Peroxisomal APX knockdown triggers antioxidant mechanisms favourable for coping with high photorespiratory H2O2 induced by CAT deficiency in rice

The physiological role of peroxisomal ascorbate peroxidases (pAPX) is unknown; therefore, we utilized pAPX4 knockdown rice and catalase (CAT) inhibition to assess its role in CAT compensation under high photorespiration. pAPX4 knockdown induced co‐suppression in the expression of pAPX3. The rice mutants exhibited metabolic changes such as lower CAT and glycolate oxidase (GO) activities and reduced glyoxylate content; however, APX activity was not altered. CAT inhibition triggered different changes in the expression of CAT, APX and glutathione peroxidase (GPX) isoforms between non‐transformed (NT) and silenced plants. These responses were associated with alterations in APX, GPX and GO activities, suggesting redox homeostasis differences. The glutathione oxidation‐reduction states were modulated differently in mutants, and the ascorbate redox state was greatly affected in both genotypes. The pAPX suffered less oxidative stress and photosystem II (PSII) damage and displayed higher photosynthesis than the NT plants. The improved acclimation exhibited by the pAPX plants was indicated by lower H₂O₂ accumulation, which was associated with lower GO activity and glyoxylate content. The suppression of both pAPXs and/or its downstream metabolic and molecular effects may trigger favourable antioxidant and compensatory mechanisms to cope with CAT deficiency. This physiological acclimation may involve signalling by peroxisomal H₂O₂, which minimized the photorespiration.     

干旱胁迫对3种不同光合类型荒漠植物叶绿体和线粒体超微结构的影响

以荒漠木本C_3植物天山猪毛菜、C_3-C_4中间型植物松叶猪毛菜、C_4植物木本猪毛菜为研究对象,采用盆栽控水试验,设置正常供水和轻度、中度和重度干旱处理(土壤含水量分别为田间持水量的80%、60%、45%和35%),研究不同程度干旱胁迫对3种不同光合类型荒漠植物叶片超微结构的影响。结果表明:(1)正常水分条件下,叶肉细胞中各细胞器结构完整。(2)轻度干旱胁迫下,3种植物叶片超微结构未受损伤,无明显变化。(3)中度干旱胁迫下,天山猪毛菜和松叶猪毛菜叶肉细胞壁界限不清晰,类囊体片层扩张且排列不紧密,不同之处在于,天山猪毛菜线粒体最先出现降解,内含物流失,而松叶猪毛菜线粒体外膜轮廓变形,嵴减少;木本猪毛菜线粒体无明显变化,叶绿体轻微扩张。(4)重度干旱胁迫下,天山猪毛菜和松叶猪毛菜叶绿体受损且结构混乱,线粒体出现降解;木本猪毛菜叶绿体出现膨胀,线粒体外膜轮廓模糊,嵴减少且结构模糊不清楚。研究认为,不同程度干旱胁迫下木本猪毛菜叶绿体和线粒体的受损程度都最低;干旱胁迫下天山猪毛菜和松叶猪毛菜叶绿体的受损程度大致相似;松叶猪毛菜和木本猪毛菜线粒体对干旱胁迫的耐受力要比叶绿体强。     

Chloroplast-derived photo-oxidative stress causes changes in H2O2 and EGSH in other subcellular compartments

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers, such as the highly reduced glutathione pool, serve as reservoirs of reducing power for several ROS-scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide and a shift of the glutathione redox potential (E_GSH) toward less negative values is considered as hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis (Arabidopsis thaliana) seedlings and recorded dynamic changes in E_GSH and H₂O₂ levels with the genetically encoded biosensors Grx1-roGFP2 (for E_GSH) and roGFP2-Orp1 (for H₂O₂) targeted to chloroplasts, the cytosol, or mitochondria. Treatment of seedlings with MV caused rapid oxidation in chloroplasts and, subsequently, in the cytosol and mitochondria. MV-induced oxidation was significantly boosted by illumination with actinic light, and largely abolished by inhibitors of photosynthetic electron transport. MV also induced autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provides a basis for understanding how compartment-specific redox dynamics might operate in retrograde signaling and stress acclimation in plants.

Methyl viologen-induced photo-oxidative stress increases hydrogen peroxide and oxidation of glutathione in chloroplasts, cytosol, and mitochondria, as well as autonomous oxidation in mitochondria.     

Involvement of glutathione peroxidase 1 in growth and peroxisome formation in Saccharomyces cerevisiae in oleic acid medium

Saccharomyces cerevisiae is able to use some fatty acids, such as oleic acid, as a sole source of carbon. β-oxidation, which occurs in a single membrane-enveloped organelle or peroxisome, is responsible for the assimilation of fatty acids. In S. cerevisiae, β-oxidation occurs only in peroxisomes, and H₂O₂ is generated during this fatty acid-metabolizing pathway. S. cerevisiae has three GPX genes (GPX1, GPX2, and GPX3) encoding atypical 2-Cys peroxiredoxins. Here we show that expression of GPX1 was induced in medium containing oleic acid as a carbon source in an Msn2/Msn4-dependent manner. We found that Gpx1 was located in the peroxisomal matrix. The peroxisomal Gpx1 showed peroxidase activity using thioredoxin or glutathione as a reducing power. Peroxisome biogenesis was induced when cells were cultured with oleic acid. Peroxisome biogenesis was impaired in gpx1? cells, and subsequently, the growth of gpx1? cells was lowered in oleic acid-containing medium. Gpx1 contains six cysteine residues. Of the cysteine-substituted mutants of Gpx1, Gpx1^C36S was not able to restore growth and peroxisome formation in oleic acid-containing medium, therefore, redox regulation of Gpx1 seems to be involved in the mechanism of peroxisome formation.     

水稻谷胱甘肽过氧化物酶基因OsGPX3 和OsGPX4的分子特性研究

植物谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)是清除体内活性氧的一种关键酶,在植物抗逆反应中发挥重要作用.本研究从水稻中克隆到2个GPX基因,分别为OsGPX3和OsGPX4.OsGPX3和OsGPX4分别编码238和234个氨基酸组成的蛋白质,预测分子量分别是25.84 kD和25.07 kD.两个基因都包含5个内含子,但是两个基因所对应的内含子长度具有较大变异.组织表达谱分析发现这2个基因在根、茎、叶和叶鞘中均表达,是组成型表达基因.在大肠杆菌中表达并纯化了这2个基因的重组蛋白,酶活性分析显示OsGPX3和OsGPX4蛋白对底物H₂O₂、tBOOH和COOH具有较高活性,但是OsGPX3对3种底物的活性均高于OsGPX4,蛋白质酶活性的差异预示着这2个基因可能存在功能上的分化.     

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

The physiological role of plant mitochondrial glutathione peroxidases is scarcely known. This study attempted to elucidate the role of a rice mitochondrial isoform(GPX1) in photosynthesis under normal growth and salinity conditions. GPX1 knockdown rice lines(GPX1s) were tested in absence and presence of 100 mM NaCl for 6 d.Growth reduction of GPX1 s line under non-stressful conditions, compared with non-transformed(NT) plants occurred in parallel to increased H_2O_2 and decreased GSH contents. These changes occurred concurrently with photosynthesis impairment, particularly in Calvin cycle's reactions, since photochemical efficiency did not change.Thus, GPX1 silencing and downstream molecular/metabolic changes modulated photosynthesis differentially. In contrast, salinity induced reduction in both phases of photosynthesis, which were more impaired in silenced plants.These changes were associated with root morphology alterations but not shoot growth. Both studied lines displayed increased GPX activity but H_2O_2 content did not change in response to salinity. Transformed plants exhibited lower photorespiration, water use efficiency and root growth, indicating that GPX1 could be important to salt tolerance. Growth reduction of GPX1 s line might be related to photosynthesis impairment, which in turn could have involved a cross talk mechanism between mitochondria and chloroplast originated from redox changes due to GPX1 deficiency.     

Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme

 NADP-dependent malic enzyme (NADP-ME) is a major decarboxylating enzyme in NADP-ME-type C₄ species such as maize and Flaveria. In this study, chloroplastic NADP-ME was transferred to rice (Oryza sativa L.) using a chimeric gene composed of maize NADP-ME cDNA under the control of rice light-harvesting chlorophyll-a/b-binding protein (Cab) promoter. There was a 20- to 70-fold increase in the NADP-ME activity in leaves of transgenic rice compared to that in wild-type rice plants. Immunocytochemical studies by electron microscopy showed that maize NADP-ME was mostly localized in chloroplasts in transgenic rice plants, and that the chloroplasts were agranal without thylakoid stacking. Chlorophyll content and photosystem II activity were inversely correlated with the level of NADP-ME activity. These results suggest that aberrant chloroplasts in transgenic plants may be caused by excessive NADP-ME activity. Based on these results and the known fact that only bundle sheath cells of NADP-ME species, among all three C₄ subgroups, have agranal chloroplasts, we postulate that a high level of chloroplastic NADP-ME activity could strongly affect the development of chloroplasts. Received: 27 January 1999 / Accepted: 20 January 2000     

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa)

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23–26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes.     

Molecular and Catalytic Properties of Glutathione Peroxidase Family Proteins from Pinus tabulaeformis

Glutathione peroxidase (GPX) is one of the key enzymes that protect cells against oxidative damage caused by reactive oxygen species. Previous studies of plant GPXs focused mainly on angiosperms. In contrast, little information is available on the molecular characteristics of this gene family in gymnosperms. In this study, four GPX genes (PtaGPX1, 2, 3, and 4) were cloned from the gymnosperm Pinus tabulaeformis, which showed high protein sequence identity and similar expression patterns in various tissues. The four Pinus GPX proteins were expressed in Escherichia coli, and the purified proteins used thioredoxin, but not glutathione, as an electron donor. The four Pinus GPXs showed different enzymatic activities and kinetic characteristics, suggesting functional divergence. Two conserved Cys residues (corresponding to Cys44 and Cys92 of PtaGPX3) were identified in all plant GPXs, and their functions were assessed using site-directed mutagenesis. Cys44 and Cys92 of PtaGPX3 could form an intramolecular disulfide bond under oxidizing conditions. These two residues were critical components of active sites and contributed to catalytic activity. This study provides novel insights into the functional divergence and catalytic properties of the GPX family in gymnosperms.     

Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VIII. Ultrastructural Characteristics of Panicum Species in the Laxa Group

Ultrastructural studies of leaves of seven Panicum species in or closely related to the Laxa group and classified as C₃, C₄ or C₃-C₄ intermediate were undertaken to examine features associated with C₃ and C₄ photosynthesis. The C₃ species Panicum rivulare Trin. had few organelles in bundle sheath cell profiles (2 chloroplasts, 1.1 mitochondria, and 0.3 peroxisomes per cell section) compared to an average of 10.6 chloroplasts, 17.7 mitochondria, and 3.2 peroxisomes per bundle sheath cell profile for three C₃-C₄ species, Panicum milioides Nees ex Trin., Panicum decipiens Nees ex Trin. and Panicum schenckii Hack. However, two other C₃ species, Panicum laxum Sw. and Panicum hylaeicum Mez, contained about 0.7, 0.5, and 0.3 as many chloroplasts, mitochondria, and peroxisomes, respectively, as in bundle sheath cell profiles of the C₃-C₄ species. Chloroplasts and mitochondria in bundle sheath cells were larger than those in mesophyll cells for the C₄ species Panicum prionitis Griseb. and the C₃-C₄ species, but in C₃ species the organelles were similar in size or were smaller in the bundle sheath cells. The C₃-C₄ species and P. laxum and P. hylaeicum exhibited an unusually close association of organelles in bundle sheath cells with mitochondria frequently surrounded in profile by chloroplasts. The high concentrations in bundle sheath cells of somewhat larger organelles than in mesophyll cells correlates with the reduced photorespiration of the C₃-C₄ species.     

Hydrogen peroxide detoxifying enzymes show different activity patterns in host and non-host plant interactions with Magnaporthe oryzae Triticum pathotype

Wheat blast caused by the hemibiotroph fungal pathogen Magnaporthe oryzae Triticum (MoT) pathotype is a destructive disease of wheat in South America, Bangladesh and Zambia. This study aimed to determine and compare the activities of antioxidant enzymes in susceptible (wheat, maize, barley and swamp rice grass) and resistant (rice) plants when interacting with MoT. The activities of reactive oxygen species-detoxifying enzymes; catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S-transferase (GST), peroxidase (POX) were increased in all plants in response to MoT inoculation with a few exceptions. Interestingly, an early and very high activity of CAT was observed within 24 h after inoculation in wheat, barley, maize and swamp rice grass with lower H₂O₂ concentration. In contrast, an early and high accumulation of H₂O₂ was observed in rice at 48 hai with little CAT activity only at a later stage of MoT inoculation. The activities of APX, GST and POD were also high at an early stage of infection in rice. However, these enzymes activities were very high at a later stage in wheat, barley, maize and swamp rice grass. The activity of GPX gradually decreased with the increase of time in rice. Taken together, our results suggest that late and early inductions of most of the antioxidant enzyme activities occurs in susceptible and resistant plants, respectively. This study demonstrates some insights into physiological responses of host and non-host plants when interacting with the devastating wheat blast fungus MoT, which could be useful for developing blast resistant wheat.     

Enclosure of mitochondria by chloroplasts

In Panicum species of the Laxa group, some of which have characteristics intermediate to C₃ and C₄ photosynthesis species, some mitochondria in leaf bundle sheath cells are surrounded by chloroplasts when viewed in profile. Serial sectioning of leaves of one Laxa species, Panicum schenckii Hack, shows that these mitochondria are enclosed by chloroplasts. Complete enclosure rather than invagination also is indicated by absence of two concentric chloroplast membranes surrounding the mitochondrial profiles.     

The expression of BrMDHAR gene in chloroplasts and mitochondria enhances tolerance to freezing stress in Arabidopsis thaliana

In chloroplasts and mitochondria, antioxidant mechanisms include the ascorbate-glutathione cycle, and monodehydroascorbate reductase (MDHAR) is important for regeneration of ascorbate (AsA) from monodehydroascorbate (MDHA). To improve detoxification of reactive oxygen species (ROS), we established a construct of the MDHAR gene from Brassica rapa fused to the targeting signal peptides of Pisum sativum glutathione reductase (GR), which was controlled by a stress-inducible SWPA2 promoter, and introduced this expression system into Arabidopsis thaliana. Transgenic (TG) plants overexpressing BrMDHAR targeted to chloroplasts and mitochondria through signal peptides showed an elevated MDHAR activity and an increased ratio of AsA to dehydroascorbate (DHA) when compared to wild-type (WT) plants under a freezing stress. These led to increased photosynthetic parameters, redox homeostasis, and biomass in TG plants when compared to the WT plants. Our results suggest that the overexpression of the BrMDHAR gene targeted to chloroplasts and mitochondria conferred an enhanced tolerance against the freezing stress, and a stress adaptation of dual-targeted BrMDHAR was better than that of single BrMDHAR.     

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 H₂O₂ into H₂O, 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 H₂O₂ 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.     

Reunification of sub-cellular fractions of bryopsis into viable cells

Protoplasm from Bryopsis maxima, a coenocytic green alga, was dissociated into two fractions; chloroplasts and a protoplasmic fraction without chloroplasts (PF). PF induced nuclei, mitochondria, dictyosome, endplasmic reticulum, etc. These two fractions were reunited and protoplasts with complete plasmamembrane were reformed within 10 h. Cell wall regeneration was observed 24–30 h after reuniting. New cells began budding 2–3 days after, and 1 month after reunification, they had developed into mature plants.     

Glutamine Synthetase/Glutamine: alpha-Ketoglutarate Aminotransferase in Chloroplasts from the Marine Alga Caulerpa simpliciuscula

The enzymic capacities for ammonia assimilation into amino acids have been investigated in chloroplasts from the siphonous green alga Caulerpa simpliciuscula (Turner) C. Ag. The results show that these chloroplasts differ from those of higher plants in having present simultaneously the enzymic capacities to permit assimilation of ammonia by two pathways. Glutamine synthetase (EC 6.3.1.2) activity at levels up to 4 μmoles per mg chlorophyll per hour were found in soluble extracts of the chloroplasts. Glutamine(amide):α-ketoglutarate aminotransferase (oxidoreductase ferredoxin) (EC 1.4.7.1) activity at levels up to 1.4 μmoles per mg chlorophyll per hour was detected by incubation of photosynthetically active chloroplasts either in light or with reduced ferredoxin. Together these enzymes provide the capacity for the conventional pathway of ammonium assimilation in chloroplasts via glutamine. A similar level of a glutamate dehydrogenase with an unusually low K_m for ammonia which has been described previously in these chloroplasts provides the second potential pathway.