Cytosolic ascorbate peroxidase 1 protects organelles against oxidative stress by wounding- and jasmonate-induced H2O2 in Arabidopsis plants

Background

Reactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production.

Methods

We studied the effect of wounding and MeJA treatment on the levels of H₂O₂ and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1).

Results

The KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H₂O₂ accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H₂O₂ were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment.

General significance

This is the first report demonstrating that H₂O₂-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment.     

Abscisic acid improves drought tolerance of triploid bermudagrass and involves H2O2- and NO-induced antioxidant enzyme activities

Drought is a major limiting factor for turfgrass growth. Protection of triploid bermudagrass against drought stress by abscisic acid (ABA) and its association with hydrogen peroxide (H₂O₂) and nitric oxide (NO) were investigated. ABA treatment increased relative water content, decreased ion leakage and the percentage of dead plants significantly under drought stress. Superoxide dismutase (SOD) and catalase (CAT) activities increased in both ABA-treated and control plants, but more in ABA-treated plants, under drought stress. Malondialdehyde, an indicator of plant lipid peroxidation, was lower in ABA-treated plants than in control plants, indicating that ABA alleviated drought-induced oxidative injury. ABA treatment increased H₂O₂ and NO contents. ABA-induced SOD and CAT activities could be blocked by scavengers of H₂O₂ and NO, and inhibitors of H₂O₂ and NO generation. The results indicated that H₂O₂ and NO were essential for ABA-induced SOD and CAT activities. Both H₂O₂ and NO could induce SOD and CAT activities individually. SOD and CAT induced by H₂O₂ could be blocked by scavenger of NO and inhibitors of NO generation, while SOD and CAT induced by NO could not be blocked by scavenger of H₂O₂ and inhibitor of H₂O₂. The results revealed that ABA-induced SOD and CAT activities were mediated sequentially by H₂O₂ and NO, and NO acted downstream of H₂O₂.     

In vivo and in situ visualization of early physiological events induced by heavy metals in pea root meristem

Heavy metals (HMs) are toxic pollutants, which can negatively affect the physiological processes of plants; moreover, HMs can be present in the food chain endangering people’s health. The aim of this study was to investigate the early physiological events during HM exposure in the root tips of the food plant Pisum sativum L. Ten-day-old pea plants were treated with 100 μM CdCl₂ or CuSO₄, in nutrient solution for 48 h. We studied the rapid formation of different reactive oxygen species (hydrogen peroxide H₂O₂ and superoxide radical O₂^·−) and reactive nitrogen species (nitric oxide NO· and peroxynitrite ONOO⁻) together with membrane damage and cell death in the meristem cells of pea roots using in vivo and in situ microscopic methods. In our experimental system, copper and cadmium induced the formation of H₂O₂ and NO. Two hours of heavy metal treatments resulted in an increased O₂^·− formation; however, later the level of this reactive molecule dramatically decreased. We found that high levels of NO were needed for ONOO⁻ production under HM exposure. A fast loss of membrane integrity and decreased cell viability were detected in root tips of copper-treated plants. The effects of cadmium seemed to be slower compared to copper, but this non-essential metal also caused cell death. We concluded that viability decreased when NO and H₂O₂ levels were simultaneously high in the same tissues. Using the NO scavenger it was also evidenced that NO generation is essential for cell death induction under copper or cadmium stress.     

The Role of GIGANTEA Gene in Mediating the Oxidative Stress Response and in Arabidopsis

The Arabidopsis GIGANTEA (GI) gene has been shown to be involved in the regulation of the oxidative stress response; however, little is known about the mechanism by which GI gene regulates the oxidative stress response. We show here that enhanced tolerance of the gi-3 mutant to oxidative stress is associated, at least in part, with constitutive activation of superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes. The gi-3 plants were more tolerant to parquart (PQ) or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of concentrations of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of gi-3 plants to oxidative stress was not due to defects in the uptake of PQ or the sequestration of PQ from its site of action, and that the gi-3 mutation alleviated oxidative damage of plant cells from PQ stress. Moreover, the gi-3 mutant showed constitutive activation of cytosolic Cu/ZnSOD and plastidic FeSOD as well as cytosolic APX1 and stromal APX genes, which at least in part contributed to constitutive increases in activities of anti-oxidative enzymes SOD and APX, respectively. To our knowledge, we demonstrate, for the first time, that GI gene regulates the oxidative stress response, at least in part, through modulation of SOD and APX genes.     

Overexpressing <Emphasis Type="Italic">SgNCED1</Emphasis> in Tobacco Increases ABA Level,Antioxidant Enzyme Activities,and Stress Tolerance

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. 9-cis-epoxycarotenoid dioxygenase (NCED) is the key enzyme of ABA biosynthesis in higher plants. A NCED gene, SgNCED1, was overexpressed in transgenic tobacco plants which resulted in 51–77% more accumulation of ABA in leaves. Transgenic tobacco plants decreased stomatal conductance, transpiration rate, and photosynthetic rate but induced activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX). Hydrogen peroxide (H₂O₂) and nitric oxide (NO) in leaves were also induced in the transgenic plants. Compared to the wild-type control, the transgenic plants improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl-induced salinity stress. It is suggested that the ABA-induced H₂O₂ and NO generation upregulates the stomatal closure and antioxidant enzymes, and therefore increases drought and salinity tolerance in the transgenic plants.     

Overexpression of <Emphasis Type="Italic">Populus tomentosa</Emphasis> cytosolic ascorbate peroxidase enhances abiotic stress tolerance in tobacco plants

Reactive oxygen species (ROS) play key roles in plants and are regulated by several ROS-scavenging enzymes. Ascorbate peroxidase (APX), which catalyzes the reduction of hydrogen peroxide to water, a vital part of ROS formation, plays a significant role in higher plants. In this study, a cytosolic APX gene from Populus tomentosa, named PcAPX, was identified and characterized. Recombinant PcAPX had a calculated mass of 33.24 kD and showed high activity towards ascorbic acid (ASA) and hydrogen peroxide (H₂O₂). Real-time PCR analysis showed that APX mRNA expression levels were higher in leaves than roots or stems of P. tomentosa. Compared with wild-type, transgenic tobacco plants overexpressing PcAPX showed no significant difference in morphology under normal conditions. However, the transgenic plants were more resistant to drought, salt and oxidative stress conditions, as shown by decreased levels of malondialdehyde and increased levels of chlorophyll. Moreover, decreased H₂O₂ levels, increased ASA consumption, an increase in the NADP to NADPH ratio, and higher APX activity in the transgenic plants suggested an increased ability to eliminate ROS. These data suggest that PcAPX overexpression in transgenic tobacco plants can enhance tolerance to drought, salt and oxidative stress. Therefore, APX has a crucial role in abiotic stress tolerance in plants.     

H2O2-induced Leaf Cell Death and the Crosstalk of Reactive Nitric/Oxygen Species([F])

In plants, the chloroplast is the main reactive oxygen species (ROS) producing site under high light stress. Catalase (CAT), which decomposes hydrogen peroxide (H₂O₂), is one of the controlling enzymes that maintains leaf redox homeostasis. The catalase mutants with reduced leaf catalase activity from different plant species exhibit an H₂O₂‐induced leaf cell death phenotype. This phenotype was differently affected by light intensity or photoperiod, which may be caused by plant species, leaf redox status or growth conditions. In the rice CAT mutant nitric oxide excess 1 (noe1), higher H₂O₂ levels induced the generation of nitric oxide (NO) and higher S‐nitrosothiol (SNO) levels, suggesting that NO acts as an important endogenous mediator in H₂O₂‐induced leaf cell death. As a free radical, NO could also react with other intracellular and extracellular targets and form a series of related molecules, collectively called reactive nitrogen species (RNS). Recent studies have revealed that both RNS and ROS are important partners in plant leaf cell death. Here, we summarize the recent progress on H₂O₂‐induced leaf cell death and the crosstalk of RNS and ROS signals in the plant hypersensitive response (HR), leaf senescence, and other forms of leaf cell death triggered by diverse environmental conditions. [ Chengcai Chu (Corresponding author)]     

Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone

Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide (H₂O₂) in tobacco and tomato, and superoxide (O₂^–?) together with H₂O₂ in Arabidopsis accessions. Accumulation of these reactive oxygen species (ROS) preceded the induction of cell death, and both responses co‐occurred spatially in the periveinal regions of the leaves. Re‐current ozone exposure of the sensitive tobacco cv. Bel W3 in closed chambers or in the field led to an enlargement of existing lesions by priming the border cells for H₂O₂ accumulation. Open top chamber experiments with native herbaceous plants in the field showed that Malva sylvestris L. accumulates O₂^–? at those sites that later exhibit plant cell death. Blocking of ROS accumulation markedly reduced ozone‐induced cell death in tomato, Arabidopsis and M. sylvestris. It is concluded that ozone triggers an in planta generation and accumulation of H₂O₂ and/or O₂^–? depending on the species, accession and cultivar, and that both these reactive oxygen species are involved in the induction of cell death in sensitive crop and native plants.     

Signal interaction between nitric oxide and hydrogen peroxide in heat shock-induced hypericin production of <Emphasis Type="Italic">Hypericum perforatum suspension</Emphasis> cells

Heat shock (HS, 40°C, 10 min) induces hypericin production, nitric oxide (NO) generation, and hydrogen peroxide (H₂O₂) accumulation of Hypericum perforatum suspension cells. Catalase (CAT) and NO specific scavenger 2–4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) suppress not only the HS-induced H₂O₂ generation and NO burst, but also the HS-triggered hypericin production. Hypericin contents of the cells treated with both NO and H₂O₂ are significantly higher than those of the cells treated with NO alone, although H₂O₂ per se has no effects on hypericin production of the cells, which suggests the synergistic action between H₂O₂ and NO on hypericin production. NO treatment enhances H₂O₂ levels of H. perforatum cells, while external application of H₂O₂ induces NO generation of cells. Thus, the results reveal a mutually amplifying action between H₂O₂ and NO in H. perforatum cells. CAT treatment inhibits both HS-induced H₂O₂ accumulation and NO generation, while cPTIO can also suppress H₂O₂ levels of the heat shocked cells. The results imply that H₂O₂ and NO may enhance each other’s levels by their mutually amplifying action in the heat shocked cells. Membrane NAD(P)H oxidase inhibitor diphenylene iodonium (DPI) and nitric oxide synthase (NOS) inhibitor S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU) not only inhibit the mutually amplifying action between H₂O₂ and NO but also abolish the synergistic effects of H₂O₂ and NO on hypericin production, showing that the synergism of H₂O₂ and NO on secondary metabolite biosynthesis might be dependent on their mutual amplification. Taken together, data of the present work demonstrate that both H₂O₂ and NO are essential for HS-induced hypericin production of H. perforatum suspension cells. Furthermore, the results reveal a special interaction between the two signal molecules in mediating HS-triggered secondary metabolite biosynthesis of the cells.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

Effect of salinity on antioxidant enzymes in calli of the halophyte <Emphasis Type="Italic">Nitraria tangutorum</Emphasis> Bobr.

Nitraria tangutorum Bobr., a typical desert halophyte, plays an important ecological role because of its superior tolerance to severe drought and high salinity. Very little is known about the physiological adaptative mechanism of this species to environmental stresses. The aim of this study was to investigate the changes of antioxidant enzyme activities and the regulatory mechanism of ascorbate peroxidase (APX) activity in the calli from Nitraria tangutorum Bobr. after treatment with different NaCl concentrations. The activities of superoxide dismutase (SOD) and catalase (CAT) significantly increased in the calli treated with NaCl, while the peroxidase activity decreased. APX activity was also elevated significantly in response to NaCl, but the increase was partly abolished by H₂O₂ scavenger dimethylthiourea (DMTU). Furthermore, the excitatory effect of salinity on APX could be alleviated by the addition of exogenous CAT and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium, indicating that the modulation of the APX activity in Nitraria tangutorum Bobr. calli might be associated with NADPH oxidase-dependent H₂O₂ generation. Measurement and analysis using fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate showed the increase of H₂O₂ content in salinity-treated calli. The investigation of NADPH-dependent O₂⁻ production in plasma membrane (PM) vesicles isolated from Nitraria tangutorum Bobr. calli revealed that salinity treatment stimulated NADPH oxidase activity. In conclusion, these results suggest that the higher activities of antioxidant enzymes play an important role in the salt tolerance of Nitraria tangutorum Bobr. calli and that the extracellular production of H₂O₂, depending on the excitation of PM NADPH oxidase, is responsible for enhancing the APX activity in Nitraria tangutorum Bobr. calli under salinity stress.     

Enhanced chilling tolerance at the booting stage in rice by transgenic overexpression of the ascorbate peroxidase gene, OsAPXa

Low temperatures during the booting stage reduce rice yields by causing cold-induced male sterility. To determine whether antioxidant capacity affects the ability of rice plants to withstand chilling at the booting stage, we produced transgenic rice plants that overexpress OsAPXa and have increased APX activity. The effect of increased APX activity on the levels of H₂O₂ and lipid peroxidation were determined by measuring H₂O₂ levels and malondialdehyde (MDA) contents in spikelets during cold treatments at the booting stage. The levels of H₂O₂ and the MDA content increased by 1.5-fold and twofold, respectively in WT plants subjected to a 12°C treatment for 6 days. In contrast, transgenic lines showed small changes in H₂O₂ levels and MDA content under cold stress, and H₂O₂ levels and MDA content were significantly lower than in WT plants. APX activity showed negative correlations with levels of H₂O₂ and MDA content, which increased during cold treatment. Cold tolerance at the booting stage in transgenic lines and WT plants was evaluated. Spikelet fertility was significantly higher in transgenic lines than in WT plants after a 12°C treatment for 6 days. These results indicate that higher APX activity enhances H₂O₂-scavenging capacity and protects spikelets from lipid peroxidation, thereby increasing spikelet fertility under cold stress.     

毛竹APX基因系统进化与表达分析

抗坏血酸过氧化物酶(aseorbate peroxidase, APX)是植物活性氧代谢中重要的抗氧化酶之一,尤其是叶绿体中清除H₂O₂的关键酶,也是维生素C代谢的主要酶类。该文基于生物信息学方法,利用毛竹的基因组及转录组数据鉴定毛竹中的APX基因家族成员,并对其编码的蛋白基本理化性质、基因结构、启动子元件、系统进化及共线性关系、重复串联基因、GO注释及表达模式进行综合分析,共鉴定出21种编码APX的基因。结果表明:(1)PeAPX基因家族成员多为不稳定疏水蛋白,基因结构、基序及结构域相对较为保守,大多数APX基因具有高度保守的内含子模式。(2)系统进化关系显示毛竹APX基因与水稻APX基因有着较高的同源性关系,PeAPX具有较高的进化保守性。(3)Ka/Ks分析表明PeAPX基因都经历了纯化选择压力,此外在每个APX基因的启动子序列中发现有许多与应激反应和植物激素相关的顺式作用元件,结合表达量分析,表明毛竹APX基因在毛竹生长发育中起着正向促进作用。该研究为进一步了解毛竹APX基因家族基本功能及其抗氧化机制提供了一定的参考,为毛竹APX基因功能的深层次鉴定提供了重要依据。     

Relationship between H2O2 and jasmonic acid in pea leaf wounding response

The relationship of H₂O₂ and jasmonic acid (JA) in wound-induced defense response was investigated in the leaves of pea (Pisum sativum L.) plants. The results showed that both wounding and JA treatment led to a significant increase in activities of plasma membrane NADPH oxidase and phenylalanine ammonialyase. However, such an increase was blocked by the pretreatment with plasma membrane NADPH oxidase inhibitors, O ₂ ^? scavengers, or H₂O₂ scavenger, implying that H₂O₂ functions downstream of JA. Furthermore, wounding treatment activated two key enzymes of JA biosynthesis, lipoxygenase and allene oxide synthase, while JA biosynthetic inhibitors impaired the wounding-induced H₂O₂ burst. Thus, it is suggested that H₂O₂ burst depends on JA production in plant wounding response.     

Systemic induction of H2O2 in pea seedlings pretreated by wounding and exogenous jasmonic acid

Pea seedlings (Pisum sativum L.) were used as materials to test the timings and compartments of hydrogen peroxide (H₂O₂) triggered by wounding and exogenous jasmonic acid (JA). The results showed that H₂O₂ could be systemically induced by wounding and exogenous JA. H₂O₂ increased within 1 h and reached the peak 3–5 h after wounding in either the wounded leaves or the unwounded leaves adjacent to the wounded ones and the inferior leaves far from the wounded ones. After this, H₂O₂ decreased and recovered to the control level 12 h after wounding. The activities of antioxidant enzymes, however, were rapidly increased by wounding. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, could significantly inhibit H₂O₂ burst that was mediated by wounding and exogenous JA. Assay of H₂O₂ subcellular location showed that H₂O₂ in response to wounding and exogenous JA was predominantly accumulated in plasma membrane, cell wall and apoplasmic space. Numerous JA (gold particles) was found via immunogold electron microscopy to be located in cell wall and phloem zones of mesophyll cell after wounding.     

Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of <Emphasis Type="Italic">Brassica napus</Emphasis> leaf protoplasts

Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H₂O₂ and enzymes involved in H₂O₂ generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H₂O₂ and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H₂O₂ was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H₂O₂ in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H₂O₂ generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.     

Hydrogen peroxide is involved in nitric oxide‐induced cell death in maize leaves

Recent evidence indicates that nitric oxide (NO) plays an important role in plant hypersensitive cell death. Here, we report that NO treatment led to rapid cell death and induced hydrogen peroxide (H₂O₂) accumulation in maize leaves. We also show that NO induced the expression of Zmrboh genes. Pharmacological study suggests that NO‐induced cell death is in part mediated via H₂O₂. In addition, semi‐quantitative RT‐PCR revealed that NO induced expression of the systemic acquired resistance (SAR) genes, ZmPR1 and ZmPR5.