Stromal Ascorbate Peroxidase (OsAPX7) Modulates Drought Stress Tolerance in Rice (Oryza sativa).

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.

Genome-wide, evolutionary, and functional analyses of ascorbate peroxidase (APX) family in Poaceae species.

Ascorbate peroxidases (APXs) are heme peroxidases involved in the control of hydrogen peroxide levels and signal transduction pathways related to development and stress responses. Here, a total of 238 APX, 30 APX-related (APX-R), and 34 APX-like (APX-L) genes were identified from 24 species from the Poaceae family. Phylogenetic analysis of APX indicated five distinct clades, equivalent to cytosolic (cAPX), peroxisomal (pAPX), mitochondrial (mitAPX), stromal (sAPX), and thylakoidal (tAPX) isoforms. Duplication events contributed to the expansion of this family and the divergence times. Different from other APX isoforms, the emergence of Poaceae mitAPXs occurred independently after eudicot and monocot divergence. Our results showed that the constitutive silencing of mitAPX genes is not viable in rice plants, suggesting that these isoforms are essential for rice regeneration or development. We also obtained rice plants silenced individually to sAPX isoforms, demonstrating that, different to plants double silenced to both sAPX and tAPX or single silenced to tAPX previously obtained, these plants do not show changes in the total APX activity and hydrogen peroxide content in the shoot. Among rice plants silenced to different isoforms, plants silenced to cAPX showed a higher decrease in total APX activity and an increase in hydrogen peroxide levels. These results suggest that the cAPXs are the main isoforms responsible for regulating hydrogen peroxide levels in the cell, whereas in the chloroplast, this role is provided mainly by the tAPX isoform. In addition to broadening our understanding of the core components of the antioxidant defense in Poaceae species, the present study also provides a platform for their functional characterization.

Biochemical Profile of the Soybean Seed Embryonic Axis and Its Changes during Accelerated Aging.

Seed deterioration is an important topic in plant science, as the majority of cultivated species use seeds as their means of propagation; however, due to its complexity, the process of seed deterioration has not yet been completely elucidated. Three soybean cultivars (BMX Raio, BMX Zeus, and DM 53i54) exposed to four distinct periods of accelerated aging (0, 3, 6 and 9 days) in a fully randomized experimental design. Initially, vigor and germination tests were performed. The activity of superoxide dismutase, catalase, ascorbate peroxidase enzymes, hydrogen peroxide, malonaldehyde, DNA oxidation, macromolecules and mineral content, and Maillard reactions were quantified in the embryonic axis. Results showed that DNA did not suffer degradation or oxidation. In terms of consumption of reserves, only sugars were consumed, while levels of protein, starch, and triglycerides were maintained. The Maillard reaction did show potential as an indicator of buffer capacity of protein to ROS. Additionally, levels of catalase and ascorbate peroxidase decreased during the aging process. Moreover, nutrient analysis showed that a high magnesium level in the cultivar bestowed greater resilience to deterioration, which can indicate a potential function of magnesium in the cell structure via reflex in seed aging through seed respiration.

Gene stacking as a strategy to confer characteristics of agronomic importance in plants by genetic engineering / Empilhamento gênico como estratégia para conferir características de importância agronômica em plantas via engenharia genética

ABSTRACT: Gene stacking refers to the introduction of two or more transgenes of agronomic interest in the same plant. The main methods for genetically engineering plants with gene stacking involve (i) the simultaneous introduction, by the co-transformation process, and (ii) the sequential introduction of genes using the re-transformation processes or the sexual crossing between separate transgenic events. In general, the choice of the best method varies according to the species of interest and the availability of genetic constructions and preexisting transgenic events. We also present here the use of minichromosome technology as a potential future gene stacking technology. The purpose of this review was to discuss aspects related to the methodology for gene stacking and trait stacking (a gene stacking strategy to combine characteristics of agronomical importance) by genetic engineering. In addition, we presented a list of crops and genes approved commercially that have been used in stacking strategies for combined characteristics and a discussion about the regulatory standards. An increased number of approved and released gene stacking events reached the market in the last decade. Initially, the most common combined characteristics were herbicide tolerance and insect resistance in soybean and maize. Recently, commercially available varieties were released combining these traits with drought tolerance in these commodities. New traits combinations are reaching the farmer's fields, including higher quality, disease resistant and nutritional value improved. In other words, gene stacking is growing as a strategy to contribute to food safety and sustainability.

RESUMO: O empilhamento gênico se refere a introdução de dois ou mais transgenes de interesse agronômico na mesma planta. Os principais métodos de produção de plantas geneticamente modificadas com empilhamento gênico envolvem (i) a introdução simultânea, pelo processo de co-transformação, e (ii) a introdução sequencial de genes, pelos processos de re-transformação ou por cruzamento entre eventos transgênicos. Em geral, a escolha do melhor método varia de acordo com a espécie de interesse e a disponibilidade de construções genéticas e eventos transgênicos preexistentes. Também é apresentado aqui o uso da tecnologia de minicromossomos como tecnologia potencial de empilhamento gênico. O objetivo desta revisão é discutir aspectos relacionados à metodologia para o empilhamento de genes a combinação de características (obtida via empilhamento de genes de interesse agronômico) via engenharia genética. Além de discutir, é apresentado uma lista de culturas e genes aprovados comercialmente que tem sido usado em estratégias de empilhamento e uma discussão sobre normas regulatórias. Um número maior de eventos com empilhamento de genes foi aprovado e liberado no mercado na última década. Inicialmente, a combinação das características de tolerância a herbicidas e resistência a insetos era a mais popular, principalmente em soja e milho. Recentemente, estas características combinadas com tolerância a seca nessas culturas foram liberadas comercialmente. Novas características combinadas estão entrando na lavoura, incluindo aumento da qualidade, resistência a doenças e aumento do valor nutricional. Em outras palavras, o empilhamento gênico está crescendo como tecnologia para contribuir para a segurança alimentar e sustentabilidade.

Physiologic alterations in orthodox seeds due to deterioration processes.

Seed deterioration is a partially elucidated phenomenon that happen during the life of the seed. This review describes the processes that lead to seed deterioration, including loss of seed protection capacity against reactive oxygen species (ROS), damage to the plasma membrane, consumption of reserves, and damage to genetic material. A hypothesis of how seed deterioration occurs was also addressed; in this hypothesis, seed deterioration was divided into three phases. The first is the beginning of deterioration, with a slight reduction of vigor caused by the reactions of reducing sugars with antioxidant enzymes and genetic material. In the second, the cell shows oxidative damages, causing lipid peroxidation, which leads to the leaching of solutes, the formation of malondialdehyde, and, consequently, an increase in damages to genetic material. In the third phase, there is cell collapse with mitochondrial membrane deconstruction and a high accumulation of reactive oxygen species, malondialdehyde, and reducing sugars.

Defenses Against ROS in Crops and Weeds: The Effects of Interference and Herbicides.

The antioxidant defense system acts to maintain the equilibrium between the production of reactive oxygen species (ROS) and the elimination of toxic levels of ROS in plants. Overproduction and accumulation of ROS results in metabolic disorders and can lead to the oxidative destruction of the cell. Several stress factors cause ROS overproduction and trigger oxidative stress in crops and weeds. Recently, the involvement of the antioxidant system in weed interference and herbicide treatment in crops and weeds has been the subject of investigation. In this review, we address ROS production and plant mechanisms of defense, alterations in the antioxidant system at transcriptional and enzymatic levels in crops induced by weed interference, and herbicide exposure in crops and weeds. We also describe the mechanisms of action in herbicides that lead to ROS generation in target plants. Lastly, we discuss the relations between antioxidant systems and weed biology and evolution, as well as the interactive effects of herbicide treatment on these factors.

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.

Antioxidant responses of wheat plants under stress.

Currently, food security depends on the increased production of cereals such as wheat (Triticum aestivum L.), which is an important source of calories and protein for humans. However, cells of the crop have suffered from the accumulation of reactive oxygen species (ROS), which can cause severe oxidative damage to the plants, due to environmental stresses. ROS are toxic molecules found in various subcellular compartments. The equilibrium between the production and detoxification of ROS is sustained by enzymatic and nonenzymatic antioxidants. In the present review, we offer a brief summary of antioxidant defense and hydrogen peroxide (H2O2) signaling in wheat plants. Wheat plants increase antioxidant defense mechanisms under abiotic stresses, such as drought, cold, heat, salinity and UV-B radiation, to alleviate oxidative damage. Moreover, H2O2 signaling is an important factor contributing to stress tolerance in cereals.

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.

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.

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 H(2)O(2) into H(2)O, 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.