The Experts below are selected from a list of 12465 Experts worldwide ranked by ideXlab platform
Ron Mittler - One of the best experts on this subject based on the ideXlab platform.
-
Ascorbate Peroxidase 1 plays a key role in the response of arabidopsis thaliana to stress combination
Journal of Biological Chemistry, 2008Co-Authors: Shai Koussevitzky, Vladimir Shulaev, Nobuhiro Suzuki, Serena Huntington, Leigh Armijo, Wei Sha, Diego F Cortes, Ron MittlerAbstract:Within their natural habitat plants are subjected to a combination of different abiotic stresses, each with the potential to exacerbate the damage caused by the others. One of the most devastating stress combinations for crop productivity, which frequently occurs in the field, is drought and heat stress. In this study we conducted proteomic and metabolic analysis of Arabidopsis thaliana plants subjected to a combination of drought and heat stress. We identified 45 different proteins that specifically accumulated in Arabidopsis in response to the stress combination. These included enzymes involved in reactive oxygen detoxification, malate metabolism, and the Calvin cycle. The accumulation of malic enzyme during the combined stress corresponded with enhanced malic enzyme activity, a decrease in malic acid, and lower amounts of oxaloacetate, suggesting that malate metabolism plays an important role in the response of Arabidopsis to the stress combination. Cytosolic Ascorbate Peroxidase 1 (APX1) protein and mRNA accumulated during the stress combination. When exposed to heat stress combined with drought, an APX1-deficient mutant (apx1) accumulated more hydrogen peroxide and was significantly more sensitive to the stress combination than wild type. In contrast, mutants deficient in thylakoid or stromal/mitochondrial APXs were not more sensitive to the stress combination than apx1 or wild type. Our findings suggest that cytosolic APX1 plays a key role in the acclimation of plants to a combination of drought and heat stress.
-
cytosolic Ascorbate Peroxidase 1 is a central component of the reactive oxygen gene network of arabidopsis
The Plant Cell, 2005Co-Authors: Sholpan Davletova, Ludmila Rizhsky, Hongjian Liang, Zong Shengqiang, David J Oliver, Jesse Coutu, Vladimir Shulaev, Karen Schlauch, Ron MittlerAbstract:Reactive oxygen species (ROS), such as O2 � and H2O2, play a key role in plant metabolism, cellular signaling, and defense. In leaf cells, the chloroplast is considered to be a focal point of ROS metabolism. It is a major producer of O2 � and H2O2 during photosynthesis, and it contains a large array of ROS-scavenging mechanisms that have been extensively studied. By contrast, the function of the cytosolic ROS-scavenging mechanisms of leaf cells is largely unknown. In this study, we demonstrate that in the absence of the cytosolic H2O2-scavenging enzyme Ascorbate Peroxidase 1 (APX1), the entire chloroplastic H2O2-scavenging system of Arabidopsis thaliana collapses, H2O2 levels increase, and protein oxidation occurs. We further identify specific proteins oxidized in APX1-deficient plants and characterize the signaling events that ensue in knockout-Apx1 plants in response to a moderate level of light stress. Using a dominant-negative approach, we demonstrate that heat shock transcription factors play a central role in the early sensing of H2O2 stress in plants. Using knockout plants for the NADPH oxidase D protein (knockout-RbohD), we demonstrate that RbohD might be required for ROS signal amplification during light stress. Our study points to a key role for the cytosol in protecting the chloroplast during light stress and provides evidence for cross-compartment protection of thylakoid and stromal/mitochondrial APXs by cytosolic APX1.
-
double antisense plants lacking Ascorbate Peroxidase and catalase are less sensitive to oxidative stress than single antisense plants lacking Ascorbate Peroxidase or catalase
Plant Journal, 2002Co-Authors: Ludmila Rizhsky, Elza Hallakherr, Frank Van Breusegem, Shimon Rachmilevitch, Jason E Barr, Steven R Rodermel, Dirk Inze, Ron MittlerAbstract:The plant genome is a highly redundant and dynamic genome. Here, we show that double antisense plants lacking the two major hydrogen peroxide-detoxifying enzymes, Ascorbate Peroxidase (APX) and catalase (CAT), activate an alternative/redundant defense mechanism that compensates for the lack of APX and CAT. A similar mechanism was not activated in single antisense plants that lacked APX or CAT, paradoxically rendering these plants more sensitive to oxidative stress compared to double antisense plants. The reduced susceptibility of double antisense plants to oxidative stress correlated with suppressed photosynthetic activity, the induction of metabolic genes belonging to the pentose phosphate pathway, the induction of monodehydroAscorbate reductase, and the induction of IMMUTANS, a chloroplastic homologue of mitochondrial alternative oxidase. Our results suggest that a co-ordinated induction of metabolic and defense genes, coupled with the suppression of photosynthetic activity, can compensate for the lack of APX and CAT. In addition, our findings demonstrate that the plant genome has a high degree of plasticity and will respond differently to different stressful conditions, namely, lack of APX, lack of CAT, or lack of both APX and CAT.
-
post transcriptional suppression of cytosolic Ascorbate Peroxidase expression during pathogen induced programmed cell death in tobacco
The Plant Cell, 1998Co-Authors: Ron Mittler, Xuqiao Feng, Mira CohenAbstract:As a means to eliminate pathogen-infected cells and prevent diseases, programmed cell death (PCD) appears to be a defense strategy employed by most multicellular organisms. Recent studies have indicated that reactive oxygen species, such as O2.- and H2O2, play a central role in the activation and propagation of pathogen-induced PCD in plants. However, plants contain several mechanisms that detoxify O2.- and H2O2 and may inhibit PCD. We found that during viral-induced PCD in tobacco, the expression of cytosolic Ascorbate Peroxidase (cAPX), a key H2O2 detoxifying enzyme, is post-transcriptionally suppressed. Thus, although the steady state level of transcripts encoding cAPX was induced during PCD, as expected under conditions of elevated H2O2, the level of the cAPX protein declined. In vivo protein labeling, followed by immunoprecipitation, indicated that the synthesis of the cAPX protein was inhibited. Although transcripts encoding cAPX were found to associate with polysomes during PCD, no cAPX protein was detected after in vitro polysome run-off assays. Our findings suggest that viral-induced PCD in tobacco is accompanied by the suppression of cAPX expression, possibly at the level of translation elongation. This suppression is likely to contribute to a reduction in the capability of cells to scavenge H2O2, which in turn enables the accumulation of H2O2 and the acceleration of PCD.
-
regulation of pea cytosolic Ascorbate Peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought
Plant Journal, 1994Co-Authors: Ron Mittler, Barbara A ZilinskasAbstract:Summary The molecular mechanism underlying the regulation of the expression of Apxl, the gene encoding cytosolic Ascorbate Peroxidase, as well as other antioxidant enzymes, was studied during the progression of drought stress and following recovery from drought. Increase in steady-state transcript levels of the cytosolic isozymes of Ascorbate Peroxidase and Cu/Zn-superoxide dismutase paralleled the increase in stomatal resistance during drought stress, but was even more dramatically enhanced following recovery from drought. Cytosolic Cu/Zn-superoxide dismutase and Ascorbate Peroxidase and chloroplastic Cu/Zn-superoxide dismutase protein and activity increased during drought stress and following recovery. In contrast, catalase activity increased during drought stress but returned to normal levels following recovery. During recovery from drought stress, cytosolic Ascorbate Peroxidase expression was regulated post-transcriptionally at the level of protein synthesis. The transcription rate of the Apxl gene, as determined by nuclear run-on assay, increased during drought stress and at 10 h following rewatering.
Barbara A Zilinskas - One of the best experts on this subject based on the ideXlab platform.
-
regulation of pea cytosolic Ascorbate Peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought
Plant Journal, 1994Co-Authors: Ron Mittler, Barbara A ZilinskasAbstract:Summary The molecular mechanism underlying the regulation of the expression of Apxl, the gene encoding cytosolic Ascorbate Peroxidase, as well as other antioxidant enzymes, was studied during the progression of drought stress and following recovery from drought. Increase in steady-state transcript levels of the cytosolic isozymes of Ascorbate Peroxidase and Cu/Zn-superoxide dismutase paralleled the increase in stomatal resistance during drought stress, but was even more dramatically enhanced following recovery from drought. Cytosolic Cu/Zn-superoxide dismutase and Ascorbate Peroxidase and chloroplastic Cu/Zn-superoxide dismutase protein and activity increased during drought stress and following recovery. In contrast, catalase activity increased during drought stress but returned to normal levels following recovery. During recovery from drought stress, cytosolic Ascorbate Peroxidase expression was regulated post-transcriptionally at the level of protein synthesis. The transcription rate of the Apxl gene, as determined by nuclear run-on assay, increased during drought stress and at 10 h following rewatering.
-
detection of Ascorbate Peroxidase activity in native gels by inhibition of the Ascorbate dependent reduction of nitroblue tetrazolium
Analytical Biochemistry, 1993Co-Authors: Ron Mittler, Barbara A ZilinskasAbstract:Abstract A method for the detection of Ascorbate Peroxidase activity in native electrophoretic gels is described. The assay is based on the ability of Ascorbate Peroxidase prevent the Ascorbate-dependent reduction of nitroblue tetrazolium in the presence of H 2 O 2 . The method was found to be both sensitive (detection of less than 0.01 units of Ascorbate Peroxidase activity) and specific for Ascorbate Peroxidase activity. The application of the method for the detection of Ascorbate Peroxidase activity in protein extracts from several plant sources was investigated by comparing staining for activities of Ascorbate Peroxidase, horseradish Peroxidase, and Ascorbate oxidase and by immunodetection of Ascorbate Peroxidase in these extracts.
-
molecular cloning and characterization of a gene encoding pea cytosolic Ascorbate Peroxidase
Journal of Biological Chemistry, 1992Co-Authors: Ron Mittler, Barbara A ZilinskasAbstract:A gene encoding cytosolic Ascorbate Peroxidase (ApxI) from pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. By homologous alignment between the ApxI cDNA (Mittler, R., and Zilinskas, B. (1991) FEBS Lett. 289, 257-259) and the genomic clone, positions of introns and exons were determined. The isolated ApxI gene was found to contain 9 introns, the first of which was located within the 5'-untranslated region of the mRNA. Southern blot analysis of pea genomic DNA suggests that in pea cytosolic Ascorbate Peroxidase is encoded by a single copy gene. Steady state ApxI transcript levels were found to increase in response to several stresses imposed by drought, heat, and application of ethephon, abscisic acid, and the superoxide-generating agent paraquat. Increases in Ascorbate Peroxidase activity in response to stresses were less marked than changes observed in transcript levels; cytosolic Ascorbate Peroxidase protein levels measured by immunoblot analysis remained unchanged.
-
purification and characterization of pea cytosolic Ascorbate Peroxidase
Plant Physiology, 1991Co-Authors: Ron Mittler, Barbara A ZilinskasAbstract:The cytosolic isoform of Ascorbate Peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with Ascorbate Peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant Peroxidases, the cytosolic Ascorbate Peroxidase had a very high preference for Ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic Ascorbate Peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish Peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic Ascorbate Peroxidase, the tea chloroplastic Ascorbate Peroxidase, and horseradish Peroxidase; homology was, however, found with chloroplastic Ascorbate Peroxidase isolated from spinach leaves.
-
molecular cloning and nucleotide sequence analysis of a cdna encoding pea cytosolic Ascorbate Peroxidase
FEBS Letters, 1991Co-Authors: Ron Mittler, Barbara A ZilinskasAbstract:A cDNA clone encoding the cytosolic Ascorbate Peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While Ascorbate Peroxidase shares limited overall homology with other Peroxidases, significant homology with all known Peroxidases was found in the vicinity of the putative active site.
Shigeru Shigeoka - One of the best experts on this subject based on the ideXlab platform.
-
diversity and evolution of Ascorbate Peroxidase functions in chloroplasts more than just a classical antioxidant enzyme
Plant and Cell Physiology, 2016Co-Authors: Shigeru Shigeoka, Takanori Maruta, Yoshihiro Sawa, Takahiro IshikawaAbstract:Reactive oxygen species (ROS) have dual functions in plant cells as cytotoxic molecules and emergency signals. The balance between the production and scavenging of these molecules in chloroplasts, major sites for the production of ROS, is one of the key determinants for plant acclimation to stress conditions. The water-water cycle is a crucial regulator of ROS levels in chloroplasts. In this cycle, the stromal and thylakoid membrane-attached isoforms of Ascorbate Peroxidase (sAPX and tAPX, respectively) are involved in the metabolism of H2O2 Current genome and phylogenetic analyses suggest that the first monofunctional APX was generated as sAPX in unicellular green algae, and that tAPX occurred in multicellular charophytes during plant evolution. Chloroplastic APXs, especially tAPX, have been considered to be the source of a bottleneck in the water-water cycle, at least in higher plants, because of their high susceptibility to H2O2 A number of studies have succeeded in improving plant stress resistance by reinforcing the fragile characteristics of the enzymes. However, researchers have unexpectedly failed to find a 'stress-sensitive phenotype' among loss-of-function mutants, at least in laboratory conditions. Interestingly, the susceptibility of enzymes to H2O2 may have been acquired during plant evolution, thereby allowing for the flexible use of H2O2 as a signaling molecule in plants, and this is supported by growing lines of evidence for the physiological significance of chloroplastic H2O2 as a retrograde signal in plant stress responses. By overviewing historical, biochemical, physiological and genetic studies, we herein discuss the diverse functions of chloroplastic APXs as antioxidant enzymes and signaling modulators.
-
euglena gracilis Ascorbate Peroxidase forms an intramolecular dimeric structure its unique molecular characterization
Biochemical Journal, 2010Co-Authors: Takahiro Ishikawa, Naoko Tajima, Hitoshi Nishikawa, Yongshun Gao, Madhusudhan Rapolu, Hitoshi Shibata, Yoshihiro Sawa, Shigeru ShigeokaAbstract:Euglena gracilis lacks a catalase and contains a single APX (Ascorbate Peroxidase) and enzymes related to the redox cycle of Ascorbate in the cytosol. In the present study, a full-length cDNA clone encoding the Euglena APX was isolated and found to contain an open reading frame encoding a protein of 649 amino acids with a calculated molecular mass of 70.5 kDa. Interestingly, the enzyme consisted of two entirely homologous catalytic domains, designated APX-N and APX-C, and an 102 amino acid extension in the N-terminal region, which had a typical class II signal proposed for plastid targeting in Euglena . A computer-assisted analysis indicated a novel protein structure with an intramolecular dimeric structure. The analysis of cell fractionation showed that the APX protein is distributed in the cytosol, but not the plastids, suggesting that Euglena APX becomes mature in the cytosol after processing of the precursor. The kinetics of the recombinant mature FL (full-length)-APX and the APX-N and APX-C domains with Ascorbate and H 2 O 2 were almost the same as that of the native enzyme. However, the substrate specificity of the mature FL-APX and the native enzyme was different from that of APX-N and APX-C. The mature FL-APX, but not the truncated forms, could reduce alkyl hydroperoxides, suggesting that the dimeric structure is correlated with substrate recognition. In Euglena cells transfected with double-stranded RNA, the silencing of APX expression resulted in a significant increase in the cellular level of H 2 O 2 , indicating the physiological importance of APX to the metabolism of H 2 O 2 .
-
arabidopsis chloroplastic Ascorbate Peroxidase isoenzymes play a dual role in photoprotection and gene regulation under photooxidative stress
Plant and Cell Physiology, 2010Co-Authors: Takanori Maruta, Kazuya Yoshimura, Masahiro Tamoi, Takahiro Ishikawa, Yukinori Yabuta, Aoi Tanouchi, Shigeru ShigeokaAbstract:: Though two types of chloroplastic Ascorbate Peroxidase (APX) located in the thylakoid membrane (tAPX) and stroma (sAPX) have been thought to be key regulators of intracellular levels of H(2)O(2), their physiological significance in the response to photooxidative stress is still under discussion. Here we characterized single mutants lacking either tAPX (KO-tAPX) or sAPX (KO-sAPX). Under exposure to high light or treatment with methylviologen under light, H(2)O(2) and oxidized proteins accumulated to higher levels in both mutant plants than in the wild-type plants. On the other hand, the absence of sAPX and tAPX drastically suppressed the expression of H(2)O(2)-responsive genes under photooxidative stress. Interestingly, the most marked effect of photooxidative stress on the accumulation of H(2)O(2) and oxidized protein and gene expression was observed in the KO-tAPX plants rather than the KO-sAPX plants. The present findings suggest that both chloroplastic APXs, but particularly tAPX, are important for photoprotection and gene regulation under photooxidative stress in Arabidopsis leaves.
-
thylakoid membrane bound Ascorbate Peroxidase is a limiting factor of antioxidative systems under photo oxidative stress
Plant Journal, 2002Co-Authors: Yukinori Yabuta, Kazuya Yoshimura, Toru Takeda, Takahiro Ishikawa, Takashi Motoki, Shigeru ShigeokaAbstract:To evaluate the physiological importance of thylakoid membrane-bound Ascorbate Peroxidase (tAPX) in the active oxygen species-scavenging system of chloroplasts, the level of tAPX in tobacco plants was altered by expression of the tAPX cDNA in both sense and antisense orientation. The tobacco plants transformed with constructs of antisense tAPXs from spinach and tobacco could not be obtained, suggesting that the suppression of tAPX in higher plants had a severe effect on the growth even under normal conditions. In contrast, the transgenic tobacco plants (TpTAP-12) overexpressing tAPX, which had approximately 37-fold higher activity than that of the wild-type plants, were generated. The TpTAP-12 plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV, 50 microm) under light intensity (300 and 1600 microE m(-2) sec(-1)) and by chilling stress with high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)). At 24 h after the MV treatment under illumination at 300 microE m-2 sec-1, destruction of chlorophyll was observed in the wild-type plants, but not in the TpTAP-12 plants. The activities of thiol-modulated enzymes in the Calvin cycle, the level and redox status of Ascorbate (AsA), and the activity of tAPX in the wild-type plants significantly decreased, while those in the TpTAP-12 plants were hardly changed. These observations suggest that tAPX is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts, and that the enhanced activity of tAPX functions to maintain the AsA content and the redox status of AsA under stress conditions.
-
identification of a cis element for tissue specific alternative splicing of chloroplast Ascorbate Peroxidase pre mrna in higher plants
Journal of Biological Chemistry, 2002Co-Authors: Kazuya Yoshimura, Takahiro Ishikawa, Yukinori Yabuta, Shigeru ShigeokaAbstract:Alternative splicing events in the 3'-terminal region of chloroplast Ascorbate Peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leaf in vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.
Takahiro Ishikawa - One of the best experts on this subject based on the ideXlab platform.
-
diversity and evolution of Ascorbate Peroxidase functions in chloroplasts more than just a classical antioxidant enzyme
Plant and Cell Physiology, 2016Co-Authors: Shigeru Shigeoka, Takanori Maruta, Yoshihiro Sawa, Takahiro IshikawaAbstract:Reactive oxygen species (ROS) have dual functions in plant cells as cytotoxic molecules and emergency signals. The balance between the production and scavenging of these molecules in chloroplasts, major sites for the production of ROS, is one of the key determinants for plant acclimation to stress conditions. The water-water cycle is a crucial regulator of ROS levels in chloroplasts. In this cycle, the stromal and thylakoid membrane-attached isoforms of Ascorbate Peroxidase (sAPX and tAPX, respectively) are involved in the metabolism of H2O2 Current genome and phylogenetic analyses suggest that the first monofunctional APX was generated as sAPX in unicellular green algae, and that tAPX occurred in multicellular charophytes during plant evolution. Chloroplastic APXs, especially tAPX, have been considered to be the source of a bottleneck in the water-water cycle, at least in higher plants, because of their high susceptibility to H2O2 A number of studies have succeeded in improving plant stress resistance by reinforcing the fragile characteristics of the enzymes. However, researchers have unexpectedly failed to find a 'stress-sensitive phenotype' among loss-of-function mutants, at least in laboratory conditions. Interestingly, the susceptibility of enzymes to H2O2 may have been acquired during plant evolution, thereby allowing for the flexible use of H2O2 as a signaling molecule in plants, and this is supported by growing lines of evidence for the physiological significance of chloroplastic H2O2 as a retrograde signal in plant stress responses. By overviewing historical, biochemical, physiological and genetic studies, we herein discuss the diverse functions of chloroplastic APXs as antioxidant enzymes and signaling modulators.
-
euglena gracilis Ascorbate Peroxidase forms an intramolecular dimeric structure its unique molecular characterization
Biochemical Journal, 2010Co-Authors: Takahiro Ishikawa, Naoko Tajima, Hitoshi Nishikawa, Yongshun Gao, Madhusudhan Rapolu, Hitoshi Shibata, Yoshihiro Sawa, Shigeru ShigeokaAbstract:Euglena gracilis lacks a catalase and contains a single APX (Ascorbate Peroxidase) and enzymes related to the redox cycle of Ascorbate in the cytosol. In the present study, a full-length cDNA clone encoding the Euglena APX was isolated and found to contain an open reading frame encoding a protein of 649 amino acids with a calculated molecular mass of 70.5 kDa. Interestingly, the enzyme consisted of two entirely homologous catalytic domains, designated APX-N and APX-C, and an 102 amino acid extension in the N-terminal region, which had a typical class II signal proposed for plastid targeting in Euglena . A computer-assisted analysis indicated a novel protein structure with an intramolecular dimeric structure. The analysis of cell fractionation showed that the APX protein is distributed in the cytosol, but not the plastids, suggesting that Euglena APX becomes mature in the cytosol after processing of the precursor. The kinetics of the recombinant mature FL (full-length)-APX and the APX-N and APX-C domains with Ascorbate and H 2 O 2 were almost the same as that of the native enzyme. However, the substrate specificity of the mature FL-APX and the native enzyme was different from that of APX-N and APX-C. The mature FL-APX, but not the truncated forms, could reduce alkyl hydroperoxides, suggesting that the dimeric structure is correlated with substrate recognition. In Euglena cells transfected with double-stranded RNA, the silencing of APX expression resulted in a significant increase in the cellular level of H 2 O 2 , indicating the physiological importance of APX to the metabolism of H 2 O 2 .
-
arabidopsis chloroplastic Ascorbate Peroxidase isoenzymes play a dual role in photoprotection and gene regulation under photooxidative stress
Plant and Cell Physiology, 2010Co-Authors: Takanori Maruta, Kazuya Yoshimura, Masahiro Tamoi, Takahiro Ishikawa, Yukinori Yabuta, Aoi Tanouchi, Shigeru ShigeokaAbstract:: Though two types of chloroplastic Ascorbate Peroxidase (APX) located in the thylakoid membrane (tAPX) and stroma (sAPX) have been thought to be key regulators of intracellular levels of H(2)O(2), their physiological significance in the response to photooxidative stress is still under discussion. Here we characterized single mutants lacking either tAPX (KO-tAPX) or sAPX (KO-sAPX). Under exposure to high light or treatment with methylviologen under light, H(2)O(2) and oxidized proteins accumulated to higher levels in both mutant plants than in the wild-type plants. On the other hand, the absence of sAPX and tAPX drastically suppressed the expression of H(2)O(2)-responsive genes under photooxidative stress. Interestingly, the most marked effect of photooxidative stress on the accumulation of H(2)O(2) and oxidized protein and gene expression was observed in the KO-tAPX plants rather than the KO-sAPX plants. The present findings suggest that both chloroplastic APXs, but particularly tAPX, are important for photoprotection and gene regulation under photooxidative stress in Arabidopsis leaves.
-
thylakoid membrane bound Ascorbate Peroxidase is a limiting factor of antioxidative systems under photo oxidative stress
Plant Journal, 2002Co-Authors: Yukinori Yabuta, Kazuya Yoshimura, Toru Takeda, Takahiro Ishikawa, Takashi Motoki, Shigeru ShigeokaAbstract:To evaluate the physiological importance of thylakoid membrane-bound Ascorbate Peroxidase (tAPX) in the active oxygen species-scavenging system of chloroplasts, the level of tAPX in tobacco plants was altered by expression of the tAPX cDNA in both sense and antisense orientation. The tobacco plants transformed with constructs of antisense tAPXs from spinach and tobacco could not be obtained, suggesting that the suppression of tAPX in higher plants had a severe effect on the growth even under normal conditions. In contrast, the transgenic tobacco plants (TpTAP-12) overexpressing tAPX, which had approximately 37-fold higher activity than that of the wild-type plants, were generated. The TpTAP-12 plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV, 50 microm) under light intensity (300 and 1600 microE m(-2) sec(-1)) and by chilling stress with high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)). At 24 h after the MV treatment under illumination at 300 microE m-2 sec-1, destruction of chlorophyll was observed in the wild-type plants, but not in the TpTAP-12 plants. The activities of thiol-modulated enzymes in the Calvin cycle, the level and redox status of Ascorbate (AsA), and the activity of tAPX in the wild-type plants significantly decreased, while those in the TpTAP-12 plants were hardly changed. These observations suggest that tAPX is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts, and that the enhanced activity of tAPX functions to maintain the AsA content and the redox status of AsA under stress conditions.
-
identification of a cis element for tissue specific alternative splicing of chloroplast Ascorbate Peroxidase pre mrna in higher plants
Journal of Biological Chemistry, 2002Co-Authors: Kazuya Yoshimura, Takahiro Ishikawa, Yukinori Yabuta, Shigeru ShigeokaAbstract:Alternative splicing events in the 3'-terminal region of chloroplast Ascorbate Peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leaf in vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.
Kazuya Yoshimura - One of the best experts on this subject based on the ideXlab platform.
-
arabidopsis chloroplastic Ascorbate Peroxidase isoenzymes play a dual role in photoprotection and gene regulation under photooxidative stress
Plant and Cell Physiology, 2010Co-Authors: Takanori Maruta, Kazuya Yoshimura, Masahiro Tamoi, Takahiro Ishikawa, Yukinori Yabuta, Aoi Tanouchi, Shigeru ShigeokaAbstract:: Though two types of chloroplastic Ascorbate Peroxidase (APX) located in the thylakoid membrane (tAPX) and stroma (sAPX) have been thought to be key regulators of intracellular levels of H(2)O(2), their physiological significance in the response to photooxidative stress is still under discussion. Here we characterized single mutants lacking either tAPX (KO-tAPX) or sAPX (KO-sAPX). Under exposure to high light or treatment with methylviologen under light, H(2)O(2) and oxidized proteins accumulated to higher levels in both mutant plants than in the wild-type plants. On the other hand, the absence of sAPX and tAPX drastically suppressed the expression of H(2)O(2)-responsive genes under photooxidative stress. Interestingly, the most marked effect of photooxidative stress on the accumulation of H(2)O(2) and oxidized protein and gene expression was observed in the KO-tAPX plants rather than the KO-sAPX plants. The present findings suggest that both chloroplastic APXs, but particularly tAPX, are important for photoprotection and gene regulation under photooxidative stress in Arabidopsis leaves.
-
thylakoid membrane bound Ascorbate Peroxidase is a limiting factor of antioxidative systems under photo oxidative stress
Plant Journal, 2002Co-Authors: Yukinori Yabuta, Kazuya Yoshimura, Toru Takeda, Takahiro Ishikawa, Takashi Motoki, Shigeru ShigeokaAbstract:To evaluate the physiological importance of thylakoid membrane-bound Ascorbate Peroxidase (tAPX) in the active oxygen species-scavenging system of chloroplasts, the level of tAPX in tobacco plants was altered by expression of the tAPX cDNA in both sense and antisense orientation. The tobacco plants transformed with constructs of antisense tAPXs from spinach and tobacco could not be obtained, suggesting that the suppression of tAPX in higher plants had a severe effect on the growth even under normal conditions. In contrast, the transgenic tobacco plants (TpTAP-12) overexpressing tAPX, which had approximately 37-fold higher activity than that of the wild-type plants, were generated. The TpTAP-12 plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV, 50 microm) under light intensity (300 and 1600 microE m(-2) sec(-1)) and by chilling stress with high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)). At 24 h after the MV treatment under illumination at 300 microE m-2 sec-1, destruction of chlorophyll was observed in the wild-type plants, but not in the TpTAP-12 plants. The activities of thiol-modulated enzymes in the Calvin cycle, the level and redox status of Ascorbate (AsA), and the activity of tAPX in the wild-type plants significantly decreased, while those in the TpTAP-12 plants were hardly changed. These observations suggest that tAPX is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts, and that the enhanced activity of tAPX functions to maintain the AsA content and the redox status of AsA under stress conditions.
-
identification of a cis element for tissue specific alternative splicing of chloroplast Ascorbate Peroxidase pre mrna in higher plants
Journal of Biological Chemistry, 2002Co-Authors: Kazuya Yoshimura, Takahiro Ishikawa, Yukinori Yabuta, Shigeru ShigeokaAbstract:Alternative splicing events in the 3'-terminal region of chloroplast Ascorbate Peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leaf in vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.
-
regulation and function of Ascorbate Peroxidase isoenzymes
Journal of Experimental Botany, 2002Co-Authors: Shigeru Shigeoka, Masahiro Tamoi, Toru Takeda, Takahiro Ishikawa, Yoshiko Miyagawa, Yukinori Yabuta, Kazuya YoshimuraAbstract:Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS). Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level. Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle. Ascorbate Peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H 2 O 2 in higher plants. APX is also found in eukaryotic algae. The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes. Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons. Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses. Transgenic plants expressing E. coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions. It is clear that a high level of endogenous Ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.
-
Expression of spinach Ascorbate Peroxidase isoenzymes in response to oxidative stresses
2000Co-Authors: Kazuya Yoshimura, Takahiro Ishikawa, Yukinori Yabuta, Shigeru ShigeokaAbstract:We studied the response of each Ascorbate Peroxidase (APX) isoenzyme in spinach leaves under stress conditions imposed by high light intensity, drought, salinity, and applications of methyl viologen and abscisic acid. The steady-state transcript level of cytosolic APX remarkably increased in response to high-light stress and methyl viologen treatment, but not in response to the other stress treatments. The transcript levels of the chloroplastic (stromal and thylakoid-bound) and microbody-bound APX isoenzymes were not changed in response to any of the stress treatments. To explore the responses of the APX isoenzymes to photooxidative stress, the levels of transcript and protein and activities of each isoenzyme were studied during high-light stress and following its recovery. The cytosolic APX activity increased in parallel with transcript abundance during high-light stress, while the protein level was not altered. The other isoenzymes showed no significant changes in transcript and protein levels and activities, except for the gradual decrease in chloroplastic isoenzyme activities. Even under optimal conditions, many metabolic processes, including chloroplastic, mitochondrial, and plasma membrane-linked electron transport sys-tems, produce active oxygen species such as the su-peroxide radical, H2O2, and the hydroxyl radical (Foyer et al., 1994; Asada, 1997). Furthermore, th
