Molecular Definition of the Ascorbate-Glutathione Cycle in Arabidopsis Mitochondria Reveals Dual Targeting of Antioxidant Defenses in Plants
Key components of the ascorbate-glutathione cycle in Arabidopsis cell organelles are encoded by single organellar targeted isoforms that are dual localized in the chloroplast stroma and the mitochondrion. We demonstrate the presence of the ascorbate-glutathione cycle in purified Arabidopsis mitochondria using enzymatic activity, proteomic and in vitro and in vivo subcellular targeting data that identify the gene products responsible. In vitro experiments using a dual import assay assessing mitochondrial and chloroplast imports simultaneously show dual targeting of ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase gene products to mitochondria and chloroplasts, while a putative dehydroascorbate reductase protein is only imported into mitochondria. In vivo subcellular localization using green fluorescent protein fusion proteins show clear targeting of all gene products to mitochondria. Transcript levels show these genes are induced by oxidative chemical stresses targeted to chloroplasts and/or mitochondria and are elevated during photosynthetic operation in the light. Together these data present a model of an integrated ascorbate-glutathione antioxidant defense common to plastids and mitochondria that is linked at the level of the genome in Arabidopsis. Key components of the ascorbate-glutathione cycle in Arabidopsis cell organelles are encoded by single organellar targeted isoforms that are dual localized in the chloroplast stroma and the mitochondrion. We demonstrate the presence of the ascorbate-glutathione cycle in purified Arabidopsis mitochondria using enzymatic activity, proteomic and in vitro and in vivo subcellular targeting data that identify the gene products responsible. In vitro experiments using a dual import assay assessing mitochondrial and chloroplast imports simultaneously show dual targeting of ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase gene products to mitochondria and chloroplasts, while a putative dehydroascorbate reductase protein is only imported into mitochondria. In vivo subcellular localization using green fluorescent protein fusion proteins show clear targeting of all gene products to mitochondria. Transcript levels show these genes are induced by oxidative chemical stresses targeted to chloroplasts and/or mitochondria and are elevated during photosynthetic operation in the light. Together these data present a model of an integrated ascorbate-glutathione antioxidant defense common to plastids and mitochondria that is linked at the level of the genome in Arabidopsis. The ascorbate-glutathione cycle is catalyzed by a set of four enzymes, ascorbate peroxidase (APX), 1The abbreviations used are: APXascorbate peroxidaseAscascorbateDHARglutathione-dependent dehydroascorbate reductaseGRglutathione reductaseGDXNglutaredoxinMDHARmonodehydroascorbate reductaseGFPgreen fluorescent protein.1The abbreviations used are: APXascorbate peroxidaseAscascorbateDHARglutathione-dependent dehydroascorbate reductaseGRglutathione reductaseGDXNglutaredoxinMDHARmonodehydroascorbate reductaseGFPgreen fluorescent protein. monodehydroascorbate reductase (MDHAR), glutathione-dependent dehydroascorbate reductase (DHAR), and glutathione reductase (GR). This cycle operates in chloroplasts of plants in order to remove the large amounts of H2O2 generated during photosynthetic operations in the light through thylakoid electron transport chain components with electrochemical potentials capable of direct reduction of O2 (1Asada K. Takahashi M. Kyle D. Osmond C. Arntzen C. Photoinhibition. Elsevier, Amsterdam1987: 227-287Google Scholar). The highest rates of O2 reduction occur on the reducing side of photosystem I (1Asada K. Takahashi M. Kyle D. Osmond C. Arntzen C. Photoinhibition. Elsevier, Amsterdam1987: 227-287Google Scholar). The high specific activity of these enzymes in plastids and extensive work on its operation clearly indicates that this is the major H2O2 metabolizing pathway in these photosynthetic organelles (2Noctor G. Foyer C.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 249-279Crossref PubMed Scopus (4398) Google Scholar). The primary peroxidation of ascorbate (Asc) by APX yields the monodehydroascorbate radical (MDHA) that is either directly reduced back to Asc by MDHAR (3Arrigoni O. Dipierro S. Borraccino G. FEBS Lett. 1981; 125: 242-244Crossref Scopus (189) Google Scholar) or undergoes non-enzymatic disproportionation to ascorbate and dehydroascorbate (DHA). Recovery of the DHA produced occurs via the glutathione-dependent reaction catalyzed by DHAR, and the oxidized glutathione dimers are re-reduced by the NADPH-dependent GR (4Foyer C.H. Halliwell B. Planta. 1976; 133: 21-25Crossref PubMed Scopus (2008) Google Scholar). ascorbate peroxidase ascorbate glutathione-dependent dehydroascorbate reductase glutathione reductase glutaredoxin monodehydroascorbate reductase green fluorescent protein. ascorbate peroxidase ascorbate glutathione-dependent dehydroascorbate reductase glutathione reductase glutaredoxin monodehydroascorbate reductase green fluorescent protein. The chloroplast Asc-glutathione cycle is housed primarily in the stroma (see review in Ref. 5Asada K. Ann. Rev. Plant Physiol. Mol. Biol. 1999; 150: 601-639Crossref Scopus (3126) Google Scholar). A stromal APX and a thylakoid-bound APX have been identified and purified from several plant species. An extended C-terminal sequence on the thylakoid APX facilitates binding to the membrane and makes this isoform ∼5 kDa larger than the stromal APX (5Asada K. Ann. Rev. Plant Physiol. Mol. Biol. 1999; 150: 601-639Crossref Scopus (3126) Google Scholar). Sequence analysis in a variety of plants clearly delineates the two chloroplast APX classes from the cytosolic APX isoforms (6Jespersen H.M. Kjaersgard I.V. Ostergaard L. Welinder K.G. Biochem. J. 1997; 326: 305-310Crossref PubMed Scopus (143) Google Scholar). The MDHA formed in the lumen disproportionates to DHA and penetrates the thylakoid membrane into the stroma. MDHA produced by both APX isoforms is reduced by stromal MDHAR. This enzyme is not present in the lumen, and chloroplast isoforms are differentiated from cyotosolic isoforms by the presence of N-terminal targeting sequences (5Asada K. Ann. Rev. Plant Physiol. Mol. Biol. 1999; 150: 601-639Crossref Scopus (3126) Google Scholar, 7Obara K. Sumi K. Fukuda H. Plant Cell Physiol. 2002; 43: 697-705Crossref PubMed Scopus (73) Google Scholar). DHAR and GR activities convert the DHA translocated from the lumen and the DHA generated in the stroma. Detailed enzymatic investigation and establishment of the stromal location of these enzymes have been reported (5Asada K. Ann. Rev. Plant Physiol. Mol. Biol. 1999; 150: 601-639Crossref Scopus (3126) Google Scholar, 8Hossain M.A. Asada K. Plant Cell Physiol. 1984; 25: 85-92Google Scholar, 9Creissen G. Reynolds H. Xue Y. Mullineaux P. Plant J. 1995; 8: 167-175Crossref PubMed Scopus (169) Google Scholar). A series of reports have also measured the activity of some of the enzymes of the Asc-glutathione cycle in mitochondrial preparations from plants (9Creissen G. Reynolds H. Xue Y. Mullineaux P. Plant J. 1995; 8: 167-175Crossref PubMed Scopus (169) Google Scholar, 10Prasad T.K. Anderson M.D. Stewart C.R. Plant Physiol. 1995; 108: 1597-1605Crossref PubMed Scopus (79) Google Scholar, 11Jimenez A. Hernandez J.A. Ros Barcelo A. Sandalio L.M. del Rio L.A. Sevilla F. Physiol. Plant. 1998; 104: 687-692Crossref Scopus (45) Google Scholar, 12De Leonardis S. Dipierro N. Dipierro S. Plant Physiol. Biochem. 2000; 38: 773-779Crossref Scopus (67) Google Scholar, 13De Leonardis S. De Lorenzo G. Borraccino G. Dipierro S. Plant Physiol. 1995; 109: 847-851Crossref PubMed Scopus (46) Google Scholar, 14Edwards E.A. Rawsthorne S. Mullineaux P.M. Planta. 1990; 180: 278-284Crossref PubMed Scopus (260) Google Scholar). Evidence for a complete, chloroplast-like, Asc-glutathione cycle in plant mitochondria has also been In green a mitochondrial cycle has been to with photosynthetic and oxidative A. Hernandez J.A. Rio L.A. Sevilla F. Plant Physiol. 1997; PubMed Scopus Google Scholar). In the of a mitochondrial Asc-glutathione cycle is to remove to the of L.M. L. G. Plant Physiol. PubMed Scopus Google Scholar, M.A. M. Mol. Plant PubMed Scopus Google Scholar). The of mitochondrial APX activities is a location of the mitochondrial membrane L.M. L. G. Plant Physiol. PubMed Scopus Google Scholar, M.A. M. Mol. Plant PubMed Scopus Google Scholar). APX is in plant mitochondria Leonardis S. Dipierro N. Dipierro S. Plant Physiol. Biochem. 2000; 38: 773-779Crossref Scopus (67) Google Scholar, A. Hernandez J.A. Rio L.A. Sevilla F. Plant Physiol. 1997; PubMed Scopus Google Scholar, M.A. M. Mol. Plant PubMed Scopus Google Scholar). The for DHAR, and GR presence is from (9Creissen G. Reynolds H. Xue Y. Mullineaux P. Plant J. 1995; 8: 167-175Crossref PubMed Scopus (169) Google Scholar, 14Edwards E.A. Rawsthorne S. Mullineaux P.M. Planta. 1990; 180: 278-284Crossref PubMed Scopus (260) Google Scholar, A. Hernandez J.A. Rio L.A. Sevilla F. Plant Physiol. 1997; PubMed Scopus Google Scholar) and M.A. M. Mol. Plant PubMed Scopus Google Scholar). data of the DHAR, and GR to in the mitochondrial (9Creissen G. Reynolds H. Xue Y. Mullineaux P. Plant J. 1995; 8: 167-175Crossref PubMed Scopus (169) Google Scholar, 14Edwards E.A. Rawsthorne S. Mullineaux P.M. Planta. 1990; 180: 278-284Crossref PubMed Scopus (260) Google Scholar, A. Hernandez J.A. Rio L.A. Sevilla F. Plant Physiol. 1997; PubMed Scopus Google Scholar). the proteins and the genes that are for these activities in plants have not been a has been to these data mitochondrial enzymes and to by the presence of or cytosolic In glutathione and are to in the direct of H2O2 2002; PubMed Scopus Google Scholar). A mitochondria peroxidase is also to for H2O2 in A. G. 2000; PubMed Scopus Google Scholar). In both an localized peroxidase and a peroxidase have been in H2O2 A. N. J.A. G. J. Biol. 2000; PubMed Scopus Google Scholar). We have identified a putative peroxidase in Arabidopsis its in H2O2 is Plant J. 2002; PubMed Scopus Google Scholar). an Asc-glutathione cycle in plant mitochondria two a set of genes components of this pathway responsible. of isoforms have been in Leonardis S. Dipierro N. Dipierro S. Plant Physiol. Biochem. 2000; 38: 773-779Crossref Scopus (67) Google Scholar, A. Hernandez J.A. Rio L.A. Sevilla F. Plant Physiol. 1997; PubMed Scopus Google Scholar, L.M. L. G. Plant Physiol. PubMed Scopus Google Scholar, M.A. M. Mol. Plant PubMed Scopus Google Scholar). a series of genes that are to chloroplasts and mitochondria. Evidence has been for the dual targeting of GR to both mitochondria and chloroplasts (9Creissen G. Reynolds H. Xue Y. Mullineaux P. Plant J. 1995; 8: 167-175Crossref PubMed Scopus (169) Google and the products of from a single Arabidopsis MDHAR gene have been to targeted to mitochondria and chloroplasts K. Sumi K. Fukuda H. Plant Cell Physiol. 2002; 43: 697-705Crossref PubMed Scopus (73) Google Scholar). In this have the activity of the Asc-glutathione cycle enzymes in purified mitochondria from the model plant Arabidopsis. A of a of the Arabidopsis genes Asc-glutathione cycle enzymes, analysis of proteomic data in purified mitochondrial and in vitro import experiments dual targeting of and GR gene products to mitochondria and chloroplasts, only mitochondrial targeting of specific DHAR In vivo experiments using proteins also mitochondrial subcellular Transcript levels for these genes induced by oxidative stresses on chloroplasts and/or mitochondria and elevated during photosynthetic operation in the light. Together these data used to present a model of an integrated Asc-glutathione antioxidant defense common to chloroplasts and mitochondria that is linked at the level of the genome in Arabidopsis. Plant and plants in an at The with of set to a light and Arabidopsis at a light and on and of from Arabidopsis plants and using J. Plant Physiol. PubMed Scopus Google Scholar). The used to the used for of Plant and from and chloroplasts from M. J. Plant Physiol. Google Scholar, S. J. K. Plant The Google Scholar, K. J. in Cell Scholar). Arabidopsis mitochondria purified from cell to P. Plant Physiol. PubMed Scopus Google Scholar). of mitochondria to and membrane to B. FEBS Lett. PubMed Scopus Google Scholar). The mitochondrial protein using the and the chloroplast by of Asc-glutathione measured Asc by H2O2 at in the presence of Asc and H2O2 A. Hernandez J.A. Rio L.A. Sevilla F. Plant Physiol. 1997; PubMed Scopus Google Scholar). GR measured at in the presence of and oxidized glutathione E.A. Rawsthorne S. Mullineaux P.M. Planta. 1990; 180: 278-284Crossref PubMed Scopus (260) Google Scholar). MDHAR measured at of MDHA generated using the using and of Asc DHAR measured Asc at in reduced glutathione and DHA S. A. PubMed Google Scholar). in a of and with or of both and some of these enzymes, and to electron transport chain with of enzymes in the of in from mitochondria in a of and and in of proteins for J. L. Plant Physiol. PubMed Scopus Google of S. Biochem. J. PubMed Scopus Google Arabidopsis J. Plant Physiol. PubMed Scopus Google DHAR, and using the in vitro in the presence of using the and by experiments using the dual import C. O. J. Plant J. 2002; PubMed Scopus Google Scholar). import into by the mitochondria in a B. J. Plant J. 2002; PubMed Scopus Google Scholar). products by and to a of by on an using an to from the at in a and at the the proteins with in and by and by B. FEBS Lett. PubMed Scopus Google Scholar). and data with and to identify Arabidopsis protein of Arabidopsis targeting of protein sequences with by and by on this of to Arabidopsis plants with of four to of and to and A. for Arabidopsis using a and and on using the Plant of in the Arabidopsis and the by using a and and The into a The sequence to the and with by using and The sequence from the by products and of the gene and by from in at light at The cell in an by at with and for at in the to of the of using and of using of using of The at in the of using Asc-glutathione in Arabidopsis and of DHAR, and MDHAR activities that in Arabidopsis mitochondria The for the high of mitochondria from and yields mitochondria only by and from and cytosolic on enzymes P. Plant Physiol. PubMed Scopus Google Scholar). activities for enzyme of the Asc-glutathione cycle measured in the presence of than reported in mitochondrial preparations from plant A. Hernandez J.A. Rio L.A. Sevilla F. Plant Physiol. 1997; PubMed Scopus Google Scholar, M.A. M. Mol. Plant PubMed Scopus Google clearly levels of The of these activities that GR and MDHAR activities in a In than the APX and DHAR activities by of to a of activity is on the side of the mitochondrial membrane the in of a high of membrane in mitochondrial of these in proteins and membrane proteins and membrane proteins by and by this are for the and and and in the B. FEBS Lett. PubMed Scopus Google Scholar, B. J. Plant J. 2002; PubMed Scopus Google Scholar, A. Cell Biol. PubMed Google Scholar). The the and the membrane of mitochondrial protein. on these the of activity the is a of activity in mitochondria the activities with the data the activity during this to of the APX and MDHAR activities present in the membrane with in the The of APX to the data for this enzyme that of this activity is to on the side of the In the high of MDHAR to data a side The GR and DHAR activities in the are also in the are and membrane data are with the that the of the APX the of the while an Asc-glutathione DHAR, operates in the and to a in the to enzyme activities in Arabidopsis in a for of the in Arabidopsis genes encoded the proteins for these enzymatic activities the protein set from of The for genome by to identify genes the enzymes of the Asc-glutathione cycle protein sequences used in analysis of the protein data set to identify protein sequences with to also of these gene This analysis are APX MDHAR DHAR and GR genes in Arabidopsis. is an extensive set of that are to DHAR activities in Arabidopsis data not in DHAR activities purified from are to J. Biol. 1990; PubMed Google Scholar, B. L. A. M. Biochem. J. PubMed Scopus Google the mitochondrial has DHAR activity M. C. J. M. G. J. M. A. J. Biol. PubMed Scopus Google Scholar) and DHAR activity of plant is reported J. C. Planta. 1997; PubMed Scopus Google Scholar). of these the APX in at the of this are all genes cycle enzymes in Arabidopsis in a of Asc-glutathione of of gene a that classes of protein are and that some have or C-terminal that targeting for localization the cell of the targeting of gene for and GR by targeting is in on the by have a for organellar or this analysis that only of the genes proteins that are to In the of and the DHAR only gene of has a clearly The organellar have been to chloroplast in the lumen thylakoid membrane and the stroma (6Jespersen H.M. Kjaersgard I.V. Ostergaard L. Welinder K.G. Biochem. J. 1997; 326: 305-310Crossref PubMed Scopus (143) Google Scholar). The organellar GR and MDHAR are also the chloroplast isoforms in Arabidopsis K. Sumi K. Fukuda H. Plant Cell Physiol. 2002; 43: 697-705Crossref PubMed Scopus (73) Google Scholar, A. H. K. N. K. Plant Cell Physiol. Scholar, P. K. H. L. J. N. S. M. Physiol. PubMed Scopus Google Scholar). A with mitochondrial targeting is also in Evidence for of in have also been a of Arabidopsis mitochondrial analysis Plant J. 2002; PubMed Scopus Google Scholar, A. Cell Biol. PubMed Google Scholar) using and direct analysis of mitochondrial this analysis have been identified for several Asc-glutathione cycle enzymes that are present in purified mitochondria from Arabidopsis cell A set of to APX a series of to the MDHAR from the GR has been from the DHAR and a set of to a of these by the Arabidopsis protein set that these with of than for of and of for of the to of the with the of DHAR the proteins by proteomic analysis are all to of Asc-glutathione cycle enzymes in Arabidopsis in a of and GR to and the and proteomic analysis that the for proteins APX MDHAR and GR The with DHAR not proteomic data to either or the while targeting to this all proteins used for import the only DHAR also for that than of proteins with organelles a dual import in C. O. J. Plant J. 2002; PubMed Scopus Google Scholar) for import of in vitro of the proteins and two proteins the import of APX MDHAR and GR using this that all imported and to a in both chloroplasts and mitochondria. The products from by of In the of APX an from the that in an In order to protein dual targeted the and of the in a single protein that dual targeted In to an in targeting only to mitochondria (see import of and GR proteins not and This that a of these proteins not the an is with the reports of of these enzyme activities the and for MDHAR and and the of APX activity that an of the membrane imports into mitochondria and the membrane and the of the of imported protein that to in the and used to the activity of is an membrane protein that is not to the P. 2000; PubMed Scopus Google and the membrane is is an membrane protein with the and in the of the membrane and the are the membrane are J. Plant Physiol. PubMed Scopus Google Scholar). This in a in on and that the is in of the membrane these show that while is is to a and of mitochondria imported APX protein that in this We have reported that this also from the membrane B. FEBS Lett. PubMed Scopus Google Scholar, B. J. Plant J. 2002; PubMed Scopus Google Scholar). MDHAR and GR proteins to in a direct of these products the and the during Arabidopsis proteins also imported into mitochondria from Arabidopsis cell In the of import into Arabidopsis mitochondria been in mitochondria The DHAR and not to a with either and to all the in the presence and of of these proteins not to import not The DHAR to the organellar targeted in Arabidopsis not to imported into either chloroplasts or mitochondria in vitro not The clearly imported and in mitochondria in a not imported into chloroplasts have also in vivo subcellular localization experiments using and to green fluorescent protein by into plant cell and used green plant yields high chloroplast and proteins to to mitochondria. We used the to a mitochondrial and Arabidopsis to a The produced the for than in The produced a of and of ∼5 in of the protein for and to all produced that mitochondrial only GR a that also the used a mitochondrial location for all four proteins using a for in vivo subcellular Transcript and of in and that all Asc-glutathione cycle genes in both In for the in while for the mitochondrial data that this set of gene products are to for photosynthetic in are in both and with a dual location in chloroplasts and mitochondria. of gene during oxidative A variety of and photosynthetic operation are to of the cytosolic and chloroplast enzymes of the Asc-glutathione cycle in plants (5Asada K. Ann. Rev. Plant Physiol. Mol. Biol. 1999; 150: 601-639Crossref Scopus (3126) Google Scholar, S. C. B. G. Mullineaux P.M. Plant 1997; PubMed Scopus Google Scholar, S. M. Y. Y. K. J. 2002; PubMed Google Scholar, L. K. S. Mullineaux P.M. Plant J. PubMed Scopus Google Scholar). in light and a in the light in of the only not to is the only that not to Transcript induced by specific chemical stresses in and are both capable of chloroplast oxidative by and by an electron from photosystem to and peroxidation in of the cell a in A is a specific of in the mitochondrial membrane and from a reduced in vitro and in The induced by that both and mitochondrial targeted stresses the GR and APX not by chemical and a while A and in a some and to levels by MDHAR and GR with while at DHAR with This of and at levels of on this antioxidant its dual to stresses from the two organelles a of with of this gene The for of the have used a variety of to identify the proteins in the Asc-glutathione cycle in Arabidopsis mitochondria. The set of proteins has dual activities in the and show that of these proteins are to chloroplasts and not a specific mitochondrial cycle from a of the mitochondrial antioxidant defense cycle is linked to the chloroplast antioxidant defense cycle at the level of the In all of dual targeted proteins mitochondria and chloroplasts, the chloroplast is the stroma N. PubMed Scopus Google Scholar). the organellar targeted in the stromal APX that to dual targeted to mitochondria and chloroplasts and of APX in and also have chloroplast stroma Asada K. Plant Cell Physiol. Scopus Google Scholar, Y. S. H. Physiol. Plant. PubMed Scopus Google Scholar, K. K. M. S. Biochem. J. 1997; PubMed Scopus Google Scholar). A stromal location for GR and MDHAR the for these is also in chloroplasts (2Noctor G. Foyer C.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 249-279Crossref PubMed Scopus (4398) Google Scholar, 5Asada K. Ann. Rev. Plant Physiol. Mol. Biol. 1999; 150: 601-639Crossref Scopus (3126) Google Scholar). is the chloroplast stromal antioxidant pathway that to to mitochondria in Arabidopsis. The in vivo subcellular localization only a mitochondrial localization for APX and MDHAR from reports in the and the in vitro import a localization is also This to a of the in vivo A of a from that is both in mitochondria and plastids that in mitochondria or plastids not both in the cell S. P. L. S. L.A. Plant J. 2002; PubMed Scopus Google Scholar). is from this the mitochondrial localization for APX and MDHAR in a is that the import pathway for the import of these proteins is not in the import have been for proteins S. A. P. K. J. D. P. Plant PubMed Scopus Google and is also C. K. Plant PubMed Google Scholar). these targeting not to the import of into proteins have been reported to protein import into mitochondria and plastids O. J. Plant Biol. PubMed Scopus Google Scholar). import has been that in the protein binding or E.A. J. J. Biochem. 2002; PubMed Scopus Google Scholar). the of the to these proteins this for import not for mitochondrial A on Arabidopsis MDHAR also reported dual targeting by of K. Sumi K. Fukuda H. Plant Cell Physiol. 2002; 43: 697-705Crossref PubMed Scopus (73) Google Scholar). the data in this is on for the and the not demonstrate the presence of two in from Arabidopsis data on the gene show that MDHAR proteins produced from both the and the at imported into both mitochondria and plastids and of the in and of this antioxidant cycle in plant mitochondria is mitochondria to directly with generated by electron transport through the of the in the mitochondrial to with directly at the of its and its reaction the of The of the from the of and has a in than The for the or chloroplasts to of plant mitochondrial H2O2 is In and mitochondria are clearly the major of and makes to with these at the of In and peroxidase, peroxidase, and glutathione peroxidase the 2002; PubMed Scopus Google Scholar, A. G. 2000; PubMed Scopus Google Scholar, A. N. J.A. G. J. Biol. 2000; PubMed Scopus Google C. H. B. Mol. 1999; PubMed Scopus Google Scholar). In chloroplasts and in of both and rates (2Noctor G. Foyer C.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 249-279Crossref PubMed Scopus (4398) Google Scholar). The that plant mitochondria H2O2 has been in of the to components by mitochondrial H2O2 in L.M. L. G. Plant Physiol. PubMed Scopus Google Scholar, M.A. M. Mol. Plant PubMed Scopus Google direct to mitochondrial enzymes by H2O2 Plant J. 2002; PubMed Scopus Google of J. Biol. 2002; PubMed Scopus Google Scholar) and the of mitochondria and chloroplasts for H2O2 antioxidant in to stresses Hernandez J.A. A. del Rio L.A. Sevilla F. 1999; PubMed Scopus Google Scholar). The of H2O2 and H2O2 that mitochondria to with in vivo in mitochondria is in a mitochondrial Asc-glutathione antioxidant defense H2O2 from generated by organelles defense from The cycle present in Arabidopsis has in and mitochondria A. Hernandez J.A. Rio L.A. Sevilla F. Plant Physiol. 1997; PubMed Scopus Google Scholar, M.A. M. Mol. Plant PubMed Scopus Google Scholar). APX is in the in plant is to the location of peroxidase in A. N. J.A. G. J. Biol. 2000; PubMed Scopus Google Scholar, C. H. B. Mol. 1999; PubMed Scopus Google Scholar). In plants this localization makes of the of Asc in the by that is also to the membrane Foyer C.H. Plant Physiol. 2000; PubMed Scopus Google while the reduced from the electron transport chain on the side of the the MDHA and DHA that from APX in plant mitochondria to the for reduction back to The location of DHAR, and GR are for the of in the of and to these from the cycle and/or for this are not in plant mitochondria to a of MDHA and DHA transport have been in plant (2Noctor G. Foyer C.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 249-279Crossref PubMed Scopus (4398) Google Scholar, 5Asada K. Ann. Rev. Plant Physiol. Mol. Biol. 1999; 150: 601-639Crossref Scopus (3126) Google Scholar). the of both DHAR and MDHAR not high in both and in and mitochondria A. Hernandez J.A. Rio L.A. Sevilla F. Plant Physiol. 1997; PubMed Scopus Google Scholar, M.A. M. Mol. Plant PubMed Scopus Google an for mitochondrial membrane transport of these and in and the for proteins have been reported to dual targeted using an targeting a mitochondrial and location N. PubMed Scopus Google Scholar, O. J. Plant Biol. PubMed Scopus Google and several genes dual proteins by of or an specific targeting on the protein Y. Y. M. Biochem. PubMed Scopus Google Scholar, N. M. S. S. A. J. Biol. PubMed Scopus Google Scholar, J. Cell PubMed Scopus Google Scholar). of these proteins in or or The into and I F. H. M. S. Plant J. PubMed Scopus Google Scholar, A. P.M. Plant Physiol. PubMed Scopus Google protein C. A. M. B. J. 2000; PubMed Scopus Google and activities the protein GR from (9Creissen G. Reynolds H. Xue Y. Mullineaux P. Plant J. 1995; 8: 167-175Crossref PubMed Scopus (169) Google Scholar) and of Asc-glutathione cycle This is to with the of genes in on in to A. A. Plant PubMed Scopus Google Scholar). these are common in mitochondria and that linked by a single gene in the the and of mitochondria and plastids the for to J. PubMed Scopus Google Scholar, A. J. J. PubMed Scopus Google Scholar). to occur via a at the level of protein import or We J. PubMed Scopus Google Scholar) and Annu. Rev. Plant Physiol. Plant Mol. Biol. PubMed Scopus Google Scholar, C. M. G. C. P. Foyer C.H. Plant PubMed Scopus Google Scholar) have the for to antioxidant defense in organelles in that at in the of the mitochondrial and the chloroplast Asc-glutathione this occurs by dual targeting than in experiments to both mitochondria and stresses This makes mitochondrial H2O2 defense is chloroplast H2O2 than mitochondrial or at that mitochondrial are a primary for a mitochondrial Asc-glutathione cycle in We of for with