Identification Of Copper-induced Proteins And The Role Of CuZn-SOD In Copper-induced Antioxidant Defense In Elsholtzia Haichowensis

Copper(Cu) is an essential element for plants associated with proteins and enzymes involved in electron transfer and redox reactions.However,excess Cu can be toxic to plants and induces a wide range of biochemical effects and physiological processes,such as photosynthesis,pigment synthesis,nitrogen and protein metabolism,membrane integrity, and mineral uptake.The toxicity of Cu can be considered an oxidative stress mediated by reactive oxygen species(ROS).Being a redox-active metal,Cu⁺ can catalyse superoxide anion(O₂^-) formation and subsequently results in hydrogen peroxide(H₂O₂) and hydroxyl radical(HO·) production via Fenton-type reactions.It had reported that excess Cu induced the contents of protein thiol and increased some expressed proteins,such as metallothionein (MT),copper-zinc superoxide dismutase(CuZn-SOD),pathogenesis-related protein(PR) and heat shock protein(HSP).These results need be testified because of lack of universality. It had reported that excess Cu induced hydrogen peroxide(H₂O₂) accumulation and increased antioxidant enzyme activities,but the resource of H₂O₂ and the mechanism of antioxidant defense under Cu stress in plant cells remain to be determined.In the present study,changes of Cu-induced proteins were investigated in roots and leaves of Elsholtzia haichowensis and in rice germinating embryoes.The ROS resource and the antioxidant enzymes activities were investigated in roots and leaves of E.haichowensis under Cu stress.The results as follows:Excess copper increased Cu accumulation in E.haichowensis and germinating rice embryoes under hydroponics,at the same time,excess copper inhibited root elongations and decreased chlorophyll contents in E.haichowensis,and inhibited radicel elongations in geminating rice seeds.Excess copper significantly increased the contents of protein thiol in roots and leaves of E.haichowensis and in rice germinating embryoes.The results of Sephadex G-50 showed that the increase of Cu-induced proteins significantly related with the distribution of copper. But copper-binding proteins have been not gained after further study.Protein profiles analyzed by SDS-PAGE and 2D-PAGE,Some differentially displayed protein bands and spots,represented with low molecular weight(about 10-20 kDa),were found under copper stress.Some copper-induced proteins were identified by MALDI-TOP or LCQ mass spectrometry.A metallotionein and some stress-related proteins such as PR and membrane-associated protein-like were identified by MALDI-TOP in rice germinating embryoes;PR,ascorbate peroxidase(APX) and S-adenosylmethionine synthetase were identified by MALDI-TOP in roots of E.haichowensis;CuZn-SOD,PRs and APX were identified by LCQ in leaves of E.haichowensis.The hydrogen peroxide(H₂O₂) and malondialdehyde contents and antioxidant enzyme (SOD,CAT,APX and GPOD) activities in roots of E.haichowensis were increased significantly followed with increasing Cu concentration.SOD activities and H₂O₂ contents in leaves of E.haichowensis were significantly activated followed with increasing Cu treatment days.Increases in SOD activity in roots and leaves of E.haichowensis were testified to be CuZn-SOD with isoenzyme pattern and inhibitor studies for SOD.The effects of Cu and additional treatment with inhibitors on Cu-induced H₂O₂ production in E.haichowensis were examined by using the methods of histochemistry with 3,3-diaminobenzidine(DAB) staining and cytochemistry with CeCl₃ staining and transmission electron microscopy,respectively.Experimental results showed that excess Cu greatly increased the accumulation of H₂O₂ in root cell walls and leaf apoplasts of E. haichowensis.To our knowledge this study is the first to characterize the accumulation of H₂O₂ in E.haichowensis as a consequence of Cu treatments using the cytochemical method. Experiments with N-N-diethyldithiocarbamate(DDC) as inhibitor of CuZn-SOD, diphenyleneiodonium(DPI) as inhibitor of NADPH oxidase and 1,2-dihydroxybenzene-3,5-disulphonic acid(Tiron) as O₂^- scavenger showed that these inhibitors almost inhibited all of Cu-induced H₂O² production except that DPI partly inhibited Cu-induced H₂O₂ production in leave cells.However,NaN₃ as inhibitor of peroxidase increased Cu-induced H₂O₂ production.The effects of Cu and additional treatment with additional treatment with inhibitors on Cu-induced O₂^- production in E.haichowensis were examined by using the methods of histochemistry with nitroblue tetrazolium(NBT) staining and cytochemistry with DAB staining and transmission electron microscopy,respectively.Experimental results showed that excess Cu greatly increased the accumulation of O₂^- around plasma-membrane of root cells and around chloroplast of leaf cells of E.haichowensis.Experiments with inhibitors showed that DDC greatly increased Cu-induced O₂^- production,however,Tiron,DPI and NaN₃ decreased Cu-indueed H₂O₂ production.The changes of antioxidant enzyme activity in E.haichowensis under the same conditions had shown that excess Cu significantly increased plasma-membrane NADPH oxidase,apoplastic and symplastic CuZn-SOD,apoplastic guaiacol peroxidase(GPOD) and symplastic APX activities in roots and leaves.Treatment with DDC,NAN₃,DPI and Tiron inhibited these enzyme activities differently in leaves.On the base of the results,we suggested that all Cu-induced H₂O₂ in roots and leaves of E.haichowensis almost came from CuZn-SOD dismutation.The source of O₂^·- in the root cells could mostly be NADPH oxidase,but the source of O₂^·- in the leaf cells could partly be NADPH oxidase.CuZn-SOD could dismutate the O₂^·- located in chloroplast or cytoplasm around chloroplast into H₂O₂ and,subsequently,these H₂O₂ were consumed by symplastic APX.The western blotting results of CuZn-SOD suggest that the role of CuZn-SOD in Cu-induced antioxidant defense in leaves of E.haichowensis was based on expressed CuZn-SOD protein.Taken together,our results suggested that excess Cu induced Cu-binding proteins, antioxidant enzymes or other stress-related proteins to decrease Cu damage to plant cells, and CuZn-SOD acts as the important role in Cu-induced antioxidant defense in E. haichowensis.