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Chloroplast ascorbate peroxidases exert an important role in the maintenance of hydrogen peroxide levels in chloroplasts by using ascorbate as the specific electron donor. In this work, we performed a functional study of the stromal APX in rice (OsAPX7) and demonstrated that silencing of OsAPX7 did not impact plant growth, redox state, or photosynthesis parameters. Nevertheless, when subjected to drought stress, silenced plants (APX7i) show a higher capacity to maintain stomata aperture and photosynthesis performance, resulting in a higher tolerance when compared to non-transformed plants. RNA-seq analyses indicate that the silencing of OsAPX7 did not lead to changes in the global expression of genes related to reactive oxygen species metabolism. In addition, the drought-mediated induction of several genes related to the proteasome pathway and the down-regulation of genes related to nitrogen and carotenoid metabolism was impaired in APX7i plants. During drought stress, APX7i showed an up-regulation of genes encoding flavonoid and tyrosine metabolism enzymes and a down-regulation of genes related to phytohormones signal transduction and nicotinate and nicotinamide metabolism. Our results demonstrate that OsAPX7 might be involved in signaling transduction pathways related to drought stress response, contributing to the understanding of the physiological role of chloroplast APX isoforms in rice.
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Photosynthesis involves the conversion of sunlight energy into stored chemical energy, which is achieved through electron transport along a series of redox reactions. Excess photosynthetic electron transport might be dangerous due to the risk of molecular oxygen reduction, generating reactive oxygen species (ROS) over-accumulation. Avoiding excess ROS production requires the rate of electron transport to be coordinated with the capacity of electron acceptors in the chloroplast stroma. Imbalance between the donor and acceptor sides of photosystem I (PSI) can lead to inactivation, which is called PSI photoinhibition. We used a light-inducible PSI photoinhibition system in Arabidopsis thaliana to resolve the time dynamics of inhibition and to investigate its impact on ROS production and turnover. The oxidation state of the PSI reaction center and rates of CO2 fixation both indicated strong and rapid PSI photoinhibition upon donor side/acceptor side imbalance, while the rate of inhibition eased during prolonged imbalance. PSI photoinhibition was not associated with any major changes in ROS accumulation or antioxidant activity; however, a lower level of lipid oxidation correlated with lower abundance of chloroplast lipoxygenase in PSI-inhibited leaves. The results of this study suggest that rapid activation of PSI photoinhibition under severe photosynthetic imbalance protects the chloroplast from over-reduction and excess ROS formation.
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Salinity commonly affects photosynthesis and crop production worldwide. Salt stress disrupts the fine balance between photosynthetic electron transport and the Calvin cycle reactions, leading to over-reduction and excess energy within the thylakoids. The excess energy triggers reactive oxygen species (ROS) overproduction that causes photoinhibition in both photosystems (PS) I and II. However, the role of PSI photoinhibition and its physiological mechanisms for photoprotection have not yet been fully elucidated. In the present study, we analyzed the effects of 15 consecutive days of 100 mM NaCl in Jatropha curcas plants, primarily focusing on the photosynthetic electron flow at PSI level. We found that J. curcas plants have important photoprotective mechanisms to cope with the harmful effects of salinity. We show that maintaining P700 in an oxidized state is an important photoprotector mechanism, avoiding ROS burst in J. curcas exposed to salinity. In addition, upon photoinhibition of PSI, the highly reduced electron transport chain triggers a significant increase in H2 O2 content which can lead to the production of hydroxyl radical by Mehler reactions in chloroplast, thereby increasing PSI photoinhibition.
Assuntos
Jatropha/efeitos dos fármacos , Jatropha/metabolismo , Cloreto de Sódio/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Complexo de Proteína do Fotossistema I/metabolismo , SalinidadeRESUMO
Chloroplast APX isoforms display controversial roles as H2O2 scavengers and signaling players in response to abiotic stress and conclusive results are lacking. We tested the hypothesis that thylakoidal APX displays an important role for drought tolerance, especially by regulating abundance of essential protein species. For this, OsApx8 RNAi-silenced rice (apx8) and non-transformed plants (NT) were exposed to mild water deficit. The drought-sensitivity in apx8 plants was revealed by decreases in shoot growth, relative water content and photosynthesis, which was accompanied by increased membrane damage, all compared to NT plants. This higher sensitivity of apx8 plants to mild drought stress was also related to a lower accumulation of important protein species involved in several metabolic processes, especially photosynthesis, photorespiration and redox metabolism. Despite apx8 plants have displayed an effective induction of compensatory antioxidant mechanisms in well-watered conditions, it was not enough to maintain H2O2 homeostasis and avoid oxidative and physiological disturbances under mild drought conditions. Thus, thylakoidal APX is involved in several phenotypic modifications at proteomic profile level, possibly via a H2O2-induced signaling mechanism. Consequently, this APX isoform is crucial for rice plants effectively cope with a mild drought condition. BIOLOGICAL SIGNIFICANCE: This work provides for the first time an integrative study involving proteomic, physiological and biochemical analyses directed to elucidation of thylakoidal APX roles for drought tolerance in rice plants. Our data reveal that this enzyme is crucial for maintaining of growth and photosynthesis under mild water deficit conditions. This essential role is related to maintaining of H2O2 homeostasis and accumulation of essential proteins involved in several important metabolic pathways. Remarkably, for drought resistance was essential the accumulation of proteins involved with metabolism of photosynthesis, signaling, carbohydrates, protein synthesis/degradation and stress. These results can contribute to understand the role of chloroplast ascorbate peroxidases in drought tolerance, highlighting the physiological importance of key proteins in this process.
Assuntos
Ascorbato Peroxidases/metabolismo , Oryza/enzimologia , Estresse Oxidativo , Proteínas de Plantas/metabolismo , Tilacoides/enzimologia , Desidratação , Peróxido de Hidrogênio/metabolismo , FotossínteseRESUMO
Retrograde signalling pathways that are triggered by changes in cellular redox homeostasis remain poorly understood. Transformed rice plants that are deficient in peroxisomal ascorbate peroxidase APX4 (OsAPX4-RNAi) are known to exhibit more effective protection of photosynthesis against oxidative stress than controls when catalase (CAT) is inhibited, but the mechanisms involved have not been characterized. An in-depth physiological and proteomics analysis was therefore performed on OsAPX4-RNAi CAT-inhibited rice plants. Loss of APX4 function led to an increased abundance of several proteins that are involved in essential metabolic pathways, possibly as a result of increased tissue H2O2 levels. Higher photosynthetic activities observed in the OsAPX4-RNAi plants under CAT inhibition were accompanied by higher levels of Rubisco, higher maximum rates of Rubisco carboxylation, and increased photochemical efficiencies, together with large increases in photosynthesis-related proteins. Large increases were also observed in the levels of proteins involved in the ascorbate/glutathione cycle and in other antioxidant-related pathways, and these changes may be important in the protection of photosynthesis in the OsAPX4-RNAi plants. Large increases in the abundance of proteins localized in the nuclei and mitochondria were also observed, together with increased levels of proteins involved in important cellular pathways, particularly protein translation. Taken together, the results show that OsAPX4-RNAi plants exhibit significant metabolic reprogramming, which incorporates a more effective antioxidant response to protect photosynthesis under conditions of impaired CAT activity.
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Ascorbato Peroxidases/metabolismo , Catalase/metabolismo , Oryza/metabolismo , Estresse Oxidativo , Fotossíntese , Interferência de RNARESUMO
Cowpea severe mosaic virus (CPSMV) causes significant losses in cowpea (Vigna unguiculata) production. In this present study biochemical, physiological, and proteomic analysis were done to identify pathways and defense proteins that are altered during the incompatible interaction between the cowpea genotype BRS-Marataoã and CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V) were evaluated at 2 and 6days post-inoculation (DPI). Data support the assumptions that increases in biochemical (high hydrogen peroxide, antioxidant enzymes, and secondary compounds) and physiological responses (high photosynthesis index and chlorophyll content), confirmed by label-free comparative proteomic approach, in which quantitative changes in proteasome proteins, proteins related to photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were observed may be implicated in the resistance of BRS-Marataoã to CPSMV. This pioneering study provides information for the selection of specific pathways and proteins, altered in this incompatible relationship, which could be chosen as targets for detailed studies to advance our understanding of the molecular, physiological, and biochemistry basis of the resistance mechanism of cowpea and design approachs to engineer plants that are more productive. BIOLOGICAL SIGNIFICANCE: This is a pioneering study in which an incompatible relationship between a resistant cowpea and Cowpea severe mosaic virus (CPSMV) was conducted to comparatively evaluate proteomic profiles by Gel-free/label-free methodology and some physiological and biochemical parameters to shed light on how a resistant cowpea cultivar deals with the virus attack. Specific proteins and associated pathways were altered in the cowpea plants challenged with CPSMV and will contribute to our knowledge on the biological process tailored by cowpea in response to CPSMV.
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Comovirus/imunologia , Resistência à Doença , Proteômica/métodos , Vigna/imunologia , Regulação da Expressão Gênica de Plantas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Fotossíntese , Proteínas de Plantas/análiseRESUMO
KEY MESSAGE: The seed treatment of a CPSMV-susceptible cowpea genotype with the mutagenic agent EMS generated mutagenized resistant plantlets that respond to the virus challenge by activating biochemical and physiological defense mechanisms. Cowpea is an important crop that makes major nutritional contributions particularly to the diet of the poor population worldwide. However, its production is low, because cowpea is naturally exposed to several abiotic and biotic stresses, including viral agents. Cowpea severe mosaic virus (CPSMV) drastically affects cowpea grain production. This study was conducted to compare photosynthetic and biochemical parameters of a CPSMV-susceptible cowpea (CE-31 genotype) and its derived ethyl methanesulfonate-mutagenized resistant plantlets, both challenged with CPSMV, to shed light on the mechanisms of virus resistance. CPSMV inoculation was done in the fully expanded secondary leaves, 15 days after planting. At 7 days post-inoculation, in vivo photosynthetic parameters were measured and leaves collected for biochemical analysis. CPSMV-inoculated mutagenized-resistant cowpea plantlets (MCPI) maintained higher photosynthesis index, chlorophyll, and carotenoid contents in relation to the susceptible (CE-31) CPSMV-inoculated cowpea (CPI). Visually, the MCPI leaves did not exhibit any viral symptoms neither the presence of the virus as examined by RT-PCR. In addition, MCPI showed higher SOD, GPOX, chitinase, and phenylalanine ammonia lyase activities, H2O2, phenolic contents, and cell wall lignifications, but lower CAT and APX activities in comparison to CPI. All together, these photosynthetic and biochemical changes might have contributed for the CPSMS resistance of MCPI. Contrarily, CPI plantlets showed CPSMV accumulation, severe disease symptoms, reduction in the photosynthesis-related parameters, chlorophyll, carotenoid, phenolic compound, and H2O2 contents, in addition to increased ß-1,3-glucanase, and catalase activities that might have favored viral infection.
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Comovirus/fisiologia , Resistência à Doença , Mutagênese/genética , Fotossíntese , Doenças das Plantas/virologia , Vigna/fisiologia , Vigna/virologia , Dióxido de Carbono/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Metanossulfonato de Etila , Homeostase , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , Oxirredução , Fenóis/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/virologia , Proteínas de Plantas/metabolismo , SolubilidadeRESUMO
The physiological and biochemical responses of a drought tolerant, virus-susceptible cowpea genotype exposed to drought stress (D), infected by Cowpea severe mosaic virus (CPSMV) (V), and to these two combined stresses (DV), at 2 and 6 days post viral inoculation (DPI), were evaluated. Gas exchange parameters (net photosynthesis, transpiration rate, stomatal conductance, and internal CO2 partial pressure) were reduced in D and DV at 2 and 6 DPI compared to control plants (C). Photosynthesis was reduced by stomatal and biochemical limitations. Water use efficiency increased at 2 DPI in D, DV, and V, but at 6 DPI only in D and DV compared to C. Photochemical parameters (effective quantum efficiency of photosystem II and electron transport rate) decreased in D and DV compared to C, especially at 6 DPI. The potential quantum efficiency of photosystem II did not change, indicating reversible photoinhibition of photosystem II. In DV, catalase decreased at 2 and 6 DPI, ascorbate peroxidase increased at 2 DPI, but decreased at 6 DPI. Hydrogen peroxide increased at 2 and 6 DPI. Peroxidase increased at 6 DPI and chitinase at 2 and 6 DPI. ß-1,3-glucanase decreased in DV at 6 DPI compared to V. Drought increased cowpea susceptibility to CPSMV at 2 DPI, as verified by RT-PCR. However, at 6 DPI, the cowpea plants overcome this effect. Likewise, CPSMV increased the negative effects of drought at 2 DPI, but not at 6 DPI. It was concluded that the responses to combined stresses are not additive and cannot be extrapolated from the study of individual stresses.
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Secas , Vírus do Mosaico/fisiologia , Doenças das Plantas/virologia , Vigna/virologia , Antioxidantes/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Clorofila A , Enzimas/genética , Enzimas/metabolismo , Regulação da Expressão Gênica de Plantas , Genótipo , Interações Hospedeiro-Patógeno , Peróxido de Hidrogênio/metabolismo , Fotossíntese/genética , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Doenças das Plantas/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estômatos de Plantas/genética , Estômatos de Plantas/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Vigna/genética , Vigna/metabolismo , Água/metabolismoRESUMO
The maintenance of H2O2 homeostasis and signaling mechanisms in plant subcellular compartments is greatly dependent on cytosolic ascorbate peroxidases (APX1 and APX2) and peroxisomal catalase (CAT) activities. APX1/2 knockdown plants were utilized in this study to clarify the role of increased cytosolic H2O2 levels as a signal to trigger the antioxidant defense system against oxidative stress generated in peroxisomes after 3-aminotriazole-inhibited catalase (CAT). Before supplying 3-AT, silenced APX1/2 plants showed marked changes in their oxidative and antioxidant profiles in comparison to NT plants. After supplying 3-AT, APX1/2 plants triggered up-expression of genes belonging to APX (OsAPX7 and OsAPX8) and GPX families (OsGPX1, OsGPX2, OsGPX3 and OsGPX5), but to a lower extent than in NT plants. In addition, APX1/2 exhibited lower glycolate oxidase (GO) activity, higher CO2 assimilation, higher cellular integrity and higher oxidation of GSH, whereas the H2O2 and lipid peroxidation levels remained unchanged. This evidence indicates that redox pre-acclimation displayed by silenced rice contributed to coping with oxidative stress generated by 3-AT. We suggest that APX1/2 plants were able to trigger alternative oxidative and antioxidant mechanisms involving signaling by H2O2, allowing these plants to display effective physiological responses for protection against oxidative damage generated by 3-AT, compared to non-transformed plants.
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Aclimatação/efeitos dos fármacos , Amitrol (Herbicida)/toxicidade , Ascorbato Peroxidases/metabolismo , Catalase/antagonistas & inibidores , Citosol/enzimologia , Inativação Gênica/efeitos dos fármacos , Oryza/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/metabolismo , Ácido Ascórbico/metabolismo , Catalase/metabolismo , Respiração Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Genes de Plantas , Glutationa/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Biológicos , Oryza/efeitos dos fármacos , Oryza/genética , Oryza/fisiologia , Oxirredução/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
The physiological role of plant mitochondrial glutathione peroxidases is scarcely known. This study attempted to elucidate the role of a rice mitochondrial isoform (GPX1) in photosynthesis under normal growth and salinity conditions. GPX1 knockdown rice lines (GPX1s) were tested in absence and presence of 100 mM NaCl for 6 d. Growth reduction of GPX1s line under non-stressful conditions, compared with non-transformed (NT) plants occurred in parallel to increased H2 O2 and decreased GSH contents. These changes occurred concurrently with photosynthesis impairment, particularly in Calvin cycle's reactions, since photochemical efficiency did not change. Thus, GPX1 silencing and downstream molecular/metabolic changes modulated photosynthesis differentially. In contrast, salinity induced reduction in both phases of photosynthesis, which were more impaired in silenced plants. These changes were associated with root morphology alterations but not shoot growth. Both studied lines displayed increased GPX activity but H2 O2 content did not change in response to salinity. Transformed plants exhibited lower photorespiration, water use efficiency and root growth, indicating that GPX1 could be important to salt tolerance. Growth reduction of GPX1s line might be related to photosynthesis impairment, which in turn could have involved a cross talk mechanism between mitochondria and chloroplast originated from redox changes due to GPX1 deficiency.