Andréia Caverzan

Plant responses to stresses: role of ascorbate peroxidase in the antioxidant protection

When plants are exposed to stressful environmental conditions, the production of Reactive Oxygen Species (ROS) increases and can cause significant damage to the cells. Antioxidant defenses, which can detoxify ROS, are present in plants. A major hydrogen peroxide detoxifying system in plant cells is the ascorbate-glutathione cycle, in which, ascorbate peroxidase (APX) enzymes play a key role catalyzing the conversion of H2O2 into H2O, using ascorbate as a specific electron donor. Different APX isoforms are present in distinct subcellular compartments, such as chloroplasts, mitochondria, peroxisome, and cytosol. The expression of APX genes is regulated in response to biotic and abiotic stresses as well as during plant development. The APX responses are directly involved in the protection of plant cells against adverse environmental conditions. Furthermore, mutant plants APX genes showed alterations in growth, physiology and antioxidant metabolism revealing those enzymes involvement in the normal plant development.

Cytosolic APx knockdown indicates an ambiguous redox responses in rice

Ascorbate peroxidases (APX, EC 1.1.11.1) are class I heme-peroxidases, which catalyze the conversion of H(2)O(2) into H(2)O, using ascorbate as a specific electron donor. Previously, the presence of eight Apx genes was identified in the nuclear genome of rice (Oryza sativa), encoding isoforms that are located in different sub-cellular compartments. Herein, the generation of rice transgenic plants silenced for either both or each one of the cytosolic Apx1 and Apx2 genes was carried out in order to investigate the importance of cytosolic Apx isoforms on plant development and on plant stress responses. Transgenic double Apx1/2-silenced plants exhibited normal development, even though these plants showed a global reduction of Apx activity which strongly impacts the whole antioxidant system regulation. Apx1/2-silenced plants also showed increased H(2)O(2) accumulation under control and stress situations and presented higher tolerance to toxic concentration of aluminum when compared to wild type plants. On the other hand, silencing OsApx1 and OsApx2 genes individually resulted in strong effect on plant development producing semi-dwarf phenotype. These results suggested that the double silencing of cytosolic OsApx genes induced compensatory antioxidant mechanisms in rice while single knockdown of these genes did not, which resulted in the impairing of normal plant development.

The mitochondrial glutathione peroxidase GPX3 is essential for H2O2 homeostasis and root and shoot development in rice

Glutathione (GSH) peroxidases (GPXs: EC 1.11.1.9 and EC1.11.1.12) are non-heme thiol peroxidases that catalyze the reduction of H2O2 or organic hydroperoxides to water, and they have been identified in almost all kingdoms of life. The rice glutathione peroxidase (OsGPX) gene family is comprised of 5 members spread throughout a range of sub cellular compartments. The OsGPX gene family is induced in response to exogenous H2O2 and cold stress. In contrast, they are down regulated in response to drought and UV-B light treatments. Transgenic rice plants have been generated that lack mitochondrial OsGPX3. These GPX3s plants showed shorter roots and shoots compared to non-transformed (NT) plants, and higher amounts of H2O2 mitochondrial release were observed in the roots of these plants cultivated under normal conditions. This accumulation of H2O2 is positively associated with shorter root length in GPX3s plants compared to NT ones. Moreover, GPX3 promoter analysis indicated that it is mainly expressed in root tissue. These results suggest that silencing the mitochondrial OsGPX3 gene impairs normal plant development and leads to a stress-induced morphogenic response via H2O2 accumulation.

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.

Succinate dehydrogenase (mitochondrial complex II) is a source of reactive oxygen species in plants and regulates development and stress responses.

Reactive oxygen species (ROS) are signaling molecules that regulate plant development and responses to stresses. Mitochondria are the source of most ROS in heterotrophic cells, and mitochondrial complex I and complex III are regarded as the main sites of ROS production in plant mitochondria. Recent studies have demonstrated that succinate dehydrogenase (SDH) also contributes to mitochondrial ROS production. However, the ability of SDH to generate ROS in plants is unclear. The aim of this study was to evaluate the role of SDH in mitochondrial ROS production. Our results demonstrated that SDH is a direct source of ROS in Arabidopsis thaliana and Oryza sativa, and the induction of ROS production by specific SDH inhibitors impaired plant growth. In addition, this effect was accompanied by the down-regulation of cell cycle genes and the up-regulation of stress-related genes. However, the partial inhibition of SDH by a competitive inhibitor decreased ROS production, which was associated with increased shoot and root growth, and prevented the down-regulation of cell cycle genes and the induction of stress-related genes by noncompetitive inhibitors. In conclusion, SDH plays an important role in ROS production, being a direct source of ROS in plant mitochondria and regulating plant development and stress responses.

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.

Chloroplastic and mitochondrial GPX genes play a critical role in rice development

Plant glutathione peroxidases (GPX) catalyze the reduction of H2O2 or organic hydroperoxides to water, mitigating the toxicity of these compounds to cells. In rice plants, the GPX gene family is composed of five members that are distributed in a range of sub-cellular compartments including cytosol, mitochondria, chloroplasts, or endoplasmic reticulum. Of these, OsGPX1 and OsGPX4 are located in mitochondria and chloroplasts, respectively. To understand the role of these GPX in rice, the effect of knockdown of OsGPX1 and OsGPX4 in rice plants was evaluated. Our data show that OsGPX4 was essential for in vitro rice regeneration because no plants were obtained from calli carrying a hairpin construct against OsGPX4. Although the knockdown of OsGPX1 did not impair plant regeneration, the plants with silenced OsGPX1 (GPX1s plants) showed reduced shoot length and a reduced number of seeds compared to the non-transformed rice plants. These results indicate that OsGPX1 and OsGPX4 are essential for redox homeostasis which leads to normal growth and development of rice.

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.