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MadhuraBabu Kunta and Eliezer Louzada*

Ascorbate Peroxidase (APX) is a heme-containing, non-glycosylated enzyme that destroys harmful hydrogen peroxide via the ascorbate glutathione pathway in plants. This enzyme is considered to be an indispensable part of the electron-scavenging pathway and is involved in preventing oxidative damage in plants. Using differential display RT-PCR and 5' RACE a full length c-DNA clone was isolated, from citrus, with very high similarity at the nucleotide and amino acid level, to ascorbate peroxidases from several plant species. It is well known that APXs have highly conserved motifs like the Arg-38, Ars-71, Glu-65 and Asp-208 residues around the distal Hist-42 and proximal His-163. These residues are essential for binding the ligand heme. Additionally, Trp-179 is conserved in most APXs and is the third participant in hydrogen bonding network, together with His-163 and Asp-208. All these conserved motifs were present in the putative APX from citrus in addition of the presence of the peroxidase active site motif residues (APITLRLAWHSA) and the peroxidase heme-ligand motif (DIVVLSGGHTL). Expression analysis in E. Coli reviewed a recombinant protein of 27 Kda.

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Yueju Wang, Michael Wisniewski, Richard Meilan, Minggang Cui, Robert Webb and Leslie Fuchigami

Ascorbate peroxidase (APX) plays an important role in the metabolism of hydrogen peroxide in higher plants, affording them protection against oxidative stress. We studied the effect of overexpressing a cytosolic ascorbate peroxidase (cAPX) gene—derived from pea (Pisum sativum L.)—in transgenic tomato plants (Lycopersicon esculentum L.). Transformants were selected in vitro using kanamycin resistance and confirmed by polymerase chain reaction (PCR) and northern analyses. An APX native-gel assay indicated that, in the absence of stress, APX activity in transgenic plants was several times greater than that measured in wild-type (WT) plants. Several independently transformed lines were propagated and evaluated for resistance to chilling and salt stress. After placing seeds at 9 °C for 5 weeks, percent germination was greater for seeds obtained from transgenic lines (26% to 37%) compared to the WT (3%). Plants from transgenic lines also had lower electrolyte leakage (20% to 23%) than WT (44%) after exposure to 4 °C. Visual assessment of transgenic and WT lines exposed to salinity stress (200 or 250 mm) confirmed that overexpression of APX minimized leaf damage. Moreover, APX activity was nearly 25- and 10-fold higher in the leaves of transgenic plants in response to chilling and salt stresses, respectively. Our results substantiate that increased levels of APX activity brought about by overexpression of a cytosolic APX gene may play an important role in ameliorating oxidative injury induced by chilling and salt stress.

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Madhurababu Kunta, H. Sonia del Rio and Eliezer Louzada

Reactive oxygen species (ROS) are continuously produced during the normal aerobic metabolism and also under environmental stress conditions. They are the major damaging factors to the photosynthetic machinery under stress conditions and need to be scavenged for the normal growth of the plant. Ascorbate peroxidase (APX) is the key enzyme in detoxifying H2O2, one of ROS from chloroplast and cytosol. A cDNA encoding a putative APXcit was isolated from mature `Dancy' tangerine (Citrus reticulata Blanco) juice vesicles using differential display reverse transcription-polymerase chain reaction (RT-PCR). Subsequently, full-length APXcit cDNA clone and genomic clone were obtained and sequenced. The full-length APXcit sequence is composed by 1082-bp nucleotides, including an open reading frame (ORF) of 753 bp, encoding a protein of 250 amino acids (27 kDa). The 5' un-translated region (UTR) of the APXcit gene consisted of 91 nucleotides and the 3' UTR consisted of 238 nucleotides. Homology search for APXcit at GenBank database showed high similarity to APX from several plant species.

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Yali He and Bingru Huang

). Ascorbate peroxidase (APX) is characterized by its high degree of specificity for ascorbate as the electron donor and by its physiological role in the scavenging of hydrogen peroxide ( Asada, 1992 ; Asada et al., 1993 ). Guaiacol peroxidases (POD) are

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Natalia Falagán, Francisco Artés, Perla A. Gómez, Francisco Artés-Hernández, Alejandro Pérez-Pastor, Jose M. de la Rosa and Encarna Aguayo

determined spectrophotometrically as the amount of enzyme that produced 1 µmol·min −1 cinnamic acid ( = 0.977 m m −1 ·cm −1 ) at 290 nm at 40 °C. Ascorbate peroxidase (EC 1.11.1.11). The extraction of APX was carried out as described by Nakano and Asada

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Michelle DaCosta and Bingru Huang

nonenzymatic components that can be divided into two different types of repair mechanisms: 1) production of antioxidants or antioxidant enzymes that directly react with and scavenge AOS, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase

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Li-Song Chen and Lailiang Cheng

To determine the cause of a characteristic zonal chlorosis of `Honeycrisp' apple (Malus ×domestica Borkh.) leaves, we compared CO2 assimilation, carbohydrate metabolism, the xanthophyll cycle and the antioxidant system between chlorotic leaves and normal leaves. Chlorotic leaves accumulated higher levels of nonstructural carbohydrates, particularly starch, sorbitol, sucrose, and fructose at both dusk and predawn, and no difference was found in total nonstructural carbohydrates between predawn and dusk. This indicates that carbon export was inhibited in chlorotic leaves. CO2 assimilation and the key enzymes in the Calvin cycle, ribulose 1,5-bisphosphate carboxylase/oxygenase, NADP-glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, stromal fructose-1,6-bisphosphatase, and the key enzymes in starch and sorbitol synthesis, ADP-glucose pyrophosphorylase, cytosolic fructose-1,6-bisphosphatase, and aldose 6-phosphate reductase were significantly lower in chlorotic leaves than in normal leaves. However, sucrose phosphate synthase activity was higher in chlorotic leaves. In response to a reduced demand for photosynthetic electron transport, thermal dissipation of excitation energy (measured as nonphotochemical quenching of chlorophyll fluorescence) was enhanced in chlorotic leaves under full sun, lowering the efficiency of excitation energy transfer to PSII reaction centers. This was accompanied by a corresponding increase in both xanthophyll cycle pool size (on a chlorophyll basis) and conversion of violaxanthin to antheraxanthin and zeaxanthin. The antioxidant system, including superoxide dismutase and ascorbate peroxidase and the ascorbate pool and glutathione pool, was up-regulated in chlorotic leaves in response to the increased generation of reactive oxygen species via photoreduction of oxygen. These findings support the hypothesis that phloem loading and/or transport is partially or completely blocked in chlorotic leaves, and that excessive accumulation of nonstructural carbohydrates may cause feedback suppression of CO2 assimilation via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes.

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Zohar Shaham, Amnon Lers and Susan Lurie

`Granny Smith' apples [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] were harvested in two seasons and stored at 0 °C air storage with no pretreatment (control), after heating for 4 d at 38 °C, or after treating for 16 hours at 20 °C with 1 μL·L-1 1-methylcyclopropene (1-MCP). The effects of the two treatments on superficial scald development were consistent over both seasons. Scald began to appear after 8 weeks in control fruit, after 16 weeks in heated fruit but not on 1-MCP treated fruit. α-Farnesene accumulation and oxidation were slower in the skin of heated than in control fruit, and almost entirely absent in 1-MCP treated fruit. The activities of five antioxidant enzymes, ascorbate peroxidase, catalase, glutathione reductase, peroxidase and superoxide dismutate, were measured at two-week intervals in the apple peel, quantitatively as total activity and qualitatively by isozyme analysis. Enzyme activities either increased or remained stable during 16 weeks of storage, except for superoxide dismutase activity, which decreased. Ascorbate oxidase activity was higher in heated than control apples and there was an additional peroxidase isozyme present in activity gels. The activities of antioxidant enzymes were lower in 1-MCP treated fruit except for catalase during the first month of storage. Lipid soluble antioxidant activity was higher in 1-MCP treated fruit than the fruit of the other treatments, and water soluble antioxidant activity was higher in both treatments than in control fruit during the time that scald was developing in control apples. Both free and total phenol contents in the peel fluctuated during storage but no consistent trend was detected. The differences in enzyme activity and antioxidant content of the peel of 1-MCP and heated apples may play a role in preventing or delaying the appearance of superficial scald.

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Jeffrey A. Anderson and Sonali R. Padhye

Although heat stress injury is known to be associated with membrane dysfunctions, protein structural changes, and reactions of activated forms of oxygen, the underlying mechanisms involved are poorly understood. In this study, the relationships between thermotolerance and hydrogen peroxide (H2O2) defense systems, radical scavenging capacity [based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) reduction], and protein aggregation were examined in vinca [Catharanthus roseus (L.) G. Don `Little Bright Eye'], a heat tolerant plant, and sweet pea (Lathyrus odoratus L. `Explorer Mix'), a heat susceptible plant. Vinca leaves were 5.5 °C more thermotolerant than sweet pea leaves based on electrolyte leakage analysis. Vinca leaf extracts were more resistant to protein aggregation at high temperatures than sweet pea leaf extracts, with precipitates forming at ≥40 °C in sweet pea and at ≥46 °C in vinca. Vinca leaves also had nearly three times greater DPPH radical scavenging capacity than sweet pea leaf extracts. Two enzymatic detoxifiers of H2O2, catalase (CAT) and ascorbate peroxidase (APOX), demonstrated greater activities in vinca leaves than in sweet pea leaves. In addition, CAT and APOX were more thermostable in vinca, compared with sweet pea leaves. However, tissue H2O2 levels did not differ between controls and tissues injured or killed by heat stress in either species, suggesting that H2O2 did not play a direct role in acute heat stress injury in vinca or sweet pea leaves. Greater thermotolerance in vinca, compared with sweet pea, was associated with greater DPPH radical scavenging capacity, indicating that AOS other than H2O2 may be involved in acute heat stress injury.

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Shiow Y. Wang and Miklos Faust

The ability of low and high temperatures and S-containing compounds to overcome endo- and paradormancy along with the possible mechanisms involved in these treatments for breaking `Anna' apple bud dormancy were studied. All three treatments induced budbreak in paradormant (July) and endodormant (October) buds. Cold, heat, and allyl disulfide increased ascorbic acid, the reduced form of glutathione (GSH), total glutathione, total nonprotein thiol (NPSH), and nonglutathione thiol (RSH), whereas dehydroascorbic acid and oxidized glutathione (GSSG) decreased. The treatments also increased the ratios of ascorbic acid: dehydroascorbate and GSH: GSSG and the activities of ascorbate free-radical reductase (AFR, EC 1.6.5.4), ascorbate peroxidase (EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), ascorbate oxidase (AAO, EC 1.10.3.3), and glutathione reductase (GR, EC 1.6.4.2) in the buds. These results indicate that budbreak induced by cold, heat, and allyl disulfide is associated with the removal of free radicals through activated peroxide-scavenging systems.