Control of Ascorbate Peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves

In Arabidopsis leaves, high light stress induces rapid expression of a gene encoding a cytosolic ascorbate peroxidase (APX2), whose expression is restricted to bundle sheath cells of the vascular tissue. Imaging of chlorophyll fluorescence and the production of reactive oxygen species (ROS) indicated that APX2 expression followed a localised increase in hydrogen peroxide (H2O2) resulting from photosynthetic electron transport in the bundle sheath cells. Furthermore, leaf transpiration rate also increased prior to APX2 expression, suggesting that water status may also be involved in the signalling pathway. Abscisic acid stimulated APX2 expression. Exposure of ABA-insensitive mutants (abi1-1, abi2-1) to excess light resulted in reduced levels of APX2 expression and confirmed a role for ABA in the signalling pathway. ABA appears to augment the role of H2O2 in initiating APX2 expression. This regulation of APX2 may reflect a functional organisation of the leaf to resolve two conflicting physiological requirements of protecting the sites of primary photosynthesis from ROS and, at the same time, stimulating ROS accumulation to signal responses to changes in the light environment.     

鲜切加工加速荸荠组织衰老与H2O2累积的关系

以荸荠为材料,研究了鲜切加工加速组织衰老与活性氧代谢的关系.结果表明:鲜切加工提高了荸荠切片抗氧化酶(超氧化物歧化酶、抗坏血酸-过氧化物酶和过氧化氢酶)的活性;但同时明显刺激了O2-产生,促进了H2O2累积,加速了抗坏血酸在贮藏后期的损失,加强了膜脂过氧化作用和增加了电解质渗出率.统计分析表明H2O2含量、丙二醛含量、电解质渗出率三者之间存在正相关性.H2O2组织定位结果也证实鲜切加速组织衰老与H2O2累积密切相关.完整荸荠组织O2-产生比较平稳,抗氧化酶活性维持稳定,H2O2未有明显累积.     

Induction of ASCORBATE PEROXIDASE 2 expression in wounded Arabidopsis leaves does not involve known wound-signalling pathways but is associated with changes in photosynthesis

ASCORBATE PEROXIDASE 2 (APX2) encodes a key enzyme of the antioxidant network. In excess light-stressed Arabidopsis leaves, photosynthetic electron transport (PET), hydrogen peroxide (H(2)O(2)) and abscisic acid (ABA) regulate APX2 expression. Wounded leaves showed low induction of APX2 expression, and when exposed to excess light, APX2 expression was increased synergistically. Signalling pathways dependent upon jasmonic acid (JA), chitosan and ABA were not involved in the wound-induced expression of APX2, but were shown to require PET and were preceded by a depressed rate of CO(2) fixation. This led to an accumulation of H(2)O(2) in veinal tissue. Diphenyl iodonium (DPI), which has been shown previously to be a potent inhibitor of H(2)O(2) accumulation in the veins of wounded leaves, prevented induction of APX2 expression probably by inhibition of PET. Thus, the weak induction of APX2 expression in wounded leaves may require H(2)O(2) and PET only. As in other environmental stresses, wounding of leaves resulted in decreased photosynthesis leading to increased reactive oxygen species (ROS) production. This may signal the induction of many 'wound-responsive' genes not regulated by JA-dependent or other known JA-independent pathways.     

Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress.

The Arabidopsis gene APX3 that encodes a putative peroxisomal membrane-bound ascorbate peroxidase was expressed in transgenic tobacco plants. APX3-expressing lines had substantial levels of APX3 mRNA and protein. The H2O2 can be converted to more reactive toxic molecules, e.g. .OH, if it is not quickly removed from plant cells. The expression of APX3 in tobacco could protect leaves from oxidative stress damage caused by aminotriazole which inhibits catalase activity that is found mainly in glyoxysomes and peroxisomes and leads to accumulation of H2O2 in those organelles. However, these plants did not show increased protection from oxidative damage caused by paraquat which leads to the production of reactive oxygen species in chloroplasts. Therefore, protection provided by the expression of APX3 seems to be specific against oxidative stress originated from peroxisomes, not from chloroplasts, which is consistent with the hypothesis that APX3 is a peroxisomal membrane-bound antioxidant enzyme.     

Ascorbate peroxidase 1 plays a key role in the response of Arabidopsis thaliana to stress combination

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.     

Expression of the <Emphasis Type="Italic">Apx</Emphasis> gene family during leaf senescence of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H₂O₂ scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.     

Singlet oxygen imaging in Arabidopsis thaliana leaves under photoinhibition by excess photosynthetically active radiation

Arabidopsis thaliana leaves were infiltrated with DanePy (3-( N -diethylaminoethyl)- N -dansyl)aminomethyl-2,5-dihydro-2,2,5,5-tetramethyl-1 H -pyrrole), a double, fluorescent and spin sensor of singlet oxygen. DanePy fluorescence was imaged by laser scanning microscopy. We found that DanePy penetrated into chloroplasts but did not alter the functioning of the photosynthetic electron transport as assessed by chlorophyll fluorescence induction. In imaging, DanePy fluorescence was well distinct from chlorophyll fluorescence. Photoinhibition by excess photosynthetically active radiation caused quenching of DanePy fluorescence in the chloroplasts but not in other cell compartments. When leaves were infiltrated with dansyl, the fluorescent group in DanePy, there was no fluorescence quenching during photoinhibition. This shows that the fluorescence quenching of DanePy is caused by the conversion of its pyrrol group into nitroxide, i.e. it was caused by the reaction of singlet oxygen with the double sensor and not by artifacts. These data provide direct experimental evidence for the localization of singlet oxygen production to chloroplasts in vivo.     

Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to bacterial pathogens

Reactive oxygen species (ROS) are responsible for mediating cellular defense responses in plants. Controversy has existed over the origin of ROS in plant defense. We have isolated a novel extracellular peroxidase gene, CaPO2, from pepper (Capsicum annuum). Local or systemic expression of CaPO2 is induced in pepper by avirulent Xanthomonas campestris pv vesicatoria (Xcv) infection. We examined the function of the CaPO2 gene in plant defense using the virus-induced gene silencing technique and gain-of-function transgenic plants. CaPO2-silenced pepper plants were highly susceptible to Xcv infection. Virus-induced gene silencing of the CaPO2 gene also compromised hydrogen peroxide (H(2)O(2)) accumulation and hypersensitive cell death in leaves, both locally and systemically, during avirulent Xcv infection. In contrast, overexpression of CaPO2 in Arabidopsis (Arabidopsis thaliana) conferred enhanced disease resistance accompanied by cell death, H(2)O(2) accumulation, and PR gene induction. In CaPO2-overexpression Arabidopsis leaves infected by Pseudomonas syringae pv tomato, H(2)O(2) generation was sensitive to potassium cyanide (a peroxidase inhibitor) but insensitive to diphenylene iodonium (an NADPH oxidase inhibitor), suggesting that H(2)O(2) generation depends on peroxidase in Arabidopsis. Together, these results indicate that the CaPO2 peroxidase is involved in ROS generation, both locally and systemically, to activate cell death and PR gene induction during the defense response to pathogen invasion.     

Imaging the production of singlet oxygen in vivo using a new fluorescent sensor, Singlet Oxygen Sensor Green

Singlet oxygen is known to be produced by cells in response to photo-oxidative stresses and wounding. Due to singlet oxygen being highly reactive, it is thought to have a very short half-life in biological systems and, consequently, it is difficult to detect. A new commercially available reagent (singlet oxygen sensor green, SOSG), which is highly selective for singlet oxygen, was applied to a range of biological systems that are known to generate singlet oxygen. Induction of singlet oxygen production by the addition of myoglobin to liposome preparations demonstrated that the singlet oxygen-induced increases in SOSG fluorescence closely followed the increase in the concentration of conjugated dienes, which is stoichiometrically related to singlet oxygen production. Applications of photo-oxidative stresses to diatom species and leaves, which are known to result in the production of singlet oxygen, produced large increases in SOSG fluorescence, as did the addition of 3-(3',4'-dichlorophenyl)1,1-dimethylurea (DCMU) to these systems, which inhibits electron transport in photosystem II and stimulates singlet oxygen production. The conditional fluorescent (flu) mutant of Arabidopsis produces singlet oxygen when exposed to light after a dark period, and this coincided with a large increase in SOSG fluorescence. Wounding of leaves was followed by an increase in SOSG fluorescence, even in the dark. It is concluded that SOSG is a useful in vivo probe for the detection of singlet oxygen.     

Rapid alteration of cellular redox homeostasis upon exposure to cadmium and mercury in alfalfa seedlings

Here, the kinetics of oxidative stress responses of alfalfa (Medicago sativa) seedlings to cadmium (Cd) and mercury (Hg) (0, 3, 10 and 30 microm) exposure, expanding from a few minutes to 24 h, were studied. Intracellular oxidative stress was analysed using 2',7'-dichlorofluorescin diacetate and extracellular hydrogen peroxide (H(2)O(2)) production was studied with Amplex Red. Growth inhibition, concentrations of ascorbate, glutathione (GSH), homoglutathione (hGSH), Cd and Hg, ascorbate peroxidase (APX) activity, and expression of genes related to GSH metabolism were also determined. Both Cd and Hg increased cellular reactive oxygen species (ROS) production and extracellular H(2)O(2) formation, but in different ways. The increase was mild and slow with Cd, but more rapid and transient with Hg. Hg treatments also caused a higher cell death rate, significant oxidation of hGSH, as well as increased APX activity and transient overexpression of glutathione reductase 2, glutamylcysteinyl synthetase, and homoglutathione synthetase genes. However, Cd caused minor alterations. Hg accumulation was one order of magnitude higher than Cd accumulation. The different kinetics of early physiological responses in vivo to Cd and Hg might be relevant to the characterization of their mechanisms of toxicity. Thus, high accumulation of Hg might explain the metabolism poisoning observed in Hg-treated seedlings.     

Effect of spermine treatment on the functioning of Thellungiella salsuginea antioxidant system

The effect of spermine (Spm) treatment on the content of polyamines (PAs) and activities of antioxidant enzymes in the roots and leaves of Thellungiella salsuginea (Pall.) O.E. Schulz plants grown under optimal conditions were studied. The genes encoding three forms of ascorbate peroxidase (APX; APX1, APX2, and APX4) and genes of key enzymes of proline metabolism (Pro, P5CS1, 1P5CD) were identified, and their expression intensity was measured. Six-day-old plants were treated with Spm (1 and 2 mM) and with the inhibitor of polyamine oxidase (PAO) activity, N,N-(2-hydroxyethyl)hydrazine (HEH, 1 and 2 mM) separately or in combination by adding these compounds to nutrient medium. Roots and leaves responded differently to Spm treatment. In the leaves, the content of PAs reduced due to a decreased in the spermidine (Spd) content, whereas in the roots the total pool of PAs increased due to putrescine (Put) and Spd accumulation. Treatment with Spm activated PAO in the roots but not in the leaves; HEH removed this increase, but the intercellular Spm concentration was not substantially changed. It was suggested that treatment with Spm suppressed the biosynthesis of intracellular Spm and, on the other hand, stimulated the reverse conversion of Spm into Spd and further into Put due to the activation of one of the PAO isoforms. Plant treatment with Spm was not accompanied by a noticeable activation enzymes degrading hydrogen peroxide in the roots: APX, (except of peroxidase II), and catalase. However, the activity of Cu/Zn-SOD doubled and the activity of Mn-SOD reduced. In the leaves, slight activation of peroxidases I and III, the inhibition of Cu/Zn- and Mn-SOD, differential changes in the time-coursed of gene expression of three APX isoforms, and activated gene expression of key enzymes of Pro metabolism were observed. At the same time, the level of MDA did not increase either in the leaves or in the roots. This indicates that treatment of Th. salsuginea plants with Spm under optimal growing conditions did not enhance ROS generation and did not manifest prooxidant properties.     

Activation of ascorbate-glutathione cycle inArabidopsis leaves in response to aminotriazole

Aminotriazole(AT)-induced changes in growth, hydrogen peroxide content and activities of H₂O₂-scavenging antioxidant enzymes were investigated in the growing leaves ofArabidopsis plants (Arabidopsis thaliana cv Columbia). Catalase activity of rosette leaves was reduced by 65% with an application of 0.1 mM AT (a herbicide known as a catalase inhibitor), whereas the leaf growth and H₂O₂ content were almost unaffected. However, an approximate 1.6 to 2-fold increase in cytosolic ascorbate peroxidase (APX) activity concomitant with a substantial activation of glutathione reductase (GR) (approx. 22% increase) was observed during leaf growth in the presence of 0.1 mM AT. The activity of cytosolic APX in leaves was also increased by 1.8-fold with an application of exogenous 2 mM paraquat (an inducer of H₂O₂ production in plant cells) in the absence of AT. These results collectively suggest that (a) cytosolic APX and GR operate to activate an ascorbate-glutathione cycle for the removal of H₂O₂ under severe catalase deactivation, and (b) the expression of APX seems to be regulated by a change of the endogenous H₂O₂ level in leaf cells.     

Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system

Ascorbic acid has numerous and diverse roles in plant metabolism. We have used the vtc-1 mutant of Arabidopsis, which is deficient in ascorbate biosynthesis, to investigate the role of ascorbate concentration in growth, regulation of photosynthesis, and control of the partitioning of antioxidative enyzmes. The mutant possessed 70% less ascorbate in the leaves compared with the wild type. This lesion was associated with a slight increase in total glutathione but no change in the redox state of either ascorbate or glutathione. In vtc-1, total ascorbate in the apoplast was decreased to 23% of the wild-type value. The mutant displayed much slower shoot growth than the wild type when grown in air or at high CO(2) (3 mL L(-1)), where oxidative stress is diminished. Leaves were smaller, and shoot fresh weight and dry weight were lower in the mutant. No significant differences in the light saturation curves for CO(2) assimilation were found in air or at high CO(2), suggesting that the effect on growth was not due to decreased photosynthetic capacity in the mutant. Analysis of chlorophyll a fluorescence quenching revealed only a slight effect on non-photochemical energy dissipation. Hydrogen peroxide contents were similar in the leaves of the vtc-1 mutant and the wild type. Total leaf peroxidase activity was increased in the mutant and compartment-specific differences in ascorbate peroxidase (APX) activity were observed. In agreement with the measurements of enzyme activity, the expression of cytosolic APX was increased, whereas that for chloroplast APX isoforms was either unchanged or slightly decreased. These data implicate ascorbate concentration in the regulation of the compartmentalization of the antioxidant system in Arabidopsis.     

Ascorbate peroxidase–thioredoxin interaction

Proteomics data have suggested ascorbate peroxidase (APX) to be a potential thioredoxin-interacting protein. Using recombinant enzymes, we observed that incubation of pea cytosolic APX with reduced poplar thioredoxins h drastically inactivated the peroxidase. A similar inactivation is induced by reduced glutathione and dithiothreitol, whereas diamide and oxidized glutathione have no effect. Oxygen consumption measurements, modifications of the APX visible spectrum and protection by hydrogen peroxide scavenging enzymes suggest that APX oxidizes thiols leading to the generation of thiyl radicals. These radicals can in turn react with thiyl anions to produce the disulfide radical anions, which are responsible for oxygen reduction and subsequent hydrogen peroxide production. The APX inactivation is not due solely to hydrogen peroxide since fluorimetry indicates that the environment of the APX tryptophan residues is dramatically modified only in the presence of thiol groups. The physiological implications of this interaction are discussed.     

Exploring the neutral invertase-oxidative stress defence connection in Arabidopsis thaliana

Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.