Purification and characterization of pea cytosolic ascorbate peroxidase

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.     

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H(2)O(2) levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.     

Superoxide dismutase and ascorbate peroxidase are constitutively more thermotolerant than other antioxidant enzymes in Chenopodium album

Thermal stability of antioxidant defense enzymes was investigated in leaf and inflorescence of heat adaptive weed Chenopodium album. Leaf samples were taken at early and late seedling stage in December (LD, 20 °C/4 °C) and March (LM, 31 °C/14 °C). Young inflorescence (INF) was sampled at flowering in April (40 °C/21 °C). LD, LM and INF crude protein extracts were subjected to elevated temperatures (5 to 100 °C) for 30′. Superoxide dismutase (SOD) was the most heat stable enzyme followed by Ascorbate peroxidase (APX). Two heat stable SOD isozymes were visible on native-PAGE at 100 °C in both leaf and INF. Some heat stable APX isozymes were more abundant in INF than leaf. Thermostability of catalase (CAT) increased with age and increasing ambient temperatures in leaves. CAT activity was observed up to 60 °C in leaves and INF while peroxidase (POX) retained activity up to 100 °C in INF due to one thermostable isozyme. Glutathione reductase (GR), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR) showed activity up to 70 °C in both leaves and INF. DHAR activity was stable up to 60 °C while GR and MDHAR declined sharply after 40 °C. Constitutive heat stable isozymes of SOD and APX in leaves and INF may contribute towards heat tolerance in C. album.     

Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase

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.     

Reactive oxygen intermediates and glutathione regulate the expression of cytosolic ascorbate peroxidase during iron-mediated oxidative stress in bean

Excess of free iron is thought to harm plant cells by enhancing the intracellular production of reactive oxygen intermediates (ROI). Cytosolic ascorbate peroxidase (cAPX) is an iron-containing, ROI-detoxifying enzyme induced in response to iron overload or oxidative stress. We studied the expression of cAPX in leaves of de-rooted bean plants in response to iron overload. cAPX expression, i.e., mRNA and protein, was rapidly induced in response to iron overload. This induction correlated with the increase in iron content in leaves and occurred in the light as well as in the dark. Reduced glutathione (GSH), which plays an important role in activating the ROI signal transduction pathway as well as in ROI detoxification, was found to enhance the induction of APX mRNA by iron. To determine whether cAPX induction during iron overload was due to an increase in the amount of free iron, which serves as a co-factor for cAPX synthesis, or due to iron-mediated increase in ROI production, we tested the expression of APX in leaves under low oxygen pressure. This treatment, which suppresses the formation of ROI, completely abolished the induction of cAPX mRNA during iron overload, without affecting the rate of iron uptake by plants. Taken together, our results suggest that high intracellular levels of free iron in plants lead to the enhanced production of ROI, which in turn induces the expression of cAPX, possibly using GSH as an intermediate signal. We further show, using cAPX-antisense transgenic plants, that cAPX expression is essential to prevent iron-mediated tissue damage in tobacco.     

Regulation and function of ascorbate peroxidase isoenzymes

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.     

Molecular cloning of a cytosolic ascorbate peroxidase cDNA from cell cultures of sweetpotato and its expression in response to stress

A cDNA encoding a cytosolic ascorbate peroxidase (APX), swAPX1 , was isolated from cell cultures of sweetpotato (Ipomoea batatas) by cDNA library screening, and its expression in the context of various environmental stresses was investigated. swAPX1 contains an ORF of 250 amino acids (27.5 kDa) encoding a protein with a pI value of 5.32. The swAPX1 ORF does not code for a transit peptide, suggesting that the product is a cytosolic isoform. RNA blot analysis showed that swAPX1 gene is expressed in cultured cells and mature leaves, but not in stems, non-storage or storage roots of sweetpotato. The level of swAPX1 RNA progressively increased during cell growth in suspension cultures. In leaf tissues, the gene responded differentially to various abiotic stresses, as revealed by RT-PCR analysis. swAPX1 was highly induced in leaves by wounding, and treatment with methyl viologen (50 M), hydrogen peroxide (440 mM), abscisic acid (ABA; 100 M) or exposure to high temperature (37°C). In addition, the gene was strongly induced in the leaves following inoculation with a bacterial pathogen (Pectobacterium chrysanthemi). These results indicate that swAPX1 may be involved in hydrogen peroxide-detoxification and thus help to overcome the oxidative stress induced by abiotic and biotic stresses.Communicated by G. Jürgens     

Differential responses of the enzymes involved in proline biosynthesis and degradation in drought tolerant and sensitive cotton genotypes during drought stress and recovery

The relative water content (RWC), free proline levels and the activities of enzymes involved in proline metabolism were studied in drought tolerant (Ca/H 680) and drought sensitive (Ca/H 148) genotypes of cotton (Gossypium hirsutum L.) during induction of water stress and posterior recovery. Water stress caused a significant increase in proline levels and P5CS activity in leaves of both tolerant and sensitive genotypes, whereas the activity of P5CR increased minimally and the activity of OAT remains unchanged. The activity of PDH decreased under drought stress in both the genotypes. The leaf of tolerant genotype maintained higher RWC, photosynthetic activity and proline levels, as well as higher P5CS and P5CR activities under water stress than that of drought sensitive genotype. The drought induced proline levels and activities of P5CS and P5CR declined and tend to be equal to their respective controls, during recovery, whereas the PDH activity tends to increase. These results indicate that induction of proline levels by up regulation of P5CS and down regulation of PDH may be involved in the development of drought tolerance in cotton.     

Characterization of ascorbate peroxidase in soybean under flooding and drought stresses

Flooding and drought are the two different forms of water stress that adversely affect the growth and development of soybean plant in particular at early stage. Ascorbate peroxidase (APX) is a known antioxidant enzyme that plays key role in abiotic stresses. To investigate the changes in APX in soybean under drought and flooding stresses, western blotting, enzyme activity assay and biophoton emission techniques were used. Flooding stress was imposed by adding excess amount of water in the sand and drought by withholding water supply. Under flooding stress, a decrease in APX was detected with time. Completely opposite trend was evident in hypocotyl and root of plants exposed to drought. Western blotting and APX activity results are complementary to each other. Biophoton emissions further confirmed the increasing and decreasing trend of APX under drought and flooding stress, respectively.     

Proteomic analysis of rice leaves during drought stress and recovery

Three-week old plants of rice (Oryza sativa L. cv CT9993 and cv IR62266) developed gradual water stress over 23 days of transpiration without watering, during which period the mid-day leaf water potential declined to approximately -2.4 MPa, compared with approximately -1.0 MPa in well-watered controls. More than 1000 protein spots that were detected in leaf extracts by proteomic analysis showed reproducible abundance within replications. Of these proteins, 42 spots showed a significant change in abundance under stress, with 27 of them exhibiting a different response pattern in the two cultivars. However, only one protein (chloroplast Cu-Zn superoxide dismutase) changed significantly in opposite directions in the two cultivars in response to drought. The most common difference was for proteins to be up-regulated by drought in CT9993 and unaffected in IR62266; or down-regulated by drought in IR62266 and unaffected in CT9993. By 10 days after rewatering, all proteins had returned completely or largely to the abundance of the well-watered control. Mass spectrometry helped to identify 16 of the drought-responsive proteins, including an actin depolymerizing factor, which was one of three proteins detectable under stress in both cultivars but undetectable in well-watered plants or in plants 10 days after rewatering. The most abundant protein up-regulated by drought in CT9993 and IR62266 was identified only after cloning of the corresponding cDNA. It was found to be an S-like RNase homologue but it lacked the two active site histidines required for RNase activity. Four novel drought-responsive mechanisms were revealed by this work: up-regulation of S-like RNase homologue, actin depolymerizing factor and rubisco activase, and down-regulation of isoflavone reductase-like protein.     

Regulation of the ascorbate-glutathione cycle in wild almond during drought stress

In wild species of almond (Prunus spp.), the activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), as well as the levels of ascorbate/glutathione pools and H₂O₂ were subjected to water deficit and shade conditions. After 60 days of water shortage, the species were subjected to a rewatering treatment. During water recovery, leaves exposed to sunlight and leaves under shade conditions of about 20–35% of environmental irradiance were sampled. After 70 days without irrigation, mean predawn leaf water potential of all the species fell from −0.32 to −2.30 MPa and marked decreases in CO₂ uptake and transpiration occurred. The activities of APX, MDHAR, DHAR, and GR increased in relation to the severity of drought stress in all the wild species studied. Generally, APX, MDHAR, DHAR, and GR were down-regulated during the rewatering phase and their activities decreased faster in shaded leaves than in sun-exposed leaves. The levels in total ascorbate, glutathione, and H₂O₂ were directly related to the increase in drought stress and subsequently decreased during rewatering. The antioxidant response of wild almond species to drought stress limits cellular damage caused by reactive oxygen species during periods of water deficit and may be of key importance for the selection of drought-resistant rootstocks for cultivated almond.     

Photosynthesis by six Portuguese maize cultivars during drought stress and recovery

Photosynthesis, chlorophyll fluorescence, and leaf water parameters were measured in six Portuguese maize (Zea mays L.) cultivars during and following a period of drought stress. The leaf relative water content (RWC) responded differently among cultivars but except for cultivar PB369, recovered close to initial values after watering was restored. Photosynthetic rate and stomatal conductance decreased with drought but more slowly in cultivars PB269 and PB260 than in cultivars AD3R, PB64, PB304 and PB369. Water use efficiency (WUE) decreased during the water stress treatment although with cultivar PB260 the decrease was marked only when the RWC fell below 40%. Recovery of WUE was seen with all cultivars except PB369. The maximum quantum efficiency of photosystem II, the photochemical quenching coefficient, the electron transport rate in PSII and the estimated functional plastoquinone pool tended to decrease with drought, while the non-photochemical quenching coefficient increased. The parameters estimated from chlorophyll fluorescence did not recover in PB369, during re-watering. The results show that PB260 and PB269 were the most tolerant and PB369 was the least tolerant cultivars to water stress. The variation found among the cultivars tested suggests the existence of valuable genetic resources for crop improvement in relation to drought tolerance.     

Molecular cloning, expression and mapping analysis of a novel cytosolic ascorbate peroxidase gene from tomato.

Ascorbate peroxidase (APX, EC 1.11.1.11) plays a major role in H(2)O(2)-scavenging in plants and can help to avoid reactive oxygen species (ROS) damage. A new cytosolic APX gene was cloned from tomato (designated LecAPX2) by RACE-PCR. The full-length cDNA of LecAPX2 contained a complete open reading frame (ORF) of 753 bp, which encoding 250 amino acid residues. Homology analysis of LecAPX2 showed a 94% identity with potato cAPX gene and 92% identity with another tomato cAPX gene (APX20), the deduced amino acid showed 88% homology with APX20 protein and 75-92% identity with cAPX from other plants such as potato, tobacco, broccoli, spinach, pea, rice, etc. LecAPX2 revealed the existence of a haem peroxidase and plant APX family signatures. Northern blot analysis showed that LecAPX2 was constitutively expressed in root, stem, leaf, flower and fruit of tomato, whereas the expression levels were different. LecAPX2 was mapped to 6-A using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii.