flow. Protein movements involved in the various mechanisms of non-photochemical quenching, including thermal dissipation, state transitions and the photosystem II damage–repair cycle are detailed. We highlight the importance of changes in the oligomerization state of VIPP and of the plastid terminal oxidase PTOX and discuss the factors that may be responsible for these changes. Photosynthesis-related protein movements and organization states of certain proteins all play a role in acclimation of the photosynthetic organism to the environment.

Alternative oxidase (AOX) and plastid terminal oxidase (PTOX) are terminal oxidases of electron transfer in mitochondria and chloroplasts, respectively. Here, taking advantage of the variegation phenotype of the Arabidopsis PTOX deficient mutant (im), we examined the functional relationship between PTOX and its five distantly related homologs (AOX1a, 1b, 1c, 1d, and AOX2). When engineered into chloroplasts, AOX1b, 1c, 1d, and AOX2 rescued the im defect, while AOX1a partially suppressed the mutant phenotype, indicating that AOXs could function as PQH2 oxidases. When the full length AOXs were overexpressed in im, only AOX1b and AOX2 rescued its variegation phenotype. In vivo fluorescence analysis of GFP-tagged AOXs and subcellular fractionation assays showed that AOX1b and AOX2 could partially enter chloroplasts while AOX1c and AOX1d were exclusively present in mitochondria. Surprisingly, the subcellular fractionation, but not the fluorescence analysis of GFP-tagged AOX1a, revealed that a small portion of AOX1a could sort into chloroplasts. We further fused and expressed the targeting peptides of AOXs with the mature form of PTOX in im individually; and found that targeting peptides of AOX1a, AOX1b, and AOX2, but not that of AOX1c or AOX1d, could direct PTOX into chloroplasts. It demonstrated that chloroplast-localized AOXs, but not mitochondria-localized AOXs, can functionally compensate for the PTOX deficiency in chloroplasts, providing a direct evidence for the functional relevance of AOX and PTOX, shedding light on the interaction between mitochondria and chloroplasts and the complex mechanisms of protein dual targeting in plant cells.

BackgroundIncreasingly, researchers use protein-coding genes from targeted PCR amplification or direct metagenomic sequencing in community and population ecology. Analysis of protein-coding genes presents different challenges from those encountered in traditional SSU rRNA studies. Most protein-coding sequences are annotated based on homology to other computationally-annotated sequences, which can lead to inaccurate annotations. Therefore, the results of sensitive homology searches must be validated to remove false-positives and assess functionality. Multiple lines of in silico evidence can be gathered by examining conserved domains and residues identified through biochemical investigations. However, manually validating sequences in this way can be time consuming and error prone, especially in large environmental studies.ResultsAn automated pipeline for protein active site validation (PASV) was developed to improve validation and partitioning accuracy for protein-coding sequences, combining multiple sequence alignment with expert domain knowledge. PASV was tested using commonly misannotated proteins: ribonucleotide reductase (RNR), alternative oxidase (AOX), and plastid terminal oxidase (PTOX). PASV partitioned 9,906 putative Class I alpha and Class II RNR sequences from bycatch in a global viral metagenomic investigation with >99% true positive and true negative rates. PASV predicted the class of 2,579 RNR sequences in >98% agreement with manual annotations. PASV correctly partitioned all 336 tested AOX and PTOX sequences.ConclusionsPASV provides an automated and accurate way to address post-homology search validation and partitioning of protein-coding marker genes. Source code is released under the MIT license and is found with documentation and usage examples on GitHub at https://github.com/mooreryan/pasv.

Melatonin plays an active role in neutralizing free radicals, especially by triggering the defense system and certain enzymes that work under stress in both mammals and plant systems. Exposure to ultraviolet (UV–B) stress can be deadly for plants since UV-B can induce production of reactive oxygen species and damage nucleic acids. In the present study, to uncover the possible alleviative role of melatonin against UV-B stress, Arabidopsis thaliana plants were treated with melatonin (10 μM) and were exposed to UV-B stress for 90 min and 180 min (46 and 92 kJ m−2 d−1). Plants treated with melatonin had lower lipid peroxidation levels and higher Fv/Fm values at both time points. UV-B stress-induced activities of superoxide dismutase (SOD), glutathione reductase (GR) and ascorbate peroxidase (APX), but no additional induction was observed in melatonin treated groups. Moreover, melatonin differentially regulated the expression of glutathione peroxidase 2 (GPX2) and GPX7 genes under UV-B stress. Melatonin treatment did not have any effect on glutathione biosynthesis and catabolism genes. However, expression of alternative oxidase 1a (AOX1a) and AOX1d were lower in UV-B + melatonin treated plants when compared to only UV-B treated plants, which indicates lower oxidative load in mitochondria.

An improvement in photosynthetic rate promotes the growth of crop plants. The sink-regulation of photosynthesis is crucial in optimizing nitrogen fixation and integrating it with carbon balance. Studies on these processes are essential in understanding growth inhibition in plants with ammonium ((Formula presented.)) syndrome. Hence, we sought to investigate the effects of using nitrogen sources with different states of reduction (during assimilation of (Formula presented.) versus (Formula presented.)) on the photosynthetic performance of Arabidopsis thaliana. Our results demonstrated that photosynthetic functioning during long-term (Formula presented.) nutrition was not disturbed and that no indication of photoinhibition of PSII was detected, revealing the robustness of the photosynthetic apparatus during stressful conditions. Based on our findings, we propose multiple strategies to sustain photosynthetic activity during limited reductant utilization for (Formula presented.) assimilation. One mechanism to prevent chloroplast electron transport chain overreduction during (Formula presented.) nutrition is for cyclic electron flow together with plastid terminal oxidase activity. Moreover, redox state in chloroplasts was optimized by a dedicated type II NAD(P)H dehydrogenase. In order to reduce the amount of energy that reaches the photosynthetic reaction centers and to facilitate photosynthetic protection during (Formula presented.) nutrition, non-photochemical quenching (NPQ) and ample xanthophyll cycle pigments efficiently dissipate excess excitation. Additionally, high redox load may be dissipated in other metabolic reaA ctions outside of chloroplasts due to the direct export of nucleotides through the malate/oxaloacetate valve. Mitochondrial alternative pathways can downstream support the overreduction of chloroplasts. This mechanism correlated with the improved growth of A. thaliana with the overexpression of the alternative oxidase 1a (AOX1a) during (Formula presented.) nutrition. Most remarkably, our findings demonstrated the capacity of chloroplasts to tolerate (Formula presented.) syndrome instead of providing redox poise to the cells. (Less)

Whereas photosynthetic function under steady-state light conditions has been well characterized, little is known about its changes that occur in response to light fluctuations. Chlororespiration, a simplified respiratory chain, is widespread across all photosynthetic lineages, but its role remains elusive. Here, we show that chlororespiration plays a crucial role in intermittentlight conditions in the green alga Chlamydomonas reinhardtii. Chlororespiration, which is localized in thylakoid membranes together with the photosynthetic electron transfer chain, involves plastoquinone reduction and plastoquinol oxidation by a Plastid Terminal Oxidase (PTOX). We show that PTOX activity is critical for growth under intermittent light, with severe growth defects being observed in a mutant lacking PTOX2, the major plastoquinol oxidase. We demonstrate that the hampered growth results from a major change in the kinetics of redox relaxation of the photosynthetic electron transfer chain during the dark periods. This change, in turn, has a dramatic effect on the physiology of photosynthesis during the light periods, notably stimulating cyclic electron flow at the expense of the linear electron flow.

The present work reports reactions of plastoquinol (PQH2-9) and plastoquinone (PQ-9) in organic solvents and summarizes the literature to understand similar reactions in thylakoids. In thylakoids, PQH2-9 is oxidized by the cytochrome b6/f complex (Cyt b6/f) but some PQH2-9 is also oxidized by reactions in which oxygen acts as an electron acceptor and is converted to reactive oxygen species (ROS). Furthermore, PQH2-9 reacts with ROS. Light enhances oxygen-dependent oxidation of PQH2-9. We examined the oxidation of PQH2-9 via dismutation of PQH2-9 and PQ-9 and scavenging of the superoxide anion radical (O2 −) and hydrogen peroxide (H2O2) by PQH2-9. Oxidation of PQH2-9 via dismutation to semiquinone was slow and independent of pH in organic solvents and in solvent/buffer systems, suggesting that intramembraneous oxidation of PQH2-9 in darkness mainly proceeds via reactions catalyzed by the plastid terminal oxidase and cytochrome b559. In the light, oxidation of PQH2-9 by singlet oxygen and by O2 − formed in PSI contribute significantly. In addition, Cyt b6/f forms H2O2 with a PQH2-9 dependent mechanism. Measurements of the reaction of O2 − with PQH2-9 and PQ-9 in acetonitrile showed that O2 − oxidizes PQH2-9, forming PQ-9 and several PQ-9-derived products. The rate constant of the reaction between PQH2-9 and O2 − was found to be 104 M−1 s−1. H2O2 was found to oxidize PQH2-9 to PQ-9, but failed to oxidize all PQH2-9, suggesting that the oxidation of PQH2-9 by H2O2 proceeds via deprotonation mechanisms producing PQH−-9, PQ2−-9 and the protonated hydrogen peroxide cation, H3O2+.

The plastid terminal oxidase (PTOX) is a plastohydroquinone:oxygen oxidoreductase that shares structural similarities with alternative oxidases (AOXs). Multiple roles have been attributed to PTOX, such as involvement in carotene desaturation, a safety valve function, participation in the processes of chlororespiration, and setting the redox poise for cyclic electron transport. PTOX activity has been previously shown to depend on its localization at the thylakoid membrane. Here we investigate the dynamics of PTOX localization dependent on the proton motive force. Infiltrating illuminated leaves with uncouplers led to a partial dissociation of PTOX from the thylakoid membrane. In vitro reconstitution experiments showed that the attachment of purified recombinant maltose-binding protein (MBP)–OsPTOX to liposomes and isolated thylakoid membranes was strongest at slightly alkaline pH values in the presence of lower millimolar concentrations of KCl or MgCl2. In Arabidopsis thaliana overexpressing green fluorescent protein (GFP)–PTOX, confocal microscopy images showed that PTOX formed distinct spots in chloroplasts of dark-adapted or uncoupler-treated leaves, while the protein was more equally distributed in a network-like structure in the light. We propose a dynamic PTOX association with the thylakoid membrane depending on the presence of a proton motive force.

The evolution of oxygenic photosynthesis enabled organisms to use sunlight as an energy source, allowing them to colonize new niches. At the same time, life (as we know it) places severe constraints on photosynthesis. For example, the initial reactions of photosynthesis involve highly energetic intermediates that, if not controlled, can generate highly toxic side products (especially reactive oxygen species, ROS), that can damage other essential components of the organisms it powers. Photosynthesis must therefore be tightly regulated to balance the need for efficient energy conversion with the necessity of avoiding photodamage (Gust D, Kramer D, Moore A, Moore T, Vermaas W, Mater Res Bull 33:383–389, 2008). A related constraint on photosynthesis is the need to precisely balance how much energy is stored in ATP and NADPH to precisely meet biochemical demands. If this balancing does not occur, the system will fail, leading to photodamage (Kramer DM, Evans JR, Plant Physiol 155:70–78, 2011). Consideration of these requirements is essential for efforts to improve the efficiency of photosynthesis by introducing CO2 concentrating mechanisms, altering metabolism or biosynthetic pathways to shunt energy to alternative products (Kramer DM, Evans JR, Plant Physiol 155:70–78, 2011). These balancing processes must be extremely robust to contend with the rapid and unpredictable fluctuations in environmental conditions and metabolic demands that occur in nature. A large body of work has come from model systems, especially terrestrial higher plants and the green alga Chlamydomonas reinhardtii, leading to a model for the regulation of light reactions that involves 1) sensing of the pH gradient component of the thylakoid proton motive force (pmf), and 2) the redox state of the plastoquinone- and stromal pools. Over the short term, these sensors trigger regulation of light capture by altering the activity of ATP synthase leading to adjustments in lumen pH, which fine tunes light capture through nonphotochemical quenching (NPQ) and control of electron flow by adjusting the rate of PQH2 oxidation at the b6f complex. Simultaneously, this system controls the balance of ATP/NADPH by adjusting electron flux to linear and cyclic electron flow pathways to balance ATP/NADPH. This integrated “pmf paradigm” model explains much of the existing data on plants and green algae, but may not extend to other diverse organisms. This review considers how advances in our understanding of photosynthesis over the past 7–8 years, particularly in the discoveries of diverse biochemical/biophysical mechanisms in aquatic photosynthetic species, affects the view of energy balance, including the shunting of electrons to O2 through the flavodiiron proteins (FLV), the plastid terminal oxidase, the dissipation of electric field by ion movements, and the activation of alternative electron sinks. We will introduce the basic model that has been developed for higher plant chloroplasts, then contrast these with selected aquatic systems, focusing on how the differences impact the needs to re-balance both energy input and its partitioning into energy currencies.

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.

The plastid terminal oxidase (PTOX) - an interfacial diiron carboxylate protein found in the thylakoid membranes of chloroplasts - oxidizes plastoquinol and reduces molecular oxygen to water. It is believed to play a physiologically important role in the response of some plant species to light and salt (NaCl) stress by diverting excess electrons to oxygen thereby protecting photosystem II (PSII) from photodamage. PTOX is therefore a candidate for engineering stress tolerance in crop plants. Previously, we used chloroplast transformation technology to over express PTOX1 from the green alga Chlamydomonas reinhardtii in tobacco (generating line Nt-PTOX-OE). Contrary to expectation, growth of Nt-PTOX-OE plants was more sensitive to light stress. Here we have examined in detail the effects of PTOX1 on photosynthesis in Nt-PTOX-OE tobacco plants grown at two different light intensities. Under 'low light' (50 μmol photons m-2 s-1) conditions, Nt-PTOX-OE and WT plants showed similar photosynthetic activities. In contrast, under 'high light' (125 μmol photons m-2 s-1) conditions, Nt-PTOX-OE showed less PSII activity than WT while photosystem I (PSI) activity was unaffected. Nt-PTOX-OE grown under high light also failed to increase the chlorophyll a/b ratio and the maximum rate of CO2 assimilation compared to low-light grown plants, suggesting a defect in acclimation. In contrast, Nt-PTOX-OE plants showed much better germination, root length, and shoot biomass accumulation than WT when exposed to high levels of NaCl and showed better recovery and less chlorophyll bleaching after NaCl stress when grown hydroponically. Overall, our results strengthen the link between PTOX and the resistance of plants to salt stress.

Flower colour and colour patterns are crucial traits for ornamental species, thus, a comprehensive understanding of their genetic basis is extremely significant for plant breeders. The tulip tree (Liriodendron tulipifera Linn.) is well known for its flowers, odd leave shape and tree form. However, the genetic basis of its colour inheritance remains unknown. In this study, a putative plastid terminal oxidase gene (LtuPTOX) was identified from L. tulipifera based on multiple databases of differentially expressed genes at various developmental stages. Then, the full-length cDNA of LtuPTOX was derived from tepals and leaves using RACE (rapid amplification of cDNA ends) approaches. Furthermore, gene structure and phylogenetic analyses of PTOX as well as AOXs (alternative oxidases), another highly similar homologue in the AOX family, were used to distinguish between the two subfamilies of genes. In addition, transient transformation and qPCR methods were used to determine the subcellular localization and tissue expression pattern of the LtuPTOX gene. Moreover, the expression of LtuPTOX as well as pigment contents was investigated to illustrate the function of this gene during the formation of orange bands on petals. The results showed that the LtuPTOX gene encodes a 358-aa protein that contains a complete AOX domain (PF01786). Accordingly, the Liriodendron PTOX and AOX genes were identified as only paralogs since they were rather similar in sequence. LtuPTOX showed chloroplast localization and was expressed in coloured organs such as petals and leaves. Additionally, an increasing pattern of LtuPTOX transcripts leads to carotenoid accumulation on the orange-band during flower bud development. Taken together, our results suggest that LtuPTOX is involved in petal carotenoid metabolism and orange band formation in L. tulipifera. The identification of this potentially involved gene will lay a foundation for further uncovering the genetic basis of flower colour in L. tulipifera.

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.

In the last common enzymatic step of tetrapyrrole biosynthesis, prior to the branching point leading to the biosynthesis of heme and chlorophyll, protoporphyrinogen IX (Protogen) is oxidised to protoporphyrin IX (Proto) by protoporphyrinogen IX oxidase (PPX). The absence of thylakoid-localised plastid terminal oxidase 2 (PTOX2) and cytochrome b6f complex in the ptox2 petB mutant, results in almost complete reduction of the plastoquinone pool (PQ pool) in light. Here we show that the lack of oxidised PQ impairs PPX function, leading to accumulation and subsequently uncontrolled oxidation of Protogen to non-metabolised Proto. Addition of 3(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) prevents the over-reduction of the PQ pool in ptox2 petB and decreases Proto accumulation. This observation strongly indicates the need of oxidised PQ as the electron acceptor for the PPX reaction in Chlamydomonas reinhardtii. The PPX-PQ pool interaction is proposed to function as a feedback loop between photosynthetic electron transport and chlorophyll biosynthesis. Pawel Brzezowski et al. demonstrated existence of a link between chlorophyll biosynthesis and photosynthetic electron transport in Chlamydomonas reinhardtii. The lack of oxidised plastoquinone pool impairs function of PROTOPORPHYRINOGEN IX OXIDASE, leading to accumulation of non-metabolised protoporphyrin IX.

During outdoor mass cultivation, Haematococcus pluvialis cells, especially at the green motile stage, are frequently exposed to high light. However, the photoprotective mechanisms in H. pluvialis have not been well characterized. This study aimed to confirm the photoprotection of chlororespiration in H. pluvialis at the green motile stage. Under high light, inhibition of plastid terminal oxidase (PTOX), a key enzyme of chlororespiration, increased the relative variable fluorescence at the I-step (VI), causing a decrease in the plastoquinone (PQ) pool, which further led to photosystem II (PSII) acceptor side over-reduction. This condition disturbed the balance between light absorption and energy utilization, causing the over-excitation of PSII reaction centers, which was indicated by the fact that the inhibition of PTOX increased the relative variable fluorescence at the J-step (VJ) and the light absorption flux per active reaction center (ABS/RC), but decreased the electron transport flux per active reaction center (ETo/RC) in H. pluvialis. The over-reduction of the PSII acceptor side and the over-excitation of PSII reaction centers inevitably caused more severe photoinhibition as shown by a decrease in Fv/Fm (maximal photochemical efficiency of PSII). Furthermore, the level of photoinhibition was not changed when the PTOX was inhibited in the presence of the photodamaged-PSII repair inhibitor chloramphenicol, indicating that the inhibition of PTOX did not accelerate the photodamage to PSII directly. The above results suggest that the chlororespiration pathway protects H. pluvialis at the green motile stage against photoinhibition by decreasing the inhibition of the photodamaged-PSII repair mechanism.

The nuclear-encoded plastid terminal oxidase gene (PTOX) is present in photosynthetic species and functions in the oxidation of the plastoquinone pool. It plays an important role on chlororespiration, chromorespiration and carotenoid biosynthesis. Here, we show short-term early response of carrot PTOX (DcPTOX) in leaves of two carrot inbred lines upon chilling. Analysis of the complete gene confirmed DcPTOX as a single gene and revealed an exceptionally large genomic sequence (9422 bp) in comparison to other species, comprising nine exons interrupted by eight introns. In silico analysis based on data from whole genome sequencing projects discovered that some plant species present two PTOX genes. A search for sequence variability at genomic level was performed in the heterogeneous, ancient carrot cultivar ‘Rotin’. DcPTOX revealed intron length polymorphisms (ILPs) in intron 2, due to the occurrence of two insertion/deletions (InDels) events. Prediction of pre-miRNA sequences in intronic regions of DcPTOX showed two putative locations coding for putative miRNAs, one located at intron 2 and other located at intron 6. Both putative miRNAs revealed high homology with miRNAs described in Glycine max (gma-miR1520p) and Oryza sativa (osa-miR5494), respectively. Plant disease resistance genes were identified as miRNAs target genes.