Physiological response of Symbiodiniaceae to thermal stress: Reactive oxygen species, photosynthesis, and relative cell size.

This study investigates the physiological response to heat stress of three genetically different Symbiodiniaceae strains isolated from the scleractinian coral Mussismilia braziliensis, endemic of the Abrolhos Bank, Brazil. Cultures of two Symbiodinium sp. and one Cladocopium sp. were exposed to a stepwise increase in temperature (2°C every second day) ranging from 26°C (modal temperature in Abrolhos) to 32°C (just above the maximum temperature registered in Abrolhos during the third global bleaching event-TGBE). After the cultures reached their final testing temperature, reactive oxygen species (ROS) production, single cell attributes (relative cell size and chlorophyll fluorescence), and photosynthetic efficiency (effective (Y(II)) and maximum (Fv/Fm) quantum yields) were measured within 4 h and 72 h. Non-photochemical coefficient (NPQ) was estimated based on fluorescence values. Population average ROS production was variable across strains and exposure times, reaching up a 2-fold increase at 32°C in one of the Symbiodinium sp. strains. A marked intrapopulation difference was observed in ROS production, with 5 to 25% of the cells producing up to 10 times more than the population average, highlighting the importance of single cell approaches to assess population physiology. Average cell size increases at higher temperatures, likely resulting from cell cycle arrest, whereas chlorophyll fluorescence decreased, especially in 4 h, indicating a photoacclimation response. The conditions tested do not seem to have elicited loss of photosynthetic efficiency nor the activation of non-photochemical mechanisms in the cells. Our results unveiled a generalized thermotolerance in three Symbiodiniaceae strains originated from Abrolhos' corals. Inter and intra-specific variability could be detected, likely reflecting the genetic differences among the strains.

Genome-wide identification of core components of ABA signaling and transcriptome analysis reveals gene circuits involved in castor bean (Ricinus communis L.) response to drought.

Castor bean (Ricinus communis L.) can withstand long periods of water deficit and high temperatures, and therefore has been recognized as a drought-resistant plant species, allowing the study of gene networks involved in drought response and tolerance. The identification of genes networks related to drought response in this plant may yield important information in the characterization of molecular mechanisms correlating changes in the gene expression with the physiological adaptation processes. In this context, gene families related to abscisic acid (ABA) signaling play a crucial role in developmental and environmental adaptation processes of plants to drought stress. However, the families that function as the core components of ABA signaling, as well as genes networks related to drought response, are not well understood in castor bean. In this study 7 RcPYL, 63 RcPP2C, and 6 RcSnRK2 genes were identified in castor bean genome, which was further supported by chromosomal distribution, gene structure, evolutionary relationships, and conserved motif analyses. The castor bean general expression profile was investigated by RNAseq in root and leaf tissues in response to drought stress. These analyses allowed the identification of genes differentially expressed, including genes from the ABA signaling core, genes related to photosynthesis, cell wall, energy transduction, antioxidant response, and transcription factors. These analyses provide new insights into the core components of ABA signaling in castor bean, allow the identification of several molecular responses associated with the high physiological adaptation of castor bean to drought stress, and contribute to the identification of candidate genes for genetic improvement.

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.

Chloroplastic ascorbate peroxidases targeted to stroma or thylakoid membrane: The chicken or egg dilemma.

Ascorbate peroxidases (APXs) are heme peroxidases that remove hydrogen peroxide in different subcellular compartments with concomitant ascorbate cycling. Here, we analysed and discussed phylogenetic and molecular features of the APX family. Ancient APX originated as a soluble stromal enzyme, and early during plant evolution, acquired both chloroplast-targeting and mitochondrion-targeting sequences and an alternative splicing mechanism whereby it could be expressed as a soluble or thylakoid membrane-bound enzyme. Later, independent duplication and neofunctionalization events in some angiosperm groups resulted in individual genes encoding stromal, thylakoidal and mitochondrial isoforms. These data reaffirm the complexity of plant antioxidant defenses that allow diverse plant species to acquire new means to adapt to changing environmental conditions.

Salicylic acid and adenine nucleotides regulate the electron transport system and ROS production in plant mitochondria.

Although mitochondria have a central role in energy transduction and reactive oxygen species (ROS) production, the regulatory mechanisms and their involvement in plant stress signaling are not fully established. The phytohormone salicylic acid (SA) is an important regulator of mitochondria-mediated ROS production and defense signaling. The role of SA and adenine nucleotides in the regulation of the mitochondrial succinate dehydrogenase (SDH) complex activity and ROS production was analyzed using WT, RNAi SDH1-1 and disrupted stress response 1 (dsr1) mutants, which show a point mutation in SDH1 subunit and are defective in SA signaling. Our results showed that SA and adenine nucleotides regulate SDH complex activity by distinct patterns, contributing to increased SDH-derived ROS production. As previously demonstrated, SA induces the succinate-quinone reductase activity of SDH complex, acting at or near the ubiquinone binding site. On the other hand, here we demonstrated that adenine nucleotides, such as AMP, ADP and ATP, induce the SDH activity provided by the SDH1 subunit. The regulation of SDH activity by adenine nucleotides is dependent on mitochondrial integrity and is prevented by atractyloside, an inhibitor of adenine nucleotide translocator (ANT), suggesting that the regulatory mechanism occurs on the mitochondrial matrix side of the inner mitochondrial membrane, and not in the intermembrane space, as previously suggested. On the other hand, in the intermembrane space, ADP and ATP limit mitochondrial oxygen consumption by a mechanism that appears to be related to cytochrome bc1 complex inhibition. Altogether, these results indicate that SA signaling and adenine nucleotides regulate the mitochondrial electron transport system and mitochondria-derived ROS production by direct effect in the electron transport system complexes, bringing new insights into mechanisms with direct implications in plant development and responses to different environmental responses, serving as a starting point for future physiological explorations.

Ascorbate-Glutathione Cycle Genes Families in Euphorbiaceae: Characterization and Evolutionary Analysis.

Ascorbate peroxidase (APX), Monodehydroascorbate Reductase (MDAR), Dehydroascorbate Reductase (DHAR) and Glutathione Reductase (GR) enzymes participate in the ascorbate-glutathione cycle, which exerts a central role in the antioxidant metabolism in plants. Despite the importance of this antioxidant system in different signal transduction networks related to development and response to environmental stresses, the pathway has not yet been comprehensively characterized in many crop plants. Among different eudicotyledons, the Euphorbiaceae family is particularly diverse with some species highly tolerant to drought. Here the APX, MDAR, DHAR, and GR genes in Ricinus communis, Jatropha curcas, Manihot esculenta, and Hevea brasiliensis were identified and characterized. The comprehensive phylogenetic and genomic analyses allowed the classification of the genes into different classes, equivalent to cytosolic, peroxisomal, chloroplastic, and mitochondrial enzymes, and revealed the duplication events that contribute to the expansion of these families within plant genomes. Due to the high drought stress tolerance of Ricinus communis, the expression patterns of ascorbate-glutathione cycle genes in response to drought were also analyzed in leaves and roots, indicating a differential expression during the stress. Altogether, these data contributed to the characterization of the expression pattern and evolutionary analysis of these genes, filling the gap in the proposed functions of core components of the antioxidant mechanism during stress response in an economically relevant group of plants.

Genome-wide analysis of general phenylpropanoid and monolignol-specific metabolism genes in sugarcane.

Lignin is the main component of secondary cell walls and is essential for plant development and defense. However, lignin is recognized as a major recalcitrant factor for efficiency of industrial biomass processing. Genes involved in general phenylpropanoid and monolignol-specific metabolism in sugarcane have been previously analyzed at the transcriptomic level. Nevertheless, the number of genes identified in this species is still very low. The recently released sugarcane genome sequence has allowed the genome-wide characterization of the 11 gene families involved in the monolignol biosynthesis branch of the phenylpropanoid pathway. After an exhaustive analysis of sugarcane genomes, 438 haplotypes derived from 175 candidate genes from Saccharum spontaneum and 144 from Saccharum hybrid R570 were identified as associated with this biosynthetic route. The phylogenetic analyses, combined with the search for protein conserved residues involved in the catalytic activity of the encoded enzymes, were employed to identify the family members potentially involved in developmental lignification. Accordingly, 15 candidates were identified as bona fide lignin biosynthesis genes: PTAL1, PAL2, C4H4, 4CL1, HCT1, HCT2, C3'H1, C3'H2, CCoAOMT1, COMT1, F5H1, CCR1, CCR2, CAD2, and CAD7. For this core set of lignin biosynthetic genes, we searched for the chromosomal location, the gene expression pattern, the promoter cis-acting elements, and microRNA targets. Altogether, our results present a comprehensive characterization of sugarcane general phenylpropanoid and monolignol-specific genes, providing the basis for further functional studies focusing on lignin biosynthesis manipulation and biotechnological strategies to improve sugarcane biomass utilization.

The mitochondrial isoform glutathione peroxidase 3 (OsGPX3) is involved in ABA responses in rice plants.

Different environmental conditions can lead plants to a condition termed oxidative stress, which is characterized by a disruption in the equilibrium between the production of reactive oxygen species (ROS) and antioxidant defenses. Glutathione peroxidase (GPX), an enzyme that acts as a peroxide scavenger in different organisms, has been identified as an important component in the signaling pathway during the developmental process and in stress responses in plants and yeast. Here, we demonstrate that the mitochondrial isoform of rice (Oryza sativa L. ssp. Japonica cv. Nipponbare) OsGPX3 is induced after treatment with the phytohormone abscisic acid (ABA) and is involved in its responses and in epigenetic modifications. Plants that have been silenced for OsGPX3 (gpx3i) present substantial changes in the accumulation of proteins related to these processes. These plants also have several altered ABA responses, such as germination, ROS accumulation, stomatal closure, and dark-induced senescence. This study is the first to demonstrate that OsGPX3 plays a role in ABA signaling and corroborate that redox homeostasis enzymes can act in different and complex pathways in plant cells. SIGNIFICANCE: This work proposes the mitochondrial glutathione peroxidase (OsGPX3) as a novel ABA regulatory pathway component. Our results suggest that this antioxidant enzyme is involved in ABA-responses, highlighting the complex pathways that these proteins can participate beyond the regulation of cellular redox status.

Identification of genes from the general phenylpropanoid and monolignol-specific metabolism in two sugarcane lignin-contrasting genotypes.

The phenylpropanoid pathway is an important route of secondary metabolism involved in the synthesis of different phenolic compounds such as phenylpropenes, anthocyanins, stilbenoids, flavonoids, and monolignols. The flux toward monolignol biosynthesis through the phenylpropanoid pathway is controlled by specific genes from at least ten families. Lignin polymer is one of the major components of the plant cell wall and is mainly responsible for recalcitrance to saccharification in ethanol production from lignocellulosic biomass. Here, we identified and characterized sugarcane candidate genes from the general phenylpropanoid and monolignol-specific metabolism through a search of the sugarcane EST databases, phylogenetic analysis, a search for conserved amino acid residues important for enzymatic function, and analysis of expression patterns during culm development in two lignin-contrasting genotypes. Of these genes, 15 were cloned and, when available, their loci were identified using the recently released sugarcane genomes from Saccharum hybrid R570 and Saccharum spontaneum cultivars. Our analysis points out that ShPAL1, ShPAL2, ShC4H4, Sh4CL1, ShHCT1, ShC3H1, ShC3H2, ShCCoAOMT1, ShCOMT1, ShF5H1, ShCCR1, ShCAD2, and ShCAD7 are strong candidates to be bona fide lignin biosynthesis genes. Together, the results provide information about the candidate genes involved in monolignol biosynthesis in sugarcane and may provide useful information for further molecular genetic studies in sugarcane.

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.

Fumarate reductase superfamily: A diverse group of enzymes whose evolution is correlated to the establishment of different metabolic pathways.

Fumarate and succinate are known to be present in prebiotic systems essential for the origin of life. The fumarate and succinate interconversion reactions have been conserved throughout evolution and are found in all living organisms. The fumarate and succinate interconversion is catalyzed by the enzymes succinate dehydrogenase (SDH) and fumarate reductase (FRD). In this work we show that SDH and FRD are part of a group of enzymes that we propose to designate "fumarate reductase superfamily". Our results demonstrate that these enzymes emerged from a common ancestor and were essential in the development of metabolic pathways involved in energy transduction.

3-Bromopyruvic Acid Inhibits Tricarboxylic Acid Cycle and Glutaminolysis in HepG2 Cells.

BACKGROUND/AIM: 3-bromopyruvate (3BrPA) is an antitumor agent able to inhibit aerobic glycolysis and oxidative phosphorylation, therefore inducing cell death. However, cancer cells are also highly dependent of glutaminolysis and tricarboxylic acid cycle (TCA) regarding survival and 3BrPA action in these metabolic routes is poorly understood. MATERIALS AND METHODS: The effect of 3BrPA was characterized in mice liver and kidney mitochondria, as well as in human HepG2 cells. RESULTS: Low concentration of 3-BrPA significantly affected both glutaminolysis and TCA cycle functions, through inhibition of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and succinate dehydrogenase. Additionally, 3-BrPA treatment significantly decreased the reduced status of thiol groups in HepG2 cells without proportional increase of oxidizing groups, suggesting that these chemical groups are the target of alkylation reactions induced by 3-BrPA. CONCLUSION: This work demonstrates, for the first time, the effect of 3-BrPA in glutaminolysis and TCA cycle. Our results suggest that the combined action of 3-BrPA in glutaminolysis, TCA and glycolysis, inhibiting steps downstream of the glucose and glutamine metabolism, has an antitumor effect.

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 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.

3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 µM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA.