Impairment of peroxisomal APX and CAT activities increases protection of photosynthesis under oxidative stress.

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.

The regulation of P700 is an important photoprotective mechanism to NaCl-salinity in Jatropha curcas.

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.

Photosynthetic and biochemical mechanisms of an EMS-mutagenized cowpea associated with its resistance to cowpea severe mosaic virus.

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.

Mitochondrial GPX1 silencing triggers differential photosynthesis impairment in response to salinity in rice plants.

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.

Quantifying the dynamics of light tolerance in Arabidopsis plants during ontogenesis.

The amount of light plants can tolerate during different phases of ontogenesis remains largely unknown. This was addressed here employing a novel methodology that uses the coefficient of photochemical quenching (qP) to assess the intactness of photosystem II reaction centres. Fluorescence quenching coefficients, total chlorophyll content and concentration of anthocyanins were determined weekly during the juvenile, adult, reproductive and senescent phases of plant ontogenesis. This enabled quantification of the protective effectiveness of non-photochemical fluorescence quenching (NPQ) and determination of light tolerance. The light intensity that caused photoinhibition in 50% of leaf population increased from ∼70 µmol m(-2) s(-1) , for 1-week-old seedlings, to a maximum of 1385 µmol m(-2) s(-1) for 8-week-old plants. After 8 weeks, the tolerated light intensity started to gradually decline, becoming only 332 µmol m(-2) s(-1) for 13-week-old plants. The dependency of light tolerance on plant age was well-related to the amplitude of protective NPQ (pNPQ) and the electron transport rates (ETRs). Light tolerance did not, however, show a similar trend to chlorophyll a/b ratios and content of anthocyanins. Our data suggest that pNPQ is crucial in defining the capability of high light tolerance by Arabidopsis plants during ontogenesis.

Peroxisomal APX knockdown triggers antioxidant mechanisms favourable for coping with high photorespiratory H2 O2 induced by CAT deficiency in rice.

The physiological role of peroxisomal ascorbate peroxidases (pAPX) is unknown; therefore, we utilized pAPX4 knockdown rice and catalase (CAT) inhibition to assess its role in CAT compensation under high photorespiration. pAPX4 knockdown induced co-suppression in the expression of pAPX3. The rice mutants exhibited metabolic changes such as lower CAT and glycolate oxidase (GO) activities and reduced glyoxylate content; however, APX activity was not altered. CAT inhibition triggered different changes in the expression of CAT, APX and glutathione peroxidase (GPX) isoforms between non-transformed (NT) and silenced plants. These responses were associated with alterations in APX, GPX and GO activities, suggesting redox homeostasis differences. The glutathione oxidation-reduction states were modulated differently in mutants, and the ascorbate redox state was greatly affected in both genotypes. The pAPX suffered less oxidative stress and photosystem II (PSII) damage and displayed higher photosynthesis than the NT plants. The improved acclimation exhibited by the pAPX plants was indicated by lower H2 O2 accumulation, which was associated with lower GO activity and glyoxylate content. The suppression of both pAPXs and/or its downstream metabolic and molecular effects may trigger favourable antioxidant and compensatory mechanisms to cope with CAT deficiency. This physiological acclimation may involve signalling by peroxisomal H2 O2 , which minimized the photorespiration.

Cytosolic APX knockdown rice plants sustain photosynthesis by regulation of protein expression related to photochemistry, Calvin cycle and photorespiration.

The biochemical mechanisms underlying the involvement of cytosolic ascorbate peroxidases (cAPXs) in photosynthesis are still unknown. In this study, rice plants doubly silenced in these genes (APX1/2) were exposed to moderate light (ML) and high light (HL) to assess the role of cAPXs in photosynthetic efficiency. APX1/2 mutants that were exposed to ML overexpressed seven and five proteins involved in photochemical activity and photorespiration, respectively. These plants also increased the pheophytin and chlorophyll levels, but the amount of five proteins that are important for Calvin cycle did not change. These responses in mutants were associated with Rubisco carboxylation rate, photosystem II (PSII) activity and potential photosynthesis, which were similar to non-transformed plants. The upregulation of photochemical proteins may be part of a compensatory mechanism for APX1/2 deficiency but apparently the finer-control for photosynthesis efficiency is dependent on Calvin cycle proteins. Conversely, under HL the mutants employed a different strategy, triggering downregulation of proteins related to photochemical activity, Calvin cycle and decreasing the levels of photosynthetic pigments. These changes were associated to strong impairment in PSII activity and Rubisco carboxylation. The upregulation of some photorespiratory proteins was maintained under that stressful condition and this response may have contributed to photoprotection in rice plants deficient in cAPXs. The data reveal that the two cAPXs are not essential for photosynthesis in rice or, alternatively, the deficient plants are able to trigger compensatory mechanisms to photosynthetic acclimation under ML and HL conditions. These mechanisms involve differential regulation in protein expression related to photochemistry, Calvin cycle and photorespiration.

Involvement of ASR genes in aluminium tolerance mechanisms in rice.

Among cereal crops, rice is considered the most tolerant to aluminium (Al). However, variability among rice genotypes leads to remarkable differences in the degree of Al tolerance for distinct cultivars. A number of studies have demonstrated that rice plants achieve Al tolerance through an unknown mechanism that is independent of root tip Al exclusion. We have analysed expression changes of the rice ASR gene family as a function of Al treatment. The gene ASR5 was differentially regulated in the Al-tolerant rice ssp. Japonica cv. Nipponbare. However, ASR5 expression did not respond to Al exposure in Indica cv. Taim rice roots, which are highly Al sensitive. Transgenic plants carrying RNAi constructs that targeted the ASR genes were obtained, and increased Al susceptibility was observed in T1 plants. Embryogenic calli of transgenic rice carrying an ASR5-green fluorescent protein fusion revealed that ASR5 was localized in both the nucleus and cytoplasm. Using a proteomic approach to compare non-transformed and ASR-RNAi plants, a total of 41 proteins with contrasting expression patterns were identified. We suggest that the ASR5 protein acts as a transcription factor to regulate the expression of different genes that collectively protect rice cells from Al-induced stress responses.

Cacao agroforestry systems beyond the stigmas: Biotic and abiotic stress incidence impact.

Low technological knowledge in production chains, global climate change, and misinformation are concrete threats to food security. In addition, these combined threats also trigger ecological instability in megadiverse areas of the world, especially in some cacao-producing countries in South America, where this crop plays an important socio-economic role, even being used to replace illicit crops. Accordingly, the use of agroforestry systems approaches has emerged as a good alternative to maintain productivity, add high-value commodities to producers, and provide important ecosystem services for sustainable agriculture. However, limitations associated with the competition for resources between the species composing the system, and the higher incidence of some diseases, have led many producers to abandon this strategy, opting for monoculture. In this review, we seek to gather the main information available in the literature, aiming to answer the question: what is the real scientific evidence that supports the benefits and harms of adopting agroforestry systems in cacao production? We seek to make critical scrutiny of the possible negative effects of certain associations of the agroforestry system with biotic and abiotic stress in cacao. Here, we review the possible competition for light and nutrients and discuss the main characteristics to be sought in cacao genotypes to optimize these inter-specific relationships. In addition, we review the research advances that show the behavior of the main cacao diseases (Witch's broom disease, frosty pod rot, black pod rot) in models of agroforestry systems contrasted with monoculture, as well as the optimization of agronomic practices to reduce some of these stresses. This compendium, therefore, sheds light on a major gap in establishing truly sustainable agriculture, which has been treated much more from the perspective of negative stigma than from the real technological advantages that can be combined to the benefit of a balanced ecosystem with generating income for farmers.

Role of peroxidases in the compensation of cytosolic ascorbate peroxidase knockdown in rice plants under abiotic stress.

Current studies, particularly in Arabidopsis, have demonstrated that mutants deficient in cytosolic ascorbate peroxidases (APXs) are susceptible to the oxidative damage induced by abiotic stress. In contrast, we demonstrate here that rice mutants double silenced for cytosolic APXs (APx1/2s) up-regulated other peroxidases, making the mutants able to cope with abiotic stress, such as salt, heat, high light and methyl viologen, similar to non-transformed (NT) plants. The APx1/2s mutants exhibited an altered redox homeostasis, as indicated by increased levels of H2O2 and ascorbate and glutathione redox states. Both mutant and NT plants exhibited similar photosynthesis (CO2) assimilation and photochemical efficiency) under both normal and stress conditions. Overall, the antioxidative compensatory mechanism displayed by the mutants was associated with increased expression of OsGpx genes, which resulted in higher glutathione peroxidase (GPX) activity in the cytosolic and chloroplastic fractions. The transcript levels of OsCatA and OsCatB and the activities of catalase (CAT) and guaiacol peroxidase (GPOD; type III peroxidases) were also up-regulated. None of the six studied isoforms of OsApx were up-regulated under normal growth conditions. Therefore, the deficiency in cytosolic APXs was effectively compensated for by up-regulation of other peroxidases. We propose that signalling mechanisms triggered in rice mutants could be distinct from those proposed for Arabidopsis.

Understanding photosynthesis in a spatial-temporal multiscale: The need for a systemic view.

In October 2020, at the peak of the COVID-19 pandemic, a group of young Brazilian photosynthesis researchers organized the 1st Brazilian Symposium on Photosynthesis. The event was free and online, with the presence of important guest speakers from all over the world, who discussed their recent works on topics related to the future and perspectives of photosynthesis research. Summarizing the expectations of this symposium we highlighted the importance of adopting a systemic perspective for a better understanding of photosynthesis as a complex and dynamic process. Plants are modular and self-regulating presenting metabolic redundancy and functional degeneration. Among the various biological processes, photosynthesis plays a crucial role in promoting the direct conversion of light energy into carbon skeletons for support growth and productivity. In the past decades, significant advances have been made in photosynthesis at the biophysical, biochemical, and molecular levels. However, this myriad of knowledge has been insufficient to answer crucial questions, such as: how can we understand and eventually increase photosynthetic efficiency and yield in crops subjected to adverse environment related to climate-changing? We believe that a crucial limitation to the whole comprehension of photosynthesis is associated with a vastly widespread classic reductionist view. Moreover, this perspective is commonly accompanied by non-integrative, simplistic, and descriptive approaches to investigate a complex and dynamic process as photosynthesis. Herein, we propose the use of new approaches, mostly based on the Systems Theory, which certainly comes closer to the real world, such as the complex systems that the plants represent.

What proteomics can reveal about plant-virus interactions? Photosynthesis-related proteins on the spotlight.

Plant viruses are responsible for losses in worldwide production of numerous economically important food and fuel crops. As obligate cellular parasites with very small genomes, viruses rely on their hosts for replication, assembly, intra- and intercellular movement, and attraction of vectors for dispersal. Chloroplasts are photosynthesis and are the site of replication for several viruses. When viruses replicate in chloroplasts, photosynthesis, an essential process in plant physiology, is inhibited. The mechanisms underlying molecular and biochemical changes during compatible and incompatible plants-virus interactions, are only beginning to be elucidated, including changes in proteomic profiles induced by virus infections. In this review, we highlight the importance of proteomic studies to understand plant-virus interactions, especially emphasizing the changes in photosynthesis-related protein accumulation. We focus on: (a) chloroplast proteins that differentially accumulate during viral infection; (b) the significance with respect to chloroplast-virus interaction; and (c) alterations in plant's energetic metabolism and the subsequently the plant defense mechanisms to overcome viral infection.

Proteomic and physiological approaches reveal new insights for uncover the role of rice thylakoidal APX in response to drought stress.

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.

Rice peroxisomal ascorbate peroxidase knockdown affects ROS signaling and triggers early leaf senescence.

H2O2, which is continually produced by aerobic metabolism, is a cytotoxic molecule when in high levels. However, low levels can act as a signaling molecule able to regulate the expression of stress responses, senescence, programmed cell death, plant growth, and development. Ascorbate peroxidase (APX) enzyme plays an essential role in the control of intracellular H2O2 levels. Here, the function of a gene encoding a peroxisomal APX (OsAPX4) from rice (Oryza sativa L.) was studied. OsAPX4 gene expression can be detected in roots and panicles, but the highest expression level occurs in leaves. Silencing of OsAPX4 and OsAPX3 expression in RNAiOsAPX4 did not affect the growth of plants under growth chamber conditions, but aging transgenic plants interestingly displayed an early senescence phenotype. Leaf fragments from silenced plants were also more sensitive to induced senescence conditions. RNAiOsAPX4 plants did not present detectable changes in intracellular H2O2 levels, but biochemical analyses showed that transgenic plants displayed some decreased APX activity in the chloroplastic fraction. Also, the peroxisomal enzyme glycolate oxidase exhibited lower activity, whereas catalase activity was similar to non-transformed rice. The results imply that OsAPX4 gene has an important role in leaf senescence pathway mediated by ROS signaling.

Proteomics, photosynthesis and salt resistance in crops: An integrative view.

Salinity is a stressful condition that causes a significant decrease in crop production worldwide. Salt stress affects several photosynthetic reactions, including the modulation of several important proteins. Despite these effects, few molecular-biochemical markers have been identified and evaluated for their importance in improving plant salt resistance. Proteomics is a powerful tool that allows the analysis of multigenic events at the post-translational level that has been widely used to evaluate protein modulation changes in plants exposed to salt stress. However, these studies are frequently fragmented and the results regarding photosynthesis proteins in response to salinity are limited. These constraints could be related to the low number of important photosynthetic proteins differently modulated in response to salinity, as has been commonly revealed by conventional proteomics. In this review, we present an evaluation and perspective on the integrated application of proteomics for the identification of photosynthesis proteins to improve salt resistance. We propose the use of phospho-, thiol- and redox-proteomics, associated with the utilization of isolated chloroplasts or photosynthetic sub-organellar components. This strategy may allow the characterization of essential proteins, providing a better understanding of photosynthesis regulation. Furthermore, this may contribute to the selection of molecular markers to improve salt resistance in crops.

Silenced rice in both cytosolic ascorbate peroxidases displays pre-acclimation to cope with oxidative stress induced by 3-aminotriazole-inhibited catalase.

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.

The knockdown of chloroplastic ascorbate peroxidases reveals its regulatory role in the photosynthesis and protection under photo-oxidative stress in rice.

The inactivation of the chloroplast ascorbate peroxidases (chlAPXs) has been thought to limit the efficiency of the water-water cycle and photo-oxidative protection under stress conditions. In this study, we have generated double knockdown rice (Oryza sativa L.) plants in both OsAPX7 (sAPX) and OsAPX8 (tAPX) genes, which encode chloroplastic APXs (chlAPXs). By employing an integrated approach involving gene expression, proteomics, biochemical and physiological analyses of photosynthesis, we have assessed the role of chlAPXs in the regulation of the protection of the photosystem II (PSII) activity and CO2 assimilation in rice plants exposed to high light (HL) and methyl violagen (MV). The chlAPX knockdown plants were affected more severely than the non-transformed (NT) plants in the activity and structure of PSII and CO2 assimilation in the presence of MV. Although MV induced significant increases in pigment content in the knockdown plants, the increases were apparently not sufficient for protection. Treatment with HL also caused generalized damage in PSII in both types of plants. The knockdown and NT plants exhibited differences in photosynthetic parameters related to efficiency of utilization of light and CO2. The knockdown plants overexpressed other antioxidant enzymes in response to the stresses and increased the GPX activity in the chloroplast-enriched fraction. Our data suggest that a partial deficiency of chlAPX expression modulate the PSII activity and integrity, reflecting the overall photosynthesis when rice plants are subjected to acute oxidative stress. However, under normal growth conditions, the knockdown plants exhibit normal phenotype, biochemical and physiological performance.