Ludmila Rizhsky

Within their natural habitat, plants are subjected to a combination of abiotic conditions that include stresses such as drought and heat. Drought and heat stress have been extensively studied; however, little is known about how their combination impacts plants. The response of Arabidopsis plants to a combination of drought and heat stress was found to be distinct from that of plants subjected to drought or heat stress. Transcriptome analysis of Arabidopsis plants subjected to a combination of drought and heat stress revealed a new pattern of defense response in plants that includes a partial combination of two multigene defense pathways (i.e. drought and heat stress), as well as 454 transcripts that are specifically expressed in plants during a combination of drought and heat stress. Metabolic profiling of plants subjected to drought, heat stress, or a combination of drought and heat stress revealed that plants subject to a combination of drought and heat stress accumulated sucrose and other sugars such as maltose and glucose. In contrast, Pro that accumulated in plants subjected to drought did not accumulate in plants during a combination of drought and heat stress. Heat stress was found to ameliorate the toxicity of Pro to cells, suggesting that during a combination of drought and heat stress sucrose replaces Pro in plants as the major osmoprotectant. Our results highlight the plasticity of the plant genome and demonstrate its ability to respond to complex environmental conditions that occur in the field.

In nature, plants encounter a combination of environmental conditions that may include stresses such as drought or heat shock. Although drought and heat shock have been extensively studied, little is known about how their combination affect plants. We used cDNA arrays, coupled with physiological measurements, to study the effect of drought and heat shock on tobacco (Nicotiana tabacum) plants. A combination of drought and heat shock resulted in the closure of stomata, suppression of photosynthesis, enhancement of respiration, and increased leaf temperature. Some transcripts induced during drought, e.g. those encoding dehydrin, catalase, and glycolate oxidase, and some transcripts induced during heat shock, e.g. thioredoxin peroxidase, and ascorbate peroxidase, were suppressed during a combination of drought and heat shock. In contrast, the expression of other transcripts, including alternative oxidase, glutathione peroxidase, phenylalanine ammonia lyase, pathogenesis-related proteins, a WRKY transcription factor, and an ethylene response transcriptional co-activator, was specifically induced during a combination of drought and heat shock. Photosynthetic genes were suppressed, whereas transcripts encoding some glycolysis and pentose phosphate pathway enzymes were induced, suggesting the utilization of sugars through these pathways during stress. Our results demonstrate that the response of plants to a combination of drought and heat shock, similar to the conditions in many natural environments, is different from the response of plants to each of these stresses applied individually, as typically tested in the laboratory. This response was also different from the response of plants to other stresses such as cold, salt, or pathogen attack. Therefore, improving stress tolerance of plants and crops may require a reevaluation, taking into account the effect of multiple stresses on plant metabolism and defense.

Reactive oxygen species (ROS), such as O2- and H2O2, play a key role in plant metabolism, cellular signaling, and defense. In leaf cells, the chloroplast is considered to be a focal point of ROS metabolism. It is a major producer of O2- and H2O2 during photosynthesis, and it contains a large array of ROS-scavenging mechanisms that have been extensively studied. By contrast, the function of the cytosolic ROS-scavenging mechanisms of leaf cells is largely unknown. In this study, we demonstrate that in the absence of the cytosolic H2O2-scavenging enzyme ascorbate peroxidase 1 (APX1), the entire chloroplastic H2O2-scavenging system of Arabidopsis thaliana collapses, H2O2 levels increase, and protein oxidation occurs. We further identify specific proteins oxidized in APX1-deficient plants and characterize the signaling events that ensue in knockout-Apx1 plants in response to a moderate level of light stress. Using a dominant-negative approach, we demonstrate that heat shock transcription factors play a central role in the early sensing of H2O2 stress in plants. Using knockout plants for the NADPH oxidase D protein (knockout-RbohD), we demonstrate that RbohD might be required for ROS signal amplification during light stress. Our study points to a key role for the cytosol in protecting the chloroplast during light stress and provides evidence for cross-compartment protection of thylakoid and stromal/mitochondrial APXs by cytosolic APX1.

Cytosolic ascorbate peroxidase 1 (Apx1) is a key H2O2 removal enzyme in plants. Microarray analysis of Apx1-deficient Arabidopsis plants revealed that the expression of two zinc finger proteins (Zat12 and Zat7) and a WRKY transcription factor (WRKY25) is elevated in knock-out Apx1 plants grown under controlled conditions. Because mutants lacking Apx1 accumulate H2O2, we examined the correlation between H2O2 and the expression of Zat12, Zat7, WRKY25, and Apx1. The expression of Zat12, Zat7, and WRKY25 was simultaneously elevated in cells in response to oxidative stress (i.e. H2O2 or paraquat application), heat shock, or wounding. In contrast, light or osmotic stress did not enhance the expression of these putative transcription factors. All stresses tested enhanced the expression of Apx1. Transgenic plants expressing Zat12 or Zat7 could tolerate oxidative stress. In contrast, transgenic plants expressing WRKY25 could not. Although the expression of Zat12, Zat7, or WRKY25 in transgenic plants did not enhance the expression of Apx1 under controlled conditions, Zat12-deficient plants were unable to enhance the expression of Apx1, Zat7, or WRKY25 in response to H2O2 or paraquat application. Zat12-deficient plants were also more sensitive than wild type plants to H2O2 application as revealed by a higher level of H2O2-induced protein oxidation detected in these plants by protein blots. Our results suggest that Zat12 is an important component of the oxidative stress response signal transduction network of Arabidopsis required for Zat7, WRKY25, and Apx1 expression during oxidative stress. Cytosolic ascorbate peroxidase 1 (Apx1) is a key H2O2 removal enzyme in plants. Microarray analysis of Apx1-deficient Arabidopsis plants revealed that the expression of two zinc finger proteins (Zat12 and Zat7) and a WRKY transcription factor (WRKY25) is elevated in knock-out Apx1 plants grown under controlled conditions. Because mutants lacking Apx1 accumulate H2O2, we examined the correlation between H2O2 and the expression of Zat12, Zat7, WRKY25, and Apx1. The expression of Zat12, Zat7, and WRKY25 was simultaneously elevated in cells in response to oxidative stress (i.e. H2O2 or paraquat application), heat shock, or wounding. In contrast, light or osmotic stress did not enhance the expression of these putative transcription factors. All stresses tested enhanced the expression of Apx1. Transgenic plants expressing Zat12 or Zat7 could tolerate oxidative stress. In contrast, transgenic plants expressing WRKY25 could not. Although the expression of Zat12, Zat7, or WRKY25 in transgenic plants did not enhance the expression of Apx1 under controlled conditions, Zat12-deficient plants were unable to enhance the expression of Apx1, Zat7, or WRKY25 in response to H2O2 or paraquat application. Zat12-deficient plants were also more sensitive than wild type plants to H2O2 application as revealed by a higher level of H2O2-induced protein oxidation detected in these plants by protein blots. Our results suggest that Zat12 is an important component of the oxidative stress response signal transduction network of Arabidopsis required for Zat7, WRKY25, and Apx1 expression during oxidative stress. Plants are sessile organisms that evolved a complex and specialized network of regulatory genes to control their response to changes in environmental conditions. It is likely that many of these regulatory genes were initially created by gene duplication and that they later acquired roles specifically related to individual pathways or stresses as well as their combination (1Chen W. Provart N.J. Glazebrook J. Katagiri F. Chang H.S. Eulgem T. Mauch F. Luan S. Zou G. Whitham S.A. Budworth P.R. Tao Y. Xie Z. Chen X. Lam S. Kreps J.A. Harper J.F. Si-Ammour A. Mauch-Mani B. Heinlein M. 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Cell Stress Chaperones. 2001; 6: 177-189Crossref PubMed Google Scholar), were found to control and regulate diverse processes in plants ranging from development to response to biotic or abiotic stresses (1Chen W. Provart N.J. Glazebrook J. Katagiri F. Chang H.S. Eulgem T. Mauch F. Luan S. Zou G. Whitham S.A. Budworth P.R. Tao Y. Xie Z. Chen X. Lam S. Kreps J.A. Harper J.F. Si-Ammour A. Mauch-Mani B. Heinlein M. Kobayashi K. Hohn T. Dangl J.L. Wang X. Zhu T. Plant Cell. 2002; 14: 559-574Crossref PubMed Scopus (797) Google Scholar, 2The Arabidopsis Genome Initiative Nature. 2000; 408: 796-815Crossref PubMed Scopus (7166) Google Scholar, 3Eulgem T. Rushton P.J. Robatzek S. Somssich I.E. Trends Plant Sci. 2000; 5: 199-206Abstract Full Text Full Text PDF PubMed Scopus (2120) Google Scholar, 4Stracke R. Werber M. Weisshaar B. Curr. Opin. Plant Biol. 2001; 4: 447-456Crossref PubMed Scopus (1501) Google Scholar, 5Nover L. Bharti K. Doring P. Mishra S.K. Ganguli A. Scharf K.D. Cell Stress Chaperones. 2001; 6: 177-189Crossref PubMed Google Scholar). The different regulatory networks of plants are also involved in modulating the production and scavenging of reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; Apx, ascorbate peroxidase; CaMV, cauliflower mosaic virus; KO, knock-out; MAPK, mitogen-activated kinase; Rubisco, ribulose-bisphosphate carboxylase/oxygenase. in cells. These toxic intermediates of oxygen reduction not only control different plant responses to environmental and developmental cues but also potently inhibit essential metabolic pathways and may lead to cell death (6Epple P. Mack A.A. Morris V.R. Dangl J.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6831-6836Crossref PubMed Scopus (134) Google Scholar, 7Foreman J. Demidchik V. Bothwell J.H. Mylona P. Miedema H. Torres M.A. Linstead P. Costa S. Brownlee C. Jones J.D. Davies J.M. Dolan L. 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The steady-state level of a number of different transcripts encoding transcription factors such as MYB, WRKY, heat shock transcription factors, and different zinc finger proteins is elevated in plants in response to different forms of ROS-induced stress (11Vranova E. Atichartpongkul S. Villarroel R. Van Montagu M. Inze D. Van Camp W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10870-10875Crossref PubMed Scopus (134) Google Scholar, 12Desikan R. Mackerness A.H. Hancock J.T. Neill S.J. Plant Physiol. 2001; 127: 159-172Crossref PubMed Scopus (716) Google Scholar, 13Pnueli L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar, 14Rizhsky L. Liang H. Mittler R. J. Biol. Chem. 2003; 278: 38921-38925Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). However, genetic evidence supporting a direct regulatory role for these transcripts was only presented for two zinc finger proteins, Lsd1 and Lol1, which were recently found to mediate ROS signals and control programmed cell death in Arabidopsis (6Epple P. Mack A.A. Morris V.R. Dangl J.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6831-6836Crossref PubMed Scopus (134) Google Scholar), and for heat shock transcription factor 3, which was shown to enhance cytosolic ascorbate peroxidase (Apx) expression in the absence of stress (15Panchuk I.I. Volkov R.A. Schoffl F. Plant Physiol. 2002; 129: 838-853Crossref PubMed Scopus (362) Google Scholar) We are studying the response of plants to of ROS in cells (i.e. oxidative Ref. 9Mittler R. Trends Plant Sci. 2002; 9: 405-410Abstract Full Text Full Text PDF Scopus (7856) Google Scholar). Our is to and the transcription factor network that the response of plants to oxidative stress. and the ROS signal transduction network of we are knock-out plants in key enzymes L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar, 14Rizhsky L. Liang H. Mittler R. J. Biol. Chem. 2003; 278: 38921-38925Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). These plants an to plant responses to ROS they accumulate ROS and in the absence of such as ROS, or ROS the ROS that accumulate in these mutants are ROS in cells the different and not ROS that may or abiotic stress response pathways L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar, 14Rizhsky L. Liang H. Mittler R. J. Biol. Chem. 2003; 278: 38921-38925Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). plants in cytosolic Apx1 are of a steady-state level of H2O2 in cells and ROS grown under controlled L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar). These plants are also in their and responses and an of heat shock proteins and in response to light stress. Microarray analysis of knock-out Apx1 plants grown under controlled revealed that the expression of two different zinc finger proteins and a putative WRKY transcription factor and a number of heat shock transcription factors is elevated in these plants L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar). The expression of Zat12 is also elevated in Arabidopsis cells in response to H2O2 application R. Mackerness A.H. Hancock J.T. Neill S.J. Plant Physiol. 2001; 127: 159-172Crossref PubMed Scopus (716) Google Scholar) and in Arabidopsis plants in response to or light stress Chang H.S. R. Wang X. Zhu T. Luan S. Plant Physiol. 2002; 129: PubMed Scopus Google Scholar, S. Plant Cell. 2002; 14: PubMed Scopus Google Scholar, A. T. S. H. K. Plant J. 2000; PubMed Google stresses that in ROS in direct was between Zat12 expression and the expression of different transcripts such as encoding Apx1. in to the between Apx1 expression and heat shock transcription factors L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar, I.I. Volkov R.A. Schoffl F. Plant Physiol. 2002; 129: 838-853Crossref PubMed Scopus (362) Google Scholar). The elevated expression of Zat12, Zat7, and WRKY25 in Apx1-deficient plants a between H2O2 the expression of these putative regulatory and Apx1 In we examined the between Zat12, Zat7, and WRKY25 oxidative and Apx1 Our results suggest that Zat12 is essential for Zat7, WRKY25, and Apx1 expression during oxidative stress and that Zat12, Zat7, and WRKY25 are to H2O2 stress in plants. Plant and and plants were grown in under controlled or light and a of Arabidopsis a in the Zat12 gene the were and to for and to as L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar, 14Rizhsky L. Liang H. Mittler R. J. Biol. Chem. 2003; 278: 38921-38925Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). of Zat12 knock-out and was by and L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar). for expression of Zat12 by was from and control wild type and knock-out plants 1 wounding. of plants was as L. Mittler R. Plant Biol. 2001; PubMed Scopus Google Scholar). of Arabidopsis plants was as L. Liang H. Mittler R. J. Biol. Chem. 2003; 278: 38921-38925Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, S.J. R.A. Plant The Scholar), and transgenic plants were by L. Liang H. Mittler R. J. Biol. Chem. 2003; 278: 38921-38925Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). All were in and the of which were as and and protein were and by and protein as L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar, 14Rizhsky L. Liang H. Mittler R. J. Biol. Chem. 2003; 278: 38921-38925Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). of protein oxidation was the protein oxidation as by the The of the protein in plants was by an as R. X. M. Plant Cell. PubMed Scopus Google Scholar). or a were used to control for of protein was used to control for protein Zat12 Zat7 and WRKY25 by was for WRKY25 were from and for Zat7 and Zat12 were by transcription from cells 1 L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar, L. Mittler R. Plant Biol. 2001; PubMed Scopus Google Scholar). were and of Zat12 and were by to R. B. Plant J. 5: PubMed Scopus Google Scholar) the Zat12 and Zat12 Zat7 and Zat7 and WRKY25 and WRKY25 two was from wild or plants of plants in grown under controlled as was used to the of for and analysis are L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar, 14Rizhsky L. Liang H. Mittler R. J. Biol. Chem. 2003; 278: 38921-38925Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). analysis of was the and the of the results were by blots. Stress the analysis of oxidative stress of transgenic plants expressing Zat12, Zat7, and WRKY25, of wild type and transgenic were and in and different of paraquat of a was two wild type and of transgenic plants. the different were by the were in a and and the of and were and shock and light stress were as L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar). stress and H2O2 and paraquat stresses were by grown in and and to paraquat or H2O2 for and of Zat12, Zat7, and the correlation between Zat12, Zat7, WRKY25, and Apx1 expression in response to oxidative stress or different abiotic we wild type plants to H2O2 heat shock, a level of light stress paraquat and osmotic stress. shown in the steady-state level of transcripts encoding Zat12, Zat7, and WRKY25 was elevated in cells in response to H2O2, heat shock, or paraquat application. In contrast, a level of light stress or osmotic stress did not enhance the expression of these The level of transcripts encoding Apx1 was elevated by specifically that oxidative stress or and heat shock a correlation between Zat12, Zat7, WRKY25, and Apx1 of the of Zat12, Zat7, and WRKY25, the revealed that a number of putative are genes and Apx1 These a number of light response shown in and response a response a and the heat shock transcription factor shock or In two an and were in the of Zat12, Zat7, WRKY25, and Apx1 the Because these were found in the of is that they are involved in the expression of these genes during abiotic stress or oxidative stress In the expression of Zat12, Zat7, WRKY25, and Apx1 during or heat shock could by the of the response or heat shock in the of these the expression of Zat12 was not elevated in response to a level of light stress a the expression of Zat12 during light stress Ref. A. T. S. H. K. Plant J. 2000; PubMed Google Scholar) and the of many different light response in the of Zat12 response in the of Zat7 and WRKY25 were also found to to the light stress regulatory found in the of Zat12, Zat7, WRKY25, and and regulatory of regulatory to shock in a analysis of Zat12, Zat7, WRKY25, and Apx1 expression during H2O2 stress revealed that the steady-state level of transcripts encoding Zat12, Zat7, and WRKY25 is elevated in plants to the in Apx1 In the expression of Zat12, Zat7, and WRKY25 was and of H2O2 application. The expression of Zat12 was found to to an level the application of H2O2 These are in the expression of Zat12 during and stress Chang H.S. R. Wang X. Zhu T. Luan S. Plant Physiol. 2002; 129: PubMed Scopus Google Scholar, S. Plant Cell. 2002; 14: PubMed Scopus Google Scholar, D. R. Plant Cell. 2002; 14: PubMed Scopus Google Scholar) and the expression of Zat7 during Chang H.S. R. Wang X. Zhu T. Luan S. Plant Physiol. 2002; 129: PubMed Scopus Google Scholar). of Zat12, Zat7, and WRKY25 in Transgenic the of Zat12, Zat7, and WRKY25 in we for these putative transcription factors in transgenic plants. we used the We tested the of from transgenic plants that Zat12, Zat7, or WRKY25 to oxidative stress a that and of of in the or absence of the paraquat L. Liang H. Mittler R. J. Biol. Chem. 2003; 278: 38921-38925Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). shown in 3, of transgenic plants expressing Zat12 or Zat7 were more than of wild type plants to the oxidative stress in In contrast, of transgenic plants expressing WRKY25 were not more than of wild type plants to These results were two transgenic for of the different putative transcription factors. we tested the expression of Zat12, Zat7, WRKY25, and Apx1 in transgenic grown under controlled conditions. shown in expression of Zat12, Zat7, or WRKY25 in the different transgenic plants did not in the elevated expression of Apx1 or of the putative transcription factors not controlled by the under the controlled we used to the different putative transcription factors in of the different were unable to the expression of other or the expression of Apx1. level of expression of Zat7, in the in a and development However, level of expression did not the expression of Zat12 or WRKY25 and the expression level of Apx1 was not in transgenic plants expressing Zat12 or WRKY25 Microarray of Transgenic Plants Zat12 expression enhanced the of plants to oxidative stress but did not in the enhanced expression of Zat7, WRKY25, or Apx1 we examined transgenic plants expressing Zat12 by to transcripts that may involved in the response of plants to ROS in plants in the absence of an these we used of plants grown under controlled and the of expression between transgenic plants expressing Zat12, wild type and knock-out plants in Apx1 shown in different transcripts that were elevated in transgenic plants expressing Zat12 were also elevated in plants transcripts elevated in plants as well as in plants are in It is that the expression of these transcripts in Apx1-deficient plants is controlled by Our that the expression of Zat7, WRKY25, or Apx1 is not elevated in transgenic plants expressing Zat12 The steady-state level of a number of transcripts a putative was elevated in transgenic plants expressing These a a number of putative transcription factors (i.e. MYB, and zinc finger two different and a related to ROS enhanced in plants expressing Zat12 peroxidase and related to response and and were also elevated in plants in the of transcripts elevated in transgenic plants expressing Zat12 elevated in transgenic plants expressing finger protein factor factor protein protein type protein protein protein protein protein protein response transcription factor finger zinc to to transcription factor protein in a of Plants for the of Zat12 in plants during oxidative we and Zat12-deficient knock-out grown under controlled conditions, Zat12-deficient plants were in their and to wild type plants However, the oxidative stress response of plants was that of wild type plants H2O2 or was found that the steady-state level of transcripts encoding Zat7, WRKY25, and Apx1 was not elevated in plants during oxidative stress The expression of Apx1 elevated in plants in response to a level of light stress the of Zat7, WRKY25, and Apx1 during oxidative stress in to cells during oxidative we used a protein to protein oxidation in plants. We tested protein oxidation in wild type plants during H2O2 stress 1 and found that the protein in Arabidopsis to in to that of the of of in protein from and plants that the protein is L. S. and H. in Ref. R.A. D. J. Biol. Chem. Full Text PDF PubMed Google for a of protein oxidation during oxidative shown in the H2O2-induced oxidation of a in plants was higher than that in wild type plants to the H2O2 stress. that the of Apx1 expression in knock-out Zat12 plants during oxidative stress results in oxidative to evidence that cytosolic Apx1 is involved in the from oxidative stress R. Trends Plant Sci. 2002; 9: 405-410Abstract Full Text Full Text PDF Scopus (7856) Google Scholar, 13Pnueli L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar). expression of transcripts encoding different regulatory proteins, different protein WRKY transcription factors, proteins, proteins, and was oxidative stress in plants (11Vranova E. Atichartpongkul S. Villarroel R. Van Montagu M. Inze D. Van Camp W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10870-10875Crossref PubMed Scopus (134) Google Scholar, 12Desikan R. Mackerness A.H. Hancock J.T. Neill S.J. Plant Physiol. 2001; 127: 159-172Crossref PubMed Scopus (716) Google Scholar, 13Pnueli L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar, 14Rizhsky L. Liang H. Mittler R. J. Biol. Chem. 2003; 278: 38921-38925Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). However, genetic evidence supporting a regulatory role for many of these proteins during oxidative stress was not In two different and were shown to involved in H2O2 and two different zinc finger proteins and were shown to an cytosolic expression during response (6Epple P. Mack A.A. Morris V.R. Dangl J.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6831-6836Crossref PubMed Scopus (134) Google Scholar, Y. G. J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). In expression of a heat shock transcription factor in transgenic Arabidopsis plants was shown to enhance the expression of Apx1 and in the absence of stress (15Panchuk I.I. Volkov R.A. Schoffl F. Plant Physiol. 2002; 129: 838-853Crossref PubMed Scopus (362) Google Scholar). we that the zinc finger protein Zat12 is required for cytosolic Apx1 expression during oxidative stress we that Zat12 is also essential for the expression of Zat7 and WRKY25 and that these putative transcription factors are involved in the response of plants to oxidative stress and The in Zat12, Zat7, and WRKY25 expression in cells to the in Apx1 expression during oxidative stress and the of Zat7, WRKY25, and Apx1 expression during oxidative stress in knock-out Zat12 plants evidence that Zat12, Zat7, and WRKY25 are of the oxidative stress response signal transduction of we that Zat12 of Zat7, WRKY25, and Apx1 the ROS signal transduction of Arabidopsis expression of Zat12, Zat7, or WRKY25 did not enhance the expression of Apx1 in the absence of stress that an may required to the expression of Apx1 in these plants. factor may only in cells during oxidative stress Because Zat12, Zat7, and WRKY25 are in cells during stress Chang H.S. R. Wang X. Zhu T. Luan S. Plant Physiol. 2002; 129: PubMed Scopus Google Scholar, S. Plant Cell. 2002; 14: PubMed Scopus Google Scholar, A. T. S. H. K. Plant J. 2000; PubMed Google is that their expression is that of other factors during stress and that the absence of these factors in transgenic plants grown under controlled the of Apx1 also Ref. Curr. Opin. Plant Biol. 2003; 6: PubMed Scopus Google for a expressing transcription factors in we H2O2 stress to wild type plants and transgenic plants that Zat12 to the shown in and the expression of Apx1 between wild type plants and transgenic plants that However, the expression of Apx1 in transgenic plants expressing Zat12 in response to oxidative stress was only higher than that of wild type plants higher than wild not are required to the factors involved in Apx1 expression during stress and expression of Zat12, Zat7, WRKY25 in transgenic plants in enhanced expression of Apx1 in cells in the absence of stress. expression of Zat12 in the elevated expression of different transcripts involved in ROS and The enhanced expression of transcripts encoding an gene in plants may suggest that Zat12 the production of ROS in cells. Because were shown to regulate the response of plants to different and developmental signals enhanced production of ROS in cells M.A. Dangl J.L. Jones J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, H. Trends Plant Sci. 2001; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, Plant Physiol. 2002; 129: PubMed Scopus Google Scholar), the that Zat12 the expression of an may suggest that are also involved in the response of plants to oxidative stress. The that and ROS are used to regulate the response of plants to ROS stress tested in that and production of ROS, and not a in the steady-state level of ROS in cells, is required to the response of plants oxidative stress. The expression of the gene elevated in plants was also found to elevated in plants to or stresses that enhance the expression of Zat12, as well as the expression of different enzymes J.A. Y. Chang H.S. Zhu T. Wang X. Harper J.F. Plant Physiol. 2002; PubMed Scopus Google Scholar). These the that Zat12 expression enhance the expression of gene during abiotic stress. of wild type plants or plants expressing WRKY25, of plants expressing Zat12 or Zat7 were to oxidative stress by paraquat In a of plants expressing a of the oxidative stress signal transduction protein a to in were found to more than wild type to different abiotic stresses such as heat shock, and stress Y. G. J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Our suggest that of the oxidative stress signal transduction of Arabidopsis could used in a to enhance the of plants to oxidative stress. Because Zat12 expression in transgenic plants did not in plants in the absence of stress and did not in a plant and Zat12 may an signal transduction protein to in plants and enhance their to oxidative stress other abiotic the of and plants to different abiotic stresses may these proteins could used for different such as the of plant to biotic or abiotic stress. In to many of the different transcription factors in the steady-state level of transcripts encoding Zat12 is elevated in Arabidopsis in response to a number of different biotic and abiotic These stresses such as heat shock, and light 12Desikan R. Mackerness A.H. Hancock J.T. Neill S.J. Plant Physiol. 2001; 127: 159-172Crossref PubMed Scopus (716) Google Scholar, 13Pnueli L. Liang H. Rozenberg M. Mittler R. Plant J. 2003; 34: 187-203Crossref PubMed Scopus (267) Google Scholar, and Chang H.S. R. Wang X. Zhu T. Luan S. Plant Physiol. 2002; 129: PubMed Scopus Google Scholar, S. Plant Cell. 2002; 14: PubMed Scopus Google Scholar, A. T. S. H. K. Plant J. 2000; PubMed Google Scholar, as well as a of stress response Arabidopsis results to these as well as to other stresses that not enhance Zat12 is the of ROS in cells during different of stress and stress Plant Biol. 2002; 53: PubMed Scopus Google Scholar). Although is not known which signals are involved in Zat12 expression in cells, is to that a combination of different signals such as ROS different stress response control the expression of Zat12 in cells during stress. of the Zat12, Zat7, and WRKY25 a between different stress and Zat12 However, the of the heat shock that may regulate Zat12 expression during heat shock or oxidative known for ROS responses was in the of We are Zat12 to the Zat12 and different mutants in Zat12 expression during stress. We and for their

Reactive oxygen species (ROS) play a key signaling role in plants and are controlled in cells by a complex network of ROS metabolizing enzymes found in several different cellular compartments. To study how different ROS signals, generated in different cellular compartments, are integrated in cells, we generated a double mutant lacking thylakoid ascorbate peroxidase (tylapx) and cytosolic ascorbate peroxidase1 (apx1). Our analysis suggests that two different signals are generated in plants lacking cytosolic APX1 or tylAPX. The lack of a chloroplastic hydrogen peroxide removal enzyme triggers a specific signal in cells that results in enhanced tolerance to heat stress, whereas the lack of a cytosolic hydrogen peroxide removal enzyme triggers a different signal, which results in stunted growth and enhanced sensitivity to oxidative stress. When the two signals are coactivated in cells (i.e. tylapx/apx1), a new response is detected, suggesting that the integration of the two different signals results in a new signal that manifests in late flowering, low protein oxidation during light stress, and enhanced accumulation of anthocyanins. Our results demonstrate a high degree of plasticity in ROS signaling in Arabidopsis (Arabidopsis thaliana) and suggest the existence of redundant pathways for ROS protection that compensate for the lack of classical ROS removal enzymes such as cytosolic and chloroplastic APXs. Further investigation of the enhanced heat tolerance in plants lacking tylAPX, using mutants deficient in chloroplast-to-nuclei retrograde signaling, suggests the existence of a chloroplast-generated stress signal that enhances basal thermotolerance in plants.