Photorespiratory H2O2 Research Articles

Wounding induces a peroxisomal H2 O2 decrease via glycolate oxidase-catalase switch dependent on glutamate receptor-like channel-supported Ca2+ signaling in plants.

Sensing of environmental challenges, such as mechanical injury, by a single plant tissue results in the activation of systemic signaling, which attunes the plant's physiology and morphology for better survival and reproduction. As key signals, both calcium ions (Ca2+ ) and hydrogen peroxide (H2 O2 ) interplay with each other to mediate plant systemic signaling. However, the mechanisms underlying Ca2+ -H2 O2 crosstalk are not fully revealed. Our previous study showed that the interaction between glycolate oxidase and catalase, key enzymes of photorespiration, serves as a molecular switch (GC switch) to dynamically modulate photorespiratory H2 O2 fluctuations via metabolic channeling. In this study, we further demonstrate that local wounding induces a rapid shift of the GC switch to a more interactive state in systemic leaves, resulting in a sharp decrease in peroxisomal H2 O2 levels, in contrast to a simultaneous outburst of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived apoplastic H2 O2 . Moreover, the systemic response of the two processes depends on the transmission of Ca2+ signaling, mediated by glutamate-receptor-like Ca2+ channels 3.3 and 3.6. Mechanistically, by direct binding and/or indirect mediation by some potential biochemical sensors, peroxisomal Ca2+ regulates the GC switch states in situ, leading to changes in H2 O2 levels. Our findings provide new insights into the functions of photorespiratory H2 O2 in plant systemic acclimation and an optimized systemic H2 O2 signaling via spatiotemporal interplay between the GC switch and NADPH oxidases.

Dynamic and fluctuating generation of hydrogen peroxide via photorespiratory metabolic channeling in plants.

The homeostasis of hydrogen peroxide (H2 O2 ), a key regulator of basic biological processes, is a result of the cooperation between its generation and scavenging. However, the mechanistic basis of this balance is not fully understood. We previously proposed that the interaction between glycolate oxidase (GLO) and catalase (CAT) may serve as a molecular switch that modulates H2 O2 levels in plants. In this study, we demonstrate that the GLO-CAT complex in plants exists in different states, which are dynamically interchangeable in response to various stimuli, typically salicylic acid (SA), at the whole-plant level. More crucially, changes in the state of the complex were found to be closely linked to peroxisomal H2 O2 fluctuations, which were independent of the membrane-associated NADPH oxidase. Furthermore, evidence suggested that H2 O2 channeling occurred even invitro when GLO and CAT worked cooperatively. These results demonstrate that dynamic changes in H2 O2 levels are physically created via photorespiratory metabolic channeling in plants, and that such H2 O2 fluctuations may serve as signals that are mechanistically involved in the known functions of photorespiratory H2 O2 . In addition, our study also revealed a new way for SA to communicate with H2 O2 in plants.

Hydrogen sulfide: A novel component in Arabidopsis peroxisomes which triggers catalase inhibition.

Plant peroxisomes have the capacity to generate different reactive oxygen and nitrogen species (ROS and RNS), such as H2 O2 , superoxide radical (O2 · - ), nitric oxide and peroxynitrite (ONOO- ). These organelles have an active nitro-oxidative metabolism which can be exacerbated by adverse stress conditions. Hydrogen sulfide (H2 S) is a new signaling gasotransmitter which can mediate the posttranslational modification (PTM) persulfidation. We used Arabidopsis thaliana transgenic seedlings expressing cyan fluorescent protein (CFP) fused to a canonical peroxisome targeting signal 1 (PTS1) to visualize peroxisomes in living cells, as well as a specific fluorescent probe which showed that peroxisomes contain H2 S. H2 S was also detected in chloroplasts under glyphosate-induced oxidative stress conditions. Peroxisomal enzyme activities, including catalase, photorespiratory H2 O2 -generating glycolate oxidase (GOX) and hydroxypyruvate reductase (HPR), were assayed in vitro with a H2 S donor. In line with the persulfidation of this enzyme, catalase activity declined significantly in the presence of the H2 S donor. To corroborate the inhibitory effect of H2 S on catalase activity, we also assayed pure catalase from bovine liver and pepper fruit-enriched samples, in which catalase activity was inhibited. Taken together, these data provide evidence of the presence of H2 S in plant peroxisomes which appears to regulate catalase activity and, consequently, the peroxisomal H2 O2 metabolism.

Hydrogen peroxide facilitates Arabidopsis seedling establishment by interacting with light signalling pathway in the dark.

Light is essential for the plant establishment. Arabidopsis seedlings germinated in the dark cannot grow leaf and only have closed cotyledons. However, exogenous application of H2 O2 can induce leaves (establishment) in the dark. Comparative transcriptomic analysis revealed that light-responsive genes were activated by H2 O2 treatment. These genes are functionally correlated with photosynthesis, photorespiration, and components of photosystem, such as antenna proteins and light-harvesting chlorophyll proteins. We further found that application of H2 O2 facilitates cell cycle by accelerating G2 -M checkpoint transition in shoot apical meristem. Phytochrome-mediated light signalling pathway was also involved in the H2 O2 -facilitated establishment process. The constitutive photomorphogenesis 1 and phytochrome interacting factor 3 proteins were shown to be down-regulated by H2 O2 treatment and accordingly removed their inhibitory effects on photomorphogenesis in the dark. The crosstalk between oxidation and light signal pathways explains the mechanism that H2 O2 regulates plant dark establishment. The endogenous photorespiratory H2 O2 production was mimicked by overexpression of glycolate oxidase genes and supplement of substrate glycolate. As expected, seedling establishment was also induced by the endogenously produced H2 O2 under dark condition. These findings also suggest that photorespiratory H2 O2 production is at least partially involved in postgermination establishment.

Reconstruction of Oryza sativa indica Genome Scale Metabolic Model and Its Responses to Varying RuBisCO Activity, Light Intensity, and Enzymatic Cost Conditions.

To combat decrease in rice productivity under different stresses, an understanding of rice metabolism is needed. Though there are different genome scale metabolic models (GSMs) of Oryza sativa japonica, no GSM with gene-protein-reaction association exist for Oryza sativa indica. Here, we report a GSM, OSI1136 of O.s. indica, which includes 3602 genes and 1136 metabolic reactions and transporters distributed across the cytosol, mitochondrion, peroxisome, and chloroplast compartments. Flux balance analysis of the model showed that for varying RuBisCO activity (Vc/Vo) (i) the activity of the chloroplastic malate valve increases to transport reducing equivalents out of the chloroplast under increased photorespiratory conditions and (ii) glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase can act as source of cytosolic ATP under decreased photorespiration. Under increasing light conditions we observed metabolic flexibility, involving photorespiration, chloroplastic triose phosphate and the dicarboxylate transporters of the chloroplast and mitochondrion for redox and ATP exchanges across the intracellular compartments. Simulations under different enzymatic cost conditions revealed (i) participation of peroxisomal glutathione-ascorbate cycle in photorespiratory H2O2 metabolism (ii) different modes of the chloroplastic triose phosphate transporters and malate valve, and (iii) two possible modes of chloroplastic Glu–Gln transporter which were related with the activity of chloroplastic and cytosolic isoforms of glutamine synthetase. Altogether, our results provide new insights into plant metabolism.

SHORT-ROOT Deficiency Alleviates the Cell Death Phenotype of the Arabidopsis catalase2 Mutant under Photorespiration-Promoting Conditions.

Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To analyze molecular mechanisms that regulate the response to increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2). By screening for second-site mutations that attenuate the PSII maximum efficiency (Fv'/Fm') decrease and lesion formation linked to the cat2-2 phenotype, we discovered that a mutation in SHORT-ROOT (SHR) rescued the cell death phenotype of cat2-2 plants under photorespiration-promoting conditions. SHR deficiency attenuated H2O2-dependent gene expression, oxidation of the glutathione pool, and ascorbate depletion in a cat2-2 genetic background upon exposure to photorespiratory stress. Decreased glycolate oxidase and catalase activities together with accumulation of glycolate further implied that SHR deficiency impacts the cellular redox homeostasis by limiting peroxisomal H2O2 production. The photorespiratory phenotype of cat2-2 mutants did not depend on the SHR functional interactor SCARECROW and the sugar signaling component ABSCISIC ACID INSENSITIVE4, despite the requirement for exogenous sucrose for cell death attenuation in cat2-2 shr-6 double mutants. Our findings reveal a link between SHR and photorespiratory H2O2 production that has implications for the integration of developmental and stress responses.

Peroxisomes sense and respond to environmental cues by regulating ROS and RNS signalling networks.

Peroxisomes are highly dynamic, metabolically active organelles that used to be regarded as a sink for H2O2 generated in different organelles. However, peroxisomes are now considered to have a more complex function, containing different metabolic pathways, and they are an important source of reactive oxygen species (ROS), nitric oxide (NO) and reactive nitrogen species (RNS). Over-accumulation of ROS and RNS can give rise oxidative and nitrosative stress, but when produced at low concentrations they can act as signalling molecules. This review focuses on the production of ROS and RNS in peroxisomes and their regulation by antioxidants. ROS production is associated with metabolic pathways such as photorespiration and fatty acid β-oxidation, and disturbances in any of these processes can be perceived by the cell as an alarm that triggers defence responses. Genetic and pharmacological studies have shown that photorespiratory H2O2 can affect nuclear gene expression, regulating the response to pathogen infection and light intensity. Proteomic studies have shown that peroxisomal proteins are targets for oxidative modification, S-nitrosylation and nitration and have highlighted the importance of these modifications in regulating peroxisomal metabolism and signalling networks. The morphology, size, number and speed of movement of peroxisomes can also change in response to oxidative stress, meaning that an ROS/redox receptor is required. Information available on the production and detection of NO/RNS in peroxisomes is more limited. Peroxisomal homeostasis is critical for maintaining the cellular redox balance and is regulated by ROS, peroxisomal proteases and autophagic processes. Peroxisomes play a key role in many aspects of plant development and acclimation to stress conditions. These organelles can sense ROS/redox changes in the cell and thus trigger rapid and specific responses to environmental cues involving changes in peroxisomal dynamics as well as ROS- and NO-dependent signalling networks, although the mechanisms involved have not yet been established. Peroxisomes can therefore be regarded as a highly important decision-making platform in the cell, where ROS and RNS play a determining role.

Activation of auxin signalling counteracts photorespiratory H2O2-dependent cell death.

The high metabolic flux through photorespiration constitutes a significant part of the carbon cycle. Although the major enzymatic steps of the photorespiratory pathway are well characterized, little information is available on the functional significance of photorespiration beyond carbon recycling. Particularly important in this respect is the peroxisomal catalase activity which removes photorespiratory H2O2 generated during the oxidation of glycolate to glyoxylate, thus maintaining the cellular redox homeostasis governing the perception, integration and execution of stress responses. By performing a chemical screen, we identified 34 small molecules that alleviate the negative effects of photorespiration in Arabidopsis thaliana mutants lacking photorespiratory catalase (cat2). The chlorophyll fluorescence parameter photosystem II maximum efficiency (Fv′/Fm′) was used as a high-throughput readout. The most potent chemical that could rescue the photorespiratory phenotype of cat2 is a pro-auxin that contains a synthetic auxin-like substructure belonging to the phenoxy herbicide family, which can be released in planta. The naturally occurring indole-3-acetic acid (IAA) and other chemically distinct synthetic auxins also inhibited the photorespiratory-dependent cell death in cat2 mutants, implying a role for auxin signalling in stress tolerance.

LESION SIMULATING DISEASE 1 is required for acclimation to conditions that promote excess excitation energy.

The lsd1 mutant of Arabidopsis fails to limit the boundaries of hypersensitive cell death response during avirulent pathogen infection and initiates unchecked lesions in long day photoperiod giving rise to the runaway cell death (rcd) phenotype. We link here the initiation and propagation of rcd to the activity of photosystem II, stomatal conductance and ultimately to photorespiratory H(2)O(2). A cross of lsd1 with the chlorophyll a/b binding harvesting-organelle specific (designated cao) mutant, which has a reduced photosystem II antenna, led to reduced lesion formation in the lsd1/cao double mutant. This lsd1 mutant also had reduced stomatal conductance and catalase activity in short-day permissive conditions and induced H(2)O(2) accumulation followed by rcd when stomatal gas exchange was further impeded. All of these traits depended on the defense regulators EDS1 and PAD4. Furthermore, nonphotorespiratory conditions retarded propagation of lesions in lsd1. These data suggest that lsd1 failed to acclimate to light conditions that promote excess excitation energy (EEE) and that LSD1 function was required for optimal catalase activity. Through this regulation LSD1 can influence the effectiveness of photorespiration in dissipating EEE and consequently may be a key determinant of acclimatory processes. Salicylic acid, which induces stomatal closure, inhibits catalase activity and triggers the rcd phenotype in lsd1, also impaired acclimation of wild-type plants to conditions that promote EEE. We propose that the roles of LSD1 in light acclimation and in restricting pathogen-induced cell death are functionally linked.

Photosynthesis and Seed Production under Water‐Deficit Conditions in Transgenic Tobacco Plants That Overexpress an Arabidopsis Ascorbate Peroxidase Gene

An underlying mechanism for reductions in crop yield under stress conditions is excessive production of reactive oxygen species (ROS) that can damage lipids, nucleic acids, and proteins, leading to disruption of physiological processes. The aim of this study was to determine whether overexpression of the gene for a peroxisomal antioxidant enzyme, ascorbate peroxidase 3 (APX3), could provide protection of photosynthesis during drought when the potential rises for excessive photorespiratory H2O2 production. Tobacco (Nicotiana tabacum L.) plants were transformed to constitutively overexpress the Arabidopsis thaliana gene for APX3. Following repeated water‐deficit cycles, fruit number and seed mass of transgenic plants were significantly higher than those of control plants. In another experiment, water deficit was developed gradually by reducing, in stages, the extent to which water lost was replenished. Genotypic differences in gas‐exchange parameters were observed at the 25% replenishment stage and at 5 h after severely stressed plants were rewatered. At these times, transgenic plants exhibited greater rates of CO2 assimilation (A), stomatal conductance (gs), and internal CO2 (Ci) to atmospheric CO2 (Ca) concentration than control plants, suggesting that differences in A were controlled by differences in gs Although these data did not support the idea that overexpression of the gene for APX3 enhances protection of the photosynthetic apparatus during water deficit, overexpression of APX3 may affect other cellular metabolisms that result in higher A under moderate water‐deficit conditions and therefore higher seed mass after repeated water‐deficit treatments.

Drought and oxidative load in the leaves of C3 plants: a predominant role for photorespiration?

Although active oxygen species are produced at high rates in both the chloroplasts and peroxisomes of the leaves of C3 plants, most attention has focused on the potentially damaging consequences of enhanced chloroplastic production in stress conditions such as drought. This article attempts to provide quantitative estimates of the relative contributions of the chloroplast electron transport chain and the glycolate oxidase reaction to the oxidative load placed on the photosynthetic leaf cell. Rates of photorespiratory H2O2 production were obtained from photosynthetic and photorespiratory flux rates, derived from steady-state leaf gas exchange measurements at varying irradiance and ambient CO2. Assuming a 10% allocation of photosynthetic electron flow to the Mehler reaction, photorespiratory H2O2 production would account for about 70% of total H2O2 formed at all irradiances measured. When chloroplastic CO2 concentration rates are decreased, photorespiration becomes even more predominant in H2O2 generation. At the increased flux through photorespiration observed at lower ambient CO2, the Mehler reaction would have to account for more than 35% of the total photosynthetic electron flow in order to match the rate of peroxisomal H2O2 production. The potential signalling role of H2O2 produced in the peroxisomes is emphasized, and it is demonstrated that photorespiratory H2O2 can perturb the redox states of leaf antioxidant pools. We discuss the interactions between oxidants, antioxidants and redox changes leading to modified gene expression, particularly in relation to drought, and call attention to the potential significance of photorespiratory H2O2 in signalling and acclimation.

Photorespiration and the Mehler reaction : cellular protection or redox perturbation?

Two processes associated with photosynthesis make the major contribution to the oxidative load borne by leaf tissue in C3 plants. These are the Mehler reaction in the chloroplast and the glycollate oxidase reaction in the peroxisomes, both of which generate H2O2. Although often discussed as protective mechanisms, both the Mehler reaction and photorespiration can therefore influence cellular redox balance and related signalling. We have modelled the rate of photorespiratory H2O2 production from rates of net CO2 uptake measured in different conditions of irradiance and CO2 concentration. The data suggest that production of H2O2 in photorespiration exceeds that derived from the Mehler reaction under most conditions and that the total rate of H2O2 generation can approach or exceed the rate of net CO2 fixation. To avoid accumulation of H2O2, the chloroplast and the peroxisome have high antioxidant capacities. In addition to antioxidant enzymes such as superoxide dismutase, catalase, and peroxiredoxins, photosynthetic cells accumulate two low molecular weight antioxidants, ascorbate and glutathione. These antioxidants do not always interact in a coupled manner and the acorbate/ dehydroascorbate (DHA) redox pair may vary independently of the reduced (GSH)/oxidised glutathione (GSSG) redoz couple. This suggests that signals derived from changes in the rate of H2O2 generation are transmitted through perturbations in each antioxidant.

Differential expression of catalase genes in Nicotiana plumbaginifolia (L.).

We have analyzed the expression of three catalase (Cat; EC 1.11.1.6) genes from Nicotiana plumbaginifolia by means of RNA blot and in situ hybridizations. Our data demonstrate that the expression of each catalase is associated with a particular H2O2-producing process. Cat1 appears to be specifically involved in the scavenging of photorespiratory H2O2 and is under control of a circadian rhythm, Cat2 is uniformly expressed in different organs with a cellular preference for vascular tissues, and the expression profile of Cat3 points to a role in glyoxysomal processes. Differential expression of these catalases is also manifested in response to temperature changes. DNA sequence comparison with other dicotyledonous catalases led to the identification of at least three distinct classes, which indicates that the functional organization of catalases is generally conserved in dicotyledonous plants.

Further Studies on O2-Resistant Photosynthesis and Photorespiration in a Tobacco Mutant with Enhanced Catalase Activity

The increase in net photosynthesis in M(4) progeny of an O(2)-resistant tobacco (Nicotiana tabacum) mutant relative to wild-type plants at 21 and 42% O(2) has been confirmed and further investigated. Self-pollination of an M(3) mutant produced M(4) progeny segregating high catalase phenotypes (average 40% greater than wild type) at a frequency of about 60%. The high catalase phenotype cosegregated precisely with O(2)-resistant photosynthesis. About 25% of the F(1) progeny of reciprocal crosses between the same M(3) mutant and wild type had high catalase activity, whether the mutant was used as the maternal or paternal parent, indicating nuclear inheritance. In high-catalase mutants the activity of NADH-hydroxypyruvate reductase, another peroxisomal enzyme, was the same as wild type. The mutants released 15% less photorespiratory CO(2) as a percent of net photosynthesis in CO(2)-free 21% O(2) and 36% less in CO(2)-free 42% O(2) compared with wild type. The mutant leaf tissue also released less (14)CO(2) per [1-(14)C]glycolate metabolized than wild type in normal air, consistent with less photorespiration in the mutant. The O(2)-resistant photosynthesis appears to be caused by a decrease in photorespiration especially under conditions of high O(2) where the stoichiometry of CO(2) release per glycolate metabolized is expected to be enhanced. The higher catalase activity in the mutant may decrease the nonenzymatic peroxidation of keto-acids such as hydroxypyruvate and glyoxylate by photorespiratory H(2)O(2).