Effects of Catalase and Ascorbate Peroxidase on the Root Growth of Rice under Cadmium Stress

[Objective] The research aimed to study the correlations between catalase（CAT） and ascorbate peroxidase（ASP） and the growth and development of rice roots under cadmium stress.[Method] Taking rice variety Zhonghua No.11 as materials,the changes of rice seedlings under the treatment conditions of Cd,Cd＋CAT inhibitor,Cd＋APX inhibitor were studied.[Result] Under Cd stress,inhibition of CAT activity caused the significant inhibition on the growth of aerial parts,decreased the number of adventitious roots and lateral roots,but it can significant promote the elongation growth of adventitious roots and lateral roots.Moreover,the length of the first lateral root from root tip on the primary roots and adventitious roots was also increased than control.When APX activity was inhibited,the growth changes of rice were similar with that treated by CAT inhibitor.[Conclusion] CAT and APX may play important roles in the regulation of rice root system growth in both non-stress and Cd-stressed rice

Mechanisms of Salt Tolerance in Transgenic Arabidopsis thaliana Carrying a Peroxisomal Ascorbate Peroxidase Gene from Barley

Ascorbate peroxidases （APX）, localized in the cytosol, peroxisome, mitochondria, and chloroplasts of plant cells, catalyze the reduction of H2O2 to water by using ascorbic acid as the specific electron donor. To determine the role of peroxisomal type ascorbate peroxidase （pAPX）, an antioxidant enzyme, in protection against salt-induced oxidative stress, transgenic Arabidopsis thaliana plant carrying a pAPX gene （HvAPX1） from barley （Hordeum vulgate L.） was analyzed. The transgenic line pAPX3 was found to be more tolerant to salt stress than the wild type. Irrespective of salt stress, there were no significant differences in Na^＋, K^＋, Ca^2＋, and Mg^2＋ contents and the ratio of K^＋ to Na^＋ between pAPX3 and the wild type. Clearly, the salt tolerance in pAPX3 was not due to the maintenance and reestablishment of cellular ion homeostasis. However, the degree of H2O2 and lipid peroxidation （measured as the levels of malondialdehyde） accumulation under salt stress was higher in the wild type than in pAPX3. The mechanism of salt tolerance in transgenic pAPX3 can thus be explained by reduction of oxidative stress injury. Under all conditions tested, activities of superoxide, glutathione reductase, and catalase were not significantly different between pAPX3 and the wild type. In contrast, the activity of APX was significantly higher in the transgenic plant than in wild type under salt stress. These results suggested that in higher plants, HvAPX1 played an important role in salt tolerance and was a candidate gene for developing salttolerant crop plants.

Overexpression of a Cytosolic Ascorbate Peroxidase Gene, OsAPX2, Increases Salt Tolerance in Transgenic Alfalfa

Alfalfa （Medicago sativa L.） is an important forage crop in the world and it is of great signiifcance for the improvement of its salt tolerance. To improve salt tolerance in alfalfa, a rice ascorbate peroxidase gene （OsAPX2） was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation with marker gene bar. The different T-DNA insertions in T1 transgenic alfalfa were identiifed by Southern hybridization. Three independent T2 transgenic lines were selected for stress analysis and the results showed that all of them were salt tolerant compared with wild-type plants. The transgenic plants had low levels of H2O2, malondialdehyde and relative electrical conductivity under salt and drought stresses. Moreover, the contents of chlorophyll and proline, and APX activity were high in transgenic plants under salt and drought stresses. Taken together, the overexpression of OsAPX2 enhances salt tolerance in alfalfa through scavenging reactive oxygen species.

Differential Expression of Two Cytosolic Ascorbate Peroxidases and Two Superoxide Dismutase Genes in Response to Abiotic Stress in Rice

Superoxide dismutase (SOD) and ascorbate peroxidase (APX) play central roles in the pathway for scavenging reactive oxygen species in plants, thereby contributing to the tolerance against abiotic stress. Here we report the responses of cytosolic SOD (cSOD; sodCc1 and sodCc2) and cytosolic APX (cAPX; OsAPX1 and OsAPX2) genes to oxidative and abiotic stress in rice. RNA blot analyses revealed that methyl viologen treatment caused a more prominent induction of cAPXs compared with cSODs, and hydrogen peroxide treatment induced the expression of cAPXs whereas cSODs were not affected. These results suggest that cAPXs play more important roles in defense against oxidative stress compared with cSODs. It is noted that cSODs and cAPXs showed coordinate response to abscisic acid treatment which induced both sodCc1 and OsAPX2. However, cSODs and cAPXs responded differentially to drought, salt and chilling stress, which indicates that cSOD and cAPX genes are expressed differentially in response to oxidative and abiotic stress in rice.

Kiwifruit(Actinidia chinensis 'Hongyang') cytosolic ascorbate peroxidases(AcAPX1 and AcAPX2) enhance salinity tolerance in Arabidopsis thaliana

Ascorbate peroxidase(APX) plays a key role in scavenging reactive oxygen species(ROS) in higher plants. However, there is very little information available on the APXs in kiwifruit(Actinidia), which is an economically and nutritionally important horticultural crop with exceptionally high ascorbic acid(AsA) accumulation. This study aims to identify and characterize two cytosolic APX genes(AcAPX1 and AcAPX2) derived from A. chinensis ‘Hongyang’. The constitutive expression pattern was determined for both AcAPX1 and AcAPX2, and showed relatively higher expression abundances of AcAPX1 in leaf and AcAPX2 in root. Transcript levels of AcAPX1 and AcAPX2 were increased in kiwifruit roots treated with Na Cl. Subcellular localization assays using GFP-fusion proteins in Arabidopsis protoplasts showed that both AcAPX1 and AcAPX2 are targeted to the cytosol. Recombinant AcAPX1 or AcAPX2 proteins were successfully expressed in the prokaryotic expression system and their individual ascorbate peroxidase activities were determined. Finally, constitutive over-expression of AcAPX1 or AcAPX2 could dramatically increase total As A, glutathione level and salinity tolerance under Na Cl stress in Arabidopsis thaliana. Our findings revealed that cytosolic AcAPX1/2 may play an important protective role in the responses to unfavorable environmental stimuli in kiwifruit.

Rapid Inactivation of Chloroplastic Ascorbate Peroxidase is Responsible for Oxidative Modification to Rubisco in Tomato (Lycopersicon esculentum) under Cadmium Stress

To investigate the sensitive site of antioxidant systems in chloroplast under cadmium stress and its consequence on reactive oxygen species production and action, the sub-organellar localization of chloroplast superoxide dismutases （SOD, EC 1.15.1.1） and ascorbic peroxidase （APX, EC 1.11.1.11） isoenzymes and changes of enzymes activities under cadmium stress were investigated in tomato seedlings. Two APX isoforms, one thylakoid-bound and one stromal, were detected. Cd at 50 μM induced a moderate increase of SOD activities but a rapid inactivation of both APX isoenzymes. APX inactivation was mainly related to the decrease of ascorbate concentration, as supported by in vitro treatment of exogenous ascorbate and APX kinetic properties under Cd stress. H2O2 accumulation in chloroplast, as a consequence of APX inactivation, was associated with a 60% loss of Rubisco （EC 4.1.1.39） activity, which could be partially accounted for by a 10% loss of Rubisco content. Protein oxidation assay found that the Rubisco large subunit was the most prominent carbonylated protein; the level of carbonylated Rubisco large subunit increased fivefold after Cd exposure. Thiol groups in the Rubisco large subunit were oxidized, as indicated by non-reducing electrophoresis. Treating crude extract with H2O2 resulted in a similar pattern of protein oxidation and thiols oxidation with that observed in Cd-treated plants. Our study indicates that APXs in the chloroplast is a highly sensitive site of antioxidant systems under Cd stress, and the inactivation of APX could be mainly responsible for oxidative modification to Rubisco and subsequent decrease in its activity.

Ascorbate peroxidase 1 confers resistance to southern corn leaf blight in maize

Southern corn leaf blight (SCLB), caused by Bipolarismaydis, is one of the most devastatingdiseases affecting maize production. However,only one SLCB resistance gene, conferring partialresistance, is currently known, underscoring theimportance of isolating new SCLB resistancerelatedgenes. Here, we performed a comparativeproteomic analysis and identified 258 proteinsshowing differential abundance during the maizeresponse to B. maydis. These proteins included anascorbate peroxidase (Zea mays ascorbate peroxidase1 (ZmAPX1)) encoded by a gene locatedwithin the mapping interval of a previously identifiedquantitative trait locus associated with SCLBresistance. ZmAPX1 overexpression resulted inlower H_(2)O_(2) accumulation and enhanced resistanceagainst B. maydis. Jasmonic acid (JA)contents and transcript levels for JA biosynthesisand responsive genes increased in ZmAPX1-overexpressing plants infected with B. maydis,whereas Zmapx1 mutants showed the oppositeeffects. We further determined that low levels of H_(2)O_(2) are accompanied by an accumulation of JAthat enhances SCLB resistance. These resultsdemonstrate that ZmAPX1 positively regulatesSCLB resistance by decreasing H_(2)O_(2) accumulationand activating the JA-mediated defensesignaling pathway. This study identified ZmAPX1as a potentially useful gene for increasing SCLBresistance. Furthermore, the generated datamay be relevant for clarifying the functions ofplant APXs.

Influence of heat stress on leaf ultrastructure, photosynthetic performance, and ascorbate peroxidase gene expression of two pear cultivars(Pyrus pyrifolia)

Plants encounter a variety of stresses in natural environments. One-year-old pot-grown trees of pear(Pyrus pyrifolia Nakai cv. Cuiguan and Wonhwang) were exposed to two heat stress regimes. Under constant short-term heat stress, chloroplasts and mitochondria were visibly damaged. Relative chlorophyll content and maximum photochemical efficiency of photosystem II were significantly decreased, which indicated that the leaf photosynthetic capability declined. Under chronic heat stress, mesophyll cell ultrastructure was not obviously damaged, but leaf photosynthetic capability was still restrained. As chronic heat stress was a simulation of the natural environment in summer, further study of the responses under this stress regime was undertaken. Ascorbate peroxidase(APX) activity was increased in ‘Cuiguan', but not in ‘Wonhwang'. Inducible expression of PpAPX genes in the cytoplasm, chloroplasts and peroxisomes was consistent with increased APX activity in ‘Cuiguan', whereas only weak induction of PpAPX genes was observed in ‘Wonhwang'. The isoenzymes cytosolic APX1(cAPX1) and stromal APX(sAPX) were confirmed to be localized in the cytoplasm and chloroplasts, respectively.

In Silico Characterization and Homology Modeling of Thylakoidbound Ascorbate Peroxidase from a Drought Tolerant Wheat Cultivar

Ascorbate peroxidase, a haem protein （EC 1.11.1.11）, efficiently scavenges hydrogen peroxide （H2O2） in cytosol and chloroplasts of plants. In this study, a fulllength coding sequence of thylakoid-bound ascorbate peroxidase cDNA （TatAPX） was cloned from a drought tolerant wheat cultivar C306. Homology modeling of the TatAPX protein was performed by using the template crystal structure of chloroplastic ascorbate peroxidase from tobacco plant （PDB： IIYN）. The model structure was further refined by molecular mechanics and dynamic methods using various tools such as PROCHECK, ProSA and Verify3D. The predicted model was then tested for docking with H2O2, the substrate for TatAPX enzyme. The results revealed that Arg233 and Glu255 in the predicted active site of the enzyme are two important amino acid residues responsible for strong hydrogen bonding affinity with H2O2, which might play an important role in scavenging of H2O2 from the plant system.

Linking Chloroplast Antioxidant Defense to Carbohydrate Availability： The Transcript Abundance of Stromal Ascorbate Peroxidase Is Sugar-Controlled via Ascorbate Biosynthesis

All genes encoding chloroplast antioxidant enzymes are nuclear-encoded and posttranscriptionally targeted to chloroplasts. The transcript levels of most of them decreased upon sucrose feeding like the transcript levels of many genes encoding components of the photosynthetic electron transport chain. However, the transcript abundance of stromal ascorbate peroxidase （s-APX; At4g08390） increased. Due to mild sugar application conditions, the plants kept the phosphorylation status of the ADP＋ATP pool and the redox states of the NADPH＋NADP~ and the ascorbate pools under control, which excludes them as signals in s-APX regulation. Correlation with ascorbate pool size regulation and comparison of transcript abundance regulation in the starch-biosynthetic mutant adgl, the ascorbate biosynthesis mutant vtcl, and the abscisic acid （ABA） biosynthetic mutant aba2 showed a link between sugar induction of s-APX and ascorbate biosynthesis.

Effects of exogenous salicylic acid on growth and H_2O_2-metabolizing enzymes in rice seedlings under lead stress

Salicylic acid （SA） was an essential component of the plant resistance to pathogens and also plays an important role in mediating plant response to some abiotic stress. The possible effects of SA on the growth and H2O2-metabolizing enzymes in rice seedlings under lead stress were studied. When rice seedlings grown in nutrient solution containing Pb^2＋ （0, 0.05, 0.15, 0.25 mmol/L） for 18 d, the plant biomass as well as the chlorophyll content of leaves decreased with increasing Pb concentration. The pre-treatment with SA （treated with 0.1 mmol/L SA for 48 h before Pb stress） partially protected seedlings from Pb toxicity. The chlorophyll contents were significant higher in leaves of Pb-exposed with SA pre-treatment seedlings than in Pb-exposed plants at the same Pb intensity. SA pre-treated alone could significantly increase the length of shoot and root of seedlings but the vigour difference was not marked under long-term exposure to Pb toxicity. SA pre-treated influence the H2O2 level in leaves of seedlings by up-regulating the activity of superoxide dismutase （SOD）, repressing the activity of catalase （CAT） and ascorbate peroxidase （APX） depending on the concentrations of Pb^2＋ in the growth medium. The results supported the conclusion that SA played a positive role in rice seedlings against Pb toxicity.

Ultraviolet irradiation induced oxidative stress and response of antioxidant system in an intertidal macroalgae Corallina officinalis L.

The response of the antioxidant defense system of an intertidal macroalgae Corallina officinalis L.to different dosages of UV-B irradiation was investigated.Results showed that superoxide dimutase （SOD） and peroxidase （POX） increased and then maintained at a relatively stable level when subjected to UV-B irradiation.Catalase （CAT） activity under medium dosage of UV-B irradiation （Muv） and high dosage of UV-B irradiation （Huv） treatments were significantly decreased.Ascorbate peroxidase （APX） activity first remained unaltered and then increased in Huv treatment.In addition,the assay on isozymes was carried out using non-denaturing polyacrylamide gel electrophoresis （PAGE）.The activities of some SOD isoforms were altered by UV-B.Two new bands （POX V and POX VII） appeared upon exposure to all three UV-B dosages.CAT III activity was increased by low dosage of UV-B irradiation （Luv）,whereas CAT III and CAT IV disappeared when the alga was exposed to Muv and Huv.Two bands of APX （APX VI and APX VII） were increased and a new band （APX X） was observed under Huv exposure.H 2 O 2 and thiobarbituric acid reacting substance （TBARS） increased under Muv and Huv treatments.Overall,UV-B protection mechanisms are partly inducible and to a certain extent sufficient to prevent the accumulation of damage in C.officinalis.

Nitric oxide and hydrogen sulfide share regulatory functions in higher plant events

Nitric oxide(NO)and hydrogen sulfide(HS)are two molecules that share signaling properties in plant and animal cells NO and H2S originate two farmilies of de rived mol ecules designated reactive nitrogen and sulfur species(RNS and RSS,respectively).These molecules are responsible for certain protein regulatory processes through posttranslational modifications(PTMs),being the most remarkable S nitrosation and persufidation,which afect the thiol group of cysteine residues.NO and H2S can also exert regulatory functions due to their interaction through the iron present in proteins that contain heme groups or iron-sulfur dlusters,as reported mainly in animal cells.Howewer,the available information in plant cells is still very limited thus far.In higher plants,NO and H2S are involved in a myriad of physiological events from seed germination to fruit ripening,but also the mec hanism of response to biotic and abiotic stress conditions.This vie wpoint manuscript highlights the functional regulatory parllelism of these two molecules which also interact with the metabolism of reactive oxygen species(ROS)in plant cells.

Stress Ameliorative Effects of Indole Acetic Acid on Hordeum vulgare L. Seedlings Subjected to Zinc Toxicity

The heavy metals present in the environment accumulate in the plants and affect their productivity and yield.By entering the food chain,metals cause several serious health problems in human beings as well as in other organisms.Indole acetic acid(IAA)is known to act as a signaling molecule between symbiotic association of metal accumulating plants and plant growth promoting rhizobacteria(PGPR).Present study demonstrated a protective role of IAA against surplus Zinc(Zn)-induced toxicity to Hordeum vulgare seedlings.Elevated Zn concentrations suppressed the plant growth,caused a reduction in leaf relative water contents(RWC)and elevated free proline and non-protein thiols(NPT)accumulation.Zinc treatment also led to enhanced lipid peroxidation(MDA contents)as well as the activity of ascorbate peroxidase(APX),showing the involvement of antioxidative defense mechanism to reduce Zn induced toxicity.IAA oxidase activity was also observed to increase due to Zn treatment.IAA pretreatment of H.vulgare caryopsis could partly revert the Zn-induced toxicity in seedlings.

Effect of Iron on Growth, Pigmentation and Antioxidative Activity of Bloom Forming Cyanobacteria

Cyanobacterial blooms are ubiquitous in fresh and brackish eutrophic waters in India. The cyanotoxins produced by many bloom forming cyanobacteria severely affect the health of animals, fishes, birds and human beings. Different physical and chemical factors contribute towards bloom formation. Ten bloom forming cyanobacteria were isolated from natural blooms of northern India. The strains were purified and enriched in the laboratory. The aim of this study was to understand the influence of iron on growth, pigmentation and antioxidative activity of enzymes-catalase and ascorbate peroxidase of bloom forming cyanobacteria. Results show that different strains of bloom forming cyanobacteria attain optimum growth at varied concentration of iron. The cyanobacterial strains like Synechocystis aquatalis, Merismopedia glauca, Anabaena variabilis and Anabaena iyengarii exhibit maximum growth at low iron concentration （2 pM） while some species require higher concentration of iron for their optimum growth namely, Arthrospira platensis show optimum growth at 10uM, and Nostocpaludosum shows maximum growth at 100uM concentration of iron. It was also noticed that chlorophyll and phycobiliprotein content also varies with change in iron concentration. The activity of antioxidative enzymescatalase and ascorbate peroxidase was noticed in all ten cyanobacterial strains. In the light of the findings, it seems that Arthrospira platensis possess maximum catalase and ascorbate peroxidase activity. Increment in concentration beyond optimum value leads to deterioration in the growth, pigment content and enzymatic activity of the cyanobacterial strains. Knowledge about the factors influencing growth of bloom forming cyanobacteria will help to work out ways for eradication of hazardous cyanobacterial blooms.

PROMOTIVE EFFECT OF Se ON THE GROWTH AND ANTIOXIDATION OF A BLUE-GREEN ALGA SPIRULINA MAXIMA

Cultures of a blue-green algaSpirulina maxima (Setch. et Gard.) Geitler with various concentrations of Se in Zarrouk's medium showed that not higher than 40 mg/L Se could promote its growth. The present experiments showed thatS. maxima grown under normal conditions, has an oxidant stress defence system for hydrogen peroxide (H2O2) removal, which is the Halliwell-Asada pathway. When 4 to 20 mg/L Se was added to the algal medium, this pathway was replaced by a so-called Sestressed pathway containing GSH peroxidase (GSH-POD). As a result of the occurrence of both higher activity of GSH-POD and lower levels of hydroxyl radical (OH·), the Se-stressed pathway scavenged H2O2 so effectively that the growth ofS. maxima was promoted by 4 to 20 mg/L Se. While GSH-POD activity of the alga disappeared at 40 mg/L Se, the recovery of ascorbate peroxidase was observed. The lower levels of ascorbic acid and GSH made the Halliwell-Asada pathway for scavenging H2O2 less effective, while the highest activity of catalase might be responsible in part for the H2O2 removal, causing the level of OH· inS. maxima grown at 40 mg/L Se to be much higher than the OH· level in this alga grown at 4 to 20 mg/L Se, but lower than that in the control. The OH· level changes caused the growth ofS. maxima cultured at 40 mg/L Se to increase slightly to close to that of the control.

Long Term Salinity Stress Reveals Variety Specific Differences in Root Oxidative Stress Response

Salinity stress induces oxidative stress caused by reactive oxygen species （ROS）： superoxide radicals, hydrogen peroxide （H2O2） and hydroxyl radicals. Activities of both enzymatic and non-enzymatic components of the antioxidant system and related growth parameters were studied in the roots of the salt tolerant rice variety FL478 and the sensitive variety IR29 in response to long term stress （12 d）induced by 50 mmol/L NaCI. The comparative study showed that FL478 maintained higher relative growth rate and lower Na＋/K＋ in the roots than IR29 due to a higher membrane stability index that effectively exclude Na＋. Lower TBARS （thiobarbituric acid reactive substance） content in FL478 roots indicated that its membrane was relatively unaffected by ROS despite high H2O2 content recorded under the salinity stress. Relatively higher superoxide dismutase activity along with a parallel increase in transcript level of superoxide dismutase （Os07g46990） in FL478 indicated that this protein might make a vital contribution to salt stress tolerance. Although the content of ascorbic acid remained unchanged in FL478, the activity of ascorbic peroxidases （APOXs） was reduced comparably in the both varieties. Transcriptomic data showed that a larger number of peroxidase genes were upregulated in FL478 compared to IR29 and several of which might provide engineering targets to improve rice salt tolerance.

Comparative analysis of panicle proteomes of two upland rice varieties upon hyper-osmotic stress

Drought is a major environmental factor that limits the yield of rice dramatically.Upland rice is now regarded as a promising rice cultivar in water saving agriculture.Two varieties of upland rice Zhonghan 3 and IR29 were used to compare the physiological and proteomic responses to hyper-osmotic stress induced by 15%polyethyleneglycol(PEG)at the reproductive stage.Osmotic stress affected the growth development and caused the loss of production especially the grain yield.IR29 was more tolerant to PEG than Zhonghan 3 as shown by less yield loss under osmotic stress conditions.Comparative proteomic analysis of the panicle suggested that the up-regulation of glycolysis related proteins and defense proteins may contribute to the better osmotic tolerance in IR29.

Facile synthesis of enzyme functional metal-organic framework for colorimetric detecting H_2O_2 and ascorbic acid

In this work,a metal-organic frameworks material MIL-88 was prepared easily using solvent-thermal method,and was first found to have catalytic activities similar to those of biological enzymes such as catalase and peroxidase.The material was characterized by XRD,SEM,TEM,EDX,FT-IR techniques and an N2 adsorption method.It exhibited peroxidase-like activity through catalytic oxidation of the peroxidase substrate 3,3＇,5,5＇-tetramethylbenzidine（TMB） in the presence of H2O2,producing a blue-colored solution.Under optimal conditions,the absorbance at 652 nm is linearly correlated with the concentration of H2O2 from 2.0×10^-6 mol/L to 2.03×10^-5 mol/L（R-2=0.981） with a detection limit of 5.62×10^-7 mol/L（S/N=3）.More importantly,a sensitive and selective method for ascorbic acid detection was developed using this material as a catalyst.The analytical method for ascorbic acid detection was observed to have a linear range from 2.57×10^-6 mol/L to 1.01×10^-5 mol/L（R-2=0.989） with a detection limit of 1.03×10^-6 mol/L（S/N=3）.This work suggests MOFs have advantages of preparing biomimetic catalysts and extends applications of the functional MOFs in the field of biosensor.