Reactive oxygen species as signals that modulate plant stress responses and programmed cell death

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1 Reactive oxygen species as signals that modulate plant stress responses and programmed cell death Tsanko S. Gechev, 1,2 * Frank Van Breusegem, 3 Julie M. Stone, 4 Iliya Denev, 2 and Christophe Laloi 1 Summary Reactive oxygen species (ROS) are known as toxic metabolic products in plants and other aerobic organisms. An elaborate and highly redundant plant ROS network, composed of antioxidant enzymes, antioxidants and ROS-producing enzymes, is responsible for maintaining ROS levels under tight control. This allows ROS to serve as signaling molecules that coordinate an astonishing range of diverse plant processes. The specificity of the biological response to ROS depends on the chemical identity of ROS, intensity of the signal, sites of production, plant developmental stage, previous stresses encountered and interactions with other signaling molecules such as nitric oxide, lipid messengers and plant hormones. Although many components of the ROS signaling network have recently been identified, the challenge remains to understand how ROS-derived signals are integrated to eventually regulate such biological processes as plant growth, development, stress adaptation and programmed cell death. BioEssays 28: , ß 2006 Wiley Periodicals, Inc. 1 Institute of Plant Sciences, ETH Zurich, Switzerland. 2 Department of Plant Physiology and Plant Molecular Biology, University of Plovdiv, Bulgaria. 3 Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Belgium. 4 Department of Biochemistry and Plant Science Initiative, University of Nebraska, Lincoln, NE, USA. Funding agencies: International Union of Biochemistry and Molecular Biology, IUBMB, grant number: S-350; The Company of Biologists Limited, EMBO, grant number: ASFTF ; Research Fund of the Ghent University (Geconcerteerde Onderzoeksacties no ). *Correspondence to: Tsanko S. Gechev, Department of Plant Physiology and Plant Molecular Biology, University of Plovdiv, 24 Tsar Assen str, Plovdiv 4000, Bulgaria. tsangech@pu.acad.bg DOI /bies Published online in Wiley InterScience ( Abbreviations: ROS, reactive oxygen species; SOD, superoxide dismutase; APX, ascorbate peroxidase; GPX, glutathione peroxidase; ABA, abscisic acid. Introduction ROS, resulting from excitation or incomplete reduction of molecular oxygen, are unwelcome harmful by-products of normal cellular metabolism in aerobic organisms. (1) Plants, facing an even greater burden of excess ROS, initially developed various protective mechanisms, such as small antioxidant molecules and antioxidant enzymes, to keep ROS levels under control. (2) As these protective mechanisms became robust, plants further evolved an elaborate network of ROS-producing and detoxifying enzymes (represented by at least 289 genes in Arabidopsis thaliana) to adjust ROS levels according to the cellular needs in different cell types and organs at a particular time and at different developmental stages. This evolutionary advance permitted ROS to be coopted as signaling molecules that control cell proliferation and cell death to regulate plant growth and development, adaptation to abiotic stress factors and proper responses to pathogen attack. (3 5) To precisely influence such diverse processes in a range of tissues at different developmental stages, the biological response to the altered ROS levels requires remarkable specificity. This specificity is ensured by multiple interacting factors, including the chemical identity of ROS, intensity of the signal, site of ROS production, developmental stage of the plant and previous stresses encountered. Interactions with other signaling molecules such as nitric oxide, lipid messengers and plant hormones are also key determinants of the final outcome of ROS signaling. (5) Despite the obvious complexity, a clearer picture of the ROS network and its role in plant biology is slowly emerging. Recent mutational and transcriptome analyses revealed key players in the ROS network, including MAPK kinases and ROS-responsive transcription factors. In addition to highlighting ROS chemistry and metabolism, this review summarizes the latest data related to plant ROS signaling. In particular, the focus is on specific factors that influence the biological processes regulated by ROS with an emphasis on plant development, stress acclimation and cell death. BioEssays 28: , ß 2006 Wiley Periodicals, Inc. BioEssays

2 Chemistry of ROS Plants, being aerobic organisms, utilize molecular dioxygen (O 2 ) as a terminal electron acceptor. As a result of O 2 reduction, highly reactive intermediates, reactive oxygen species (ROS), are produced. (1) The first step during O 2 reduction leads to the formation of superoxide (O 2 ) or hydroperoxide (HO 2 ) radicals (Fig. 1). O 2 has a short halflife of 2 to 4 ms. The second step leads to formation of hydrogen peroxide (H 2 O 2 ), which is a relatively stable molecule with a 1 ms half-life. Because of this longer half-life, H 2 O 2 can migrate from the subcellular synthesis sites to adjacent compartments and even neighboring cells. (6,7) The oxidizing power of O 2 and H 2 O 2 makes them potentially dangerous for the surrounding cellular environment. O 2 can inactivate important metabolic enzymes containing Fe-S clusters and alter catalytic activities. (1,2) Its protonated form, HO 2, is found mainly in acidic cellular environments. HO 2 can cross biological membranes and initiate lipid oxidation by extracting protons from polyunsaturated fatty acids. In most biological systems, O 2 is rapidly converted to H 2 O 2 by the enzyme superoxide dismutase (SOD). H 2 O 2 can inactivate enzymes by oxidizing their thiol groups. (1) The destructive properties of O 2 and H 2 O 2 are more prominent when they interact in the presence of metal ions to form the highly reactive hydroxyl radical (HO ) during the so-called Haber-Weiss reaction. (8) HO can react with and damage virtually anything with which it comes into contact. (1) Because HO is highly reactive, cells do not possess enzymatic mechanisms for HO detoxification and rely on mechanisms that prevent its formation. These mechanisms include the preceding elimination of O 2 and H 2 O 2 and/or sequestering metal ions that catalyze the Haber- Weiss reaction with metal-binding proteins, such as ferritins or metallothioneins. (9,10) Singlet oxygen ( 1 O 2 ) is a non-radical ROS produced by spin reversal of one electron of the ground state triplet oxygen ( 3 O 2 ). (11) Such spin reversals occur under input of energy and are most often caused by reaction with the highly energized triplet-state chlorophyll. (11) If not quenched by carotenoids, 1 O 2 can in turn transfer its energy to other molecules and damage them, like the rapid peroxidation of polyunsaturated fatty acids. (1) An important feature of ROS chemistry is the conversion of one ROS into another. In addition to reacting with H 2 O 2 and Figure 1. Production of ROS by multistep reduction of molecular oxygen. forming HO,O 2 can react with nitric oxide radical (NO )to form peroxynitrite (ONOO ). Peroxynitrite is rapidly protonated to peroxynitrous acid (ONOOH), which is a powerful oxidizing agent. It can damage all biomolecules and initiate reactions leading to formation of several other destructive radical- and non-radical reactive species. (1) ROS homeostasis Sites and sources of ROS production The multiple sites and sources of ROS production increase the complexity of ROS. ROS are normal products of metabolism and are produced in all cellular compartments within a variety of processes (Fig. 2). (4) In general, they are maintained at constant basal levels in healthy cells, but their levels transiently or persistently increase under different stress conditions or in response to developmental signals. Chloroplasts are a major site of ROS generation in plants. (12) Photosynthetic electron transfer chains produce O 2, especially under conditions leading to overenergization of the electron transfer chains. (4) O 2 is formed mainly by electron leakage from Fe-S centers of photosystem I or reduced ferredoxin to O 2 (Mehler reaction), which is then quickly metabolized to H 2 O 2 by SOD. Although excessive production of ROS is dangerous, in this case the ability of oxygen to accept electrons prevents overreduction of the electron transport chains, thus minimizing the chance of 1 O 2 production. (4) 1 O 2 is produced by energy transfer to 3 O 2 from the excited triplet state chlorophyll in photosystem II, especially under high light intensities. (11) Carotenoids can quench 1 O 2 directly, a role that is shared with tocopherols, or prevent 1 O 2 formation by quenching the excited triplet state chlorophyll. (12) Peroxisomes and glyoxysomes are other major sites of ROS generation during photorespiration and fatty acid oxidation, respectively. (13) Photorespiration is a complex process tightly linked to photosynthesis. Under conditions that impair CO 2 fixation in chloroplasts, the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase increases and the produced glycolate moves to peroxisomes, where it is oxidized by glycolate oxidase forming H 2 O 2. Fatty acid oxidation in glyoxysomes of germinating seeds generates H 2 O 2 as a by-product of the enzyme acyl-coa-oxidase. Mitochondrial respiration is another process leading to O 2 and H 2 O 2 formation. (14) The main sources of ROS production in mitochondria are NADH dehydrogenase, ubiquinone radical and complex III. (14) Although mitochondrial ROS production is much lower compared to chloroplasts (lack of light energyabsorbing chlorophyll pigments), mitochondrial ROS are important regulators of a number of cellular processes, including stress adaptation and programmed cell death. (15) The estimated H 2 O 2 production in mitochondria may be 20 times lower than in the chloroplasts, at least in C 3 plants. (16) 1092 BioEssays 28.11

3 Figure 2. Schematic representation of a generalized plant cell depicting major sources of ROS generation and scavenging enzymes described in the text. Much of the ROS generated in photosynthetic plant cells is produced in chloroplasts. Chloroplasts produce singlet oxygen ( 1 O 2 ) from the excited triplet state chlorophyll (primary source Photosystem II, PSII) and superoxide anion (O 2 ) in the Mehler reaction (primary source PSI). Mitochondria produce O 2 due to electron leakage from the mitochondrial electron transport chain. O 2 from both organelles is then rapidly converted to hydrogen peroxide (H 2 O 2 ) by superoxide dismutases (SOD). H 2 O 2, in turn, is detoxified by ascorbate peroxidases (APX) with the ascorbate (AsA) as an electron donor. AsA, oxidized to monodehydroascorbate radical (MDA ) and eventually to dehydroascorbate (DHA), is then recycled in the Halliwell-Asada pathway (4) via a series of enzymatic reactions involving monodehydroascorbate reductase (MDHAR), reduced ferredoxin (Fd), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and non-enzymatic antioxidant glutathione (reduced form GSH, oxidized form GSSG). Peroxisomes and glyoxysomes produce large amounts of H 2 O 2 during photorespiration and fatty acid oxidation, respectively. This H 2 O 2 is rapidly scavenged by catalases (CAT). Plasma membrane-bound NADPH oxidases generate superoxide anion in the apoplast, which then dismutates to H 2 O 2. The movement of H 2 O 2 between different cellular compartments is facilitated by peroxoporins (specialized aquaporins). The excess H 2 O 2 leaking into cytosol from different compartments is metabolized by various peroxidases or may eventually be transported and detoxified into the vacuole. Plasmalemma-bound NAD(P)H oxidases as well as cellwall-associated peroxidases are the main O 2 and H 2 O 2 producing apoplastic enzymes. (17) These are regulated by various developmental and environmental stimuli. (4) Apoplastic ROS accumulation participates in the so-called oxidative burst observed as a part of the hypersensitive response to pathogens but also regulates cell growth, development and cell death. (3,5,17 19) O 2 and H 2 O 2 are produced also by xanthine oxidase during purine catabolism, ribonucleotide reductase during deoxyribonucleotide synthesis and various other oxidases induced by biotic and abiotic stresses. (4) ROS detoxification A strict control of ROS levels is essential to prevent their toxicity and to ensure an accurate execution of their signaling functions. Therefore, plants have evolved an elaborate enzymatic and non-enzymatic antioxidant system, which together with the ROS-producing enzymes maintains ROS homeostasis in all cellular compartments and regulates the adjustment of ROS levels according to the cellular need at a particular time (Table 1). (10) SODs are the only plant enzymes capable of scavenging O 2, whereas H 2O 2 can be catabolized directly by catalases or with the help of various reductants by ascorbate peroxidases BioEssays

4 Table I. Major plant ROS-associated enzymes and antioxidants Enzyme/antioxidant (in brackets: number of genes in A. thaliana) Function Localization Reference Superoxide dismutases (SOD) (8) Dismutation of O 2, leads to H 2 O 2 formation cyt, chl, mit, per (10) Catalases (3) Detoxifies H 2 O 2 ; no reductor required mit, per, gly (4) Ascorbate peroxidases (APX) (9) Detoxifies H 2 O 2 with ascorbate as reductor cyt, chl, mit, per, (12) Monodehydroascorbate Reduces monodehydroascorbate radicals with NAD(P)H cyt, chl, mit (10) reductases (MDHAR) (5) as reductor Dehydroascorbate reductases Reduces dehydroascorbate radicals with GSH as reductor cyt, chl, mit (10) (DHAR) (5) Glutathione reductases (GR) (2) Reduces oxidized glutathione with NADPH as reductor cyt, chl, mit, per (10) Guaiacol peroxidases (POX) (73) Detoxifies H 2 O 2 with various substrates as reductors; can also cw, cyt, mit, vac (21, 24) produce ROS (O 2, HO, HOO ). Involved in lignin biosynthesis, hormone metabolism, cross-linking of cell wall polymers, pathogen defense, plant development, senescence and symbiotic interactions Glutathione peroxidases (GPX) (8) Detoxifies H 2 O 2 and lipid hydroperoxides with GSH as reductor cyt, chl, mit, er (10) Glutathione-S-transferases (GST) (53) Detoxification reactions (Degluthathionylation). Can detoxify lipid hydroperoxides and exhibit DHAR activity. Can act as non-catalythic cariers that facilitate the distribution and transport of various biomolecules apo, cyt, chl, mit, nuc (25) Peroxiredoxins (Prx) (10) Thiol-containing peroxidases, detoxify H 2 O 2 cyt, chl, mit, nuc (10) Thioredoxins (Trx) (46) Redox-control of enzymes and transcription factors, electron donor to cyt, chl, mit, nuc (22) Prx and GPX Glutaredoxins (Grx) (31) Deglutathionilation, redox-control of enzymes and transcription plasmalemma,cyt, chl, mit, (23) factors, electron donor to DHA and Prx. Protection against oxidative damage, regulation of plant development. er Ferritins (4) Binds iron, thus sequestering it in a bioavailable, non toxic form and chl, mit (9) preventing formation of HO. Iron homeostasis Alternative oxidases (AOX) (6) Channels electrons from electron transfer chains of mitochondria and chl, mit (10) chloroplasts directly to oxygen, thus minimizing O 2 production under conditions that favour electron transport chain over energization. The chloroplastic AOX homologue Immutans participates also in carotenoid biosynthesis Ascorbate Substrate for APX. Detoxifies H 2 O 2 apo, cyt, chl, mit, per, vac (12) Glutathione Substrate for various peroxidases, glutathione transferases and apo, cyt, chl, mit, per, vac (4) glutathione reductases. Detoxifies H 2 O 2, other hydroperoxides and toxic compounds a-tocopherol Protects membrane lipids from peroxidation, detoxifies membranes (20) lipid peroxides and quenches 1 O 2 Carotenoids Quench 1 O 2. Photosystem assembly, key components of the light chl, chromoplasts, (20) harvesting complex, precursors of ABA elaioplasts, amyloplasts Flavonoids Can scavenge H 2 O 2 and HO directly. vac (26) The abbreviations are: cw, cell wall; apo, apoplast; cyt, cytosol; chl, chloroplasts; mit, mitochondria; er, endoplasmatic reticulum; vac, vacuole; per, peroxisomes; gly, glyoxysomes; nuc, nucleus (APX), peroxiredoxins, glutathione peroxidases (GPX) and the heterogenous group of guaiacol peroxidases. (4) Nonenzymatic antioxidants also contribute to ROS homeostasis, with ascorbate, glutathione, tocopherol and carotenoids as the most-abundant water- and lipid-soluble antioxidants. (20) As catalase degrades H 2 O 2 without any reducing power, this enzyme provides plants with an energy-efficient way to remove H 2 O 2. However, catalase is active only at relatively high H 2 O 2 concentrations. Lower H 2 O 2 levels are eliminated by APX and other peroxidases with the aid of various reductants like ascorbate and glutathione. While some of the ROS network enzymes as SOD, catalase and APX are entirely dedicated to ROS homeostasis, others like guaiacol peroxidases, thioredoxins, ferritins and glutathione-s-transferases are involved also in other processes related to control of development, redox regulation of target proteins and detoxification reactions (Table 1). Some of the ROS-associated enzymes, like guaiacol peroxidases, thioredoxins, glutaredoxins and glutathione-s-transferases, have evolved into large multigene families with diverse functions that facilitate the adaptation of photosynthetic organisms to terrestrial life in elevated oxygen concentrations and different stressful environments. (21 25) These and other antioxidant enzymes together with the ROS-producing enzymes constitute a highly sophisticated and redundant network, which in Arabidopsis thaliana consists of at least 289 genes (Table 1) BioEssays 28.11

5 All cellular compartments are well-equipped with antioxidant enzymes and antioxidants (Fig. 2, Table 1). Therefore, ROS are normally scavenged immediately at the sites of their production by the locally present antioxidants. However, when this local antioxidant capacity cannot cope with ROS production (for example, during stress or temporarily reduced antioxidant levels due to developmental signals), H 2 O 2 can leak into the cytosol and diffuse to other compartments. Plants can also deal with excess H 2 O 2 by transporting it into vacuoles for detoxification. (7,26) Vacuoles are very rich in flavonoids, powerful antioxidants that can scavenge various ROS and peroxynitrite. (27) They also contain high levels of ascorbate, glutathione and peroxidases localized at the tonoplast inner surface. (26) ROS signaling How is ROS specificity ensured? ROS are not just toxic products that need to be eliminated. Spatial and temporal fluctuations of ROS levels are interpreted as signals required for growth, development, tolerance to abiotic stress factors, proper response to pathogens and cell death (Fig. 3). (5,18,28 30) It is of principal importance to understand how such simple molecules can regulate so many diverse processes in different cell types and organs, and at different developmental stages. It has become increasingly clear that the specificity of the biological response to the altered ROS levels depends on multiple factors: chemical identity of the ROS, intensity of the signal (dose-dependent effect), sites of production, developmental stage of the plant, pre-history of the plant cell (for example, previous stress encounters), and interaction with other signaling molecules such as nitric oxide, lipid messengers and plant hormones. (31,32) Most ROS seem to possess signaling functions that enable them to regulate specific biological processes. Initially, signaling functions were attributed to H 2 O 2 and comprehensive transcriptional analysis by microarray- or AFLP-based technologies identified H 2 O 2 -responsive genes. (33 38) Later, signaling properties and distinct transcriptional responses were confirmed for the other ROS. (39 41) A recent comparative meta-analysis of microarray datasets obtained from plants accumulating different ROS at different subcellular locations revealed specific transcriptomic footprints for O 2, 1 O 2 and H 2 O 2. (42) The biological outcome of ROS signaling is intrinsically related to the nature of the ROS signal and is exquisitely dose dependent. (39,40,43) Low doses of O 2 and H 2 O 2 have been shown to induce protective mechanisms and acclimation responses against oxidative and abiotic stress, while high doses trigger cell death. (39,43) Interestingly, cell death initiated by high doses of H 2 O 2 and 1 O 2 can be uncoupled from the necrosis caused by even higher doses of these ROS. (11,44,45) Figure 3. Plant processes regulated by ROS. Developmental processes, stress responses and biotic interactions regulated by ROS include root growth, elongation and gravitropism, stress tolerance and systemic acquired acclimation (SAA), tracheary elements development (TE), senescence, hypersensitive response (HR) to pathogens, systemic acquired resistance (SAR) and plant plant allelopathic interactions. (3,18,86,91) How specificity in plant cells is transduced is unclear but it could be due to activation of different MAP kinases and transcription factors, as it is proposed for Schizosaccharomyces pombe. (46) Aside from the type, dose and duration of ROS signal, the site of ROS production is also a critical determinant. (27,47) For example, localized production of O 2 by NADPH oxidase in the root hair tip triggers Ca 2þ peaks necessary for the root hair growth. (18) This spatial regulation of NADPH oxidase activity is regulated by the Rho-like GTPases. (28) These GTPases also control tracheary elements differentiation through localized ROS production. (48) The issue of spatial control of ROS production is tightly linked with the aspect of ROS mobility and communication between different cellular compartments. While 1 O 2, O 2 and especially HO are not very mobile, BioEssays

6 H 2 O 2 can migrate quite a distance from the site of its production and cross biological membranes. (7) However, membranes are not very permeable for H 2 O 2 and the transport is most likely carried through specialized aquaporins called peroxoporins. (6,7) This transport is another way of adjusting local concentrations, thus the biological effect of H 2 O 2.An example of cross-compartment communication associated with H 2 O 2 mobility is the increased levels of H 2 O 2 produced in cytosol in the absence of the cytosolic APX, which leads to inhibition of chloroplastic APX and collapse of the chloroplastic antioxidant system. (49) Also peroxisomal catalase can act as a sink for H 2 O 2 produced in peroxisomes or elsewhere and catalase infiltrated in the extracellular space of leaves can scavenge photorespiratory H 2 O 2 produced in peroxisomes. (50) ROS and other signaling molecules Interaction with other signaling molecules such as nitric oxide (NO ), lipid messengers or plant hormones determine the outcome or help to fine-tune the biological responses to altered ROS levels. (31,32) NO interacts with O 2 and H 2 O 2 in a complex manner to regulate cell death during the hypersensitive response. (51) It has been proposed that ROS are key mediators in channeling NO into the death pathway. Indeed, Arabidopsis thaliana overexpressing the H 2 O 2 -detoxifying enzyme thylakoid APX showed increased resistance towards NO-induced cell death. (52) Recent transcriptome analyses identified genes commonly regulated by NO and H 2 O 2 as well as genes that are specifically responsive to the two stress signals in tobacco. (53) Several lipid-derived messengers that interplay with ROS have been described. ROS, in particular HO, can initiate nonenzymatic formation of hydroperoxy fatty acids and other oxidized lipids collectively known as oxylipins. (54) Phytoprostanes are a major group of prostaglandin- and jasmonate-like oxylipins that are constantly produced in healthy cells, but their levels increase under various stresses. (54,55) Phytoprostane B1, for example, can trigger detoxification and defense responses, and plants primed with phytoprostane B1 become more tolerant to oxidative stress-induced cell death. (55) Sphingolipids are other biologically active lipids that regulate plant growth and cell death. (58) Disruption of sphingolipid metabolism by the fungal AAL-toxin in AAL-toxin-sensitive plants leads to H 2 O 2 accumulation and subsequent cell death. (57) The link between sphingolipid and redox signaling is further substantiated by isolating mutants altered to fungal toxin- and ROS-induced cell death (30) (T. Gechev, M. Ferwerda, L. Bernier, J. Hille, unpublished results). Phospholipids are other bioactive molecules that can modulate ROS response. (58) These second messengers are rapidly formed in response to a variety of stimuli via the activation of lipid kinases or phospholipases. For example, the oleate-stimulated phospholipase D and phosphatidic acid can inhibit H 2 O 2 -induced cell death in Arabidopsis thaliana. (59) Interaction of ROS with plant hormones can be another determinant of specificity. For example, auxin, abscisic acid (ABA) and jasmonic acid together with ROS regulate such diverse processes as growth, stomatal closure and wounding responses. (32) Likewise, ethylene and salicylic acid act synergistically with ROS. (60) Increased synthesis of both ethylene and salicylic acid is observed under abiotic stress and pathogen attack, which can in turn potentiate ROS production. (60) Recent evidence implicate complex ethylene interactions with ABA and H 2 O 2 to regulate stomatal closure. (61) Next to the interactions with the classical hormones, a number of polypeptides, including systemin and the recently identified polypeptide AtPep1, can induce H 2 O 2 synthesis and activate defense gene expression in Arabidopsis thaliana. (62,63) The AtPep1 induces also its own precursor gene propep1, suggesting a possible amplification of the ROS signal. (63) Indeed, propep1 and one of its paralogs are highly induced in a number of stresses leading to H 2 O 2 accumulation. (57,63) Perception and transduction of ROS-derived signals While specific ROS sensors in plants remain elusive, there is ample data pointing out different components of the ROS signaling network, including kinases, phosphatases and ROSresponsive transcription factors. The conditional fluorescent (flu) mutant accumulates free protochlorophyllide in darkness. (64) Upon re-illumination, the excited protochlorophyllide acts as a photosensitizer and generates 1 O 2. The flu system seems to be very specific in generating only 1 O 2 and not O 2 or H 2O 2. (40) 1 O 2, produced in the chloroplasts, generates a signal that migrates to the nucleus where it switches on genetic programs leading to growth inhibition or/and programmed cell death. (11) The chloroplastic protein EXECUTOR1 is probably situated at the beginning of the 1 O 2 signaling cascade as exe1 flu plants show no growth inhibition or cell death upon dark to light shift. (65) The zinc finger proteins LSD1 and LOL1 are negative and positive regulators, respectively, of O 2 - induced cell death in Arabidopsis thaliana. (66,67) It has been proposed that they act together as a molecular rheostat to sense and transmit the O 2 -derived signal. The phenotype of lsd1 mutant is uncontrolled, spreading cell death, initiated by O 2.(68) Interestingly, a triple mutant between lsd1 and two ROS-generating NADPH oxidase homologues, atrbohd and atrbohf, showed uncontrolled cell death even under growth conditions that normally repress lsd1 cell death. (29) The lsd1 phenotype was restored by overexpression of AtrbohD, demonstrating that O 2 produced by NADPH oxidase and its subsequent dismutation to H 2 O 2 is somehow able to antagonize the O 2 -induced cell 1096 BioEssays 28.11

7 death in the neighboring cells. (29) In accordance with that observation, catalase overproducing tobacco plants with reduced levels of H 2 O 2 have larger hypersensitive response lesions upon challenge with tobacco mosaic virus. (69) A similar antagonistic effect has been observed between 1 O 2 and H 2 O 2. Overexpression of the H 2 O 2 -scavenging enzyme thylakoidbound APX in flu increases further 1 O 2 -dependent growth inhibition and cell death (C. Laloi and K. Apel, unpublished results), indicating that some of the H 2 O 2 -regulated genes may negatively control 1 O 2 signaling. Mitogen-activated protein kinases (MAPK) are widespread signal transmitters in eukaryotes. In Arabidopsis thaliana, a multifaceted networkof kinases is involved in relaying the H 2 O 2 signal. ANP1, a MAPK kinase kinase, is activated by H 2 O 2 and, through an unidentified intermediate kinase, in turn activates two downstream MAPKs, AtMPK3 and AtMPK6, which eventually upregulate GST6 and HSP18.2 genes. (70) Overexpression of ANP1 in transgenic plants resulted in increased tolerance to heat shock, freezing and salt stress. (70) The serine/threonine kinase OXI1 (oxidative signal-inducible1) is another essential component of the H 2 O 2 signaling network in Arabidopsis thaliana. The oxi1-null mutant has abnormal root hair growth and enhanced susceptibility to pathogen infection, two processes mediated by H 2 O 2. (71) OXI1 is activated by H 2 O 2 and abiotic stresses and OXI1 is needed for full activation of AtMPK3 and AtMPK6. (71) Another H 2 O 2 - inducible kinase is OMTK1 (oxidative stress-activated MAP triple-kinase 1) in alfalfa. (72) In contrast to OXI1, OMTK1 is H 2 O 2 specific and not activated by abiotic stresses or hormones. OMTK1 activates the downstream MAP kinase MMK3. MMK3 can also be activated by ethylene and elicitors, thus serving as a convergence point of ethylene and ROS signaling. (72) H 2 O 2 also increases expression of the Arabidopsis thaliana nucleotide diphosphate kinase 2 (AtNDPK2). (73) AtNDPK2 overexpression reduced the accumulation of H 2 O 2 and, like ANP1, resulted in enhanced tolerance to cold, salt and oxidative stress. (73) It has been shown that Arabidopsis thaliana nucleotide diphosphate kinase 1 (AtNDK1) interacts with the three Arabidopsis thaliana catalases in a yeast twohybrid system and transgenic plants overexpressing AtNDK1 exhibited resistance to paraquat and enhanced ability to detoxify H 2 O 2. (74) Protein phosphatases, which are equally as important for modulating the signal, have also been implicated in ROS signaling recently. (75) One of the earliest events that follow elevation in H 2 O 2 levels is alteration in calcium ion fluxes. (71,76) Transient Ca 2þ oscillations are specific for different types of stress and can lead to various downstream effects through the numerous Ca 2þ -interacting proteins, including calmodulins and calciumdependent protein kinases that are involved in different, sometimes even antagonistic responses. (76) While some of them, like NAD kinase, aid in the production of H 2 O 2 by generating more substrate for NADPH oxidase, others like catalase have the opposite effect. (77) Effects mediated by Ca 2þ can be rapid and short-term, as with the activation of calcium channels by hydrogen peroxide following abscisic acid signaling in guard cells, (78) or long-term, which relies on altered gene expression. Activation of MAPK cascades, alterations in Ca 2þ fluxes and other biochemical changes associated with the relay of ROS signals ultimately lead to activation of ROS-sensitive transcription factors. Genetic and molecular data identified transcription factors that rapidly respond to different ROS. (33,36 38,40,57) A recent comparative analysis of transcriptome changes induced by different types of ROS in Arabidopsis thaliana identified transcription factors that are specifically responding to each of the different ROS as well as others that are induced by all types of ROS. (42) While several members of the ERF and Myb family transcription factors are specifically induced by 1 O 2, the heat-shock regulon seems to specifically respond to H 2 O 2. (33,42) Heat-shock transcription factors have been proposed as possible H 2 O 2 sensors and the downstream genes are involved not only in heat-shock tolerance but play more general roles in defense against variety of stresses, including oxidative stress. (79) The induction of Arabidopsis thaliana Apx1 gene whose promoter contains heat-shock-factor-binding motif substantiates these findings. (80) Two genes encoding WRKY-family transcription factors and two zinc-finger transcription factors ZAT11 and ZAT12 were commonly upregulated by O 2, 1 O 2 and H 2 O 2. ZAT12 has been attributed an important role in abiotic stress signaling as ZAT12 overexpressors have elevated transcript levels of oxidative- and light stress-responsive transcripts and ZAT12-deficient plants are more sensitive to H 2 O 2 -induced oxidative stress. (80,81) Processes regulated by ROS Plant growth and development ROS are involved in the regulation of several developmental processes, including root hair growth and elongation, apical dominance, leaf shape, tracheary elements maturation, trichome development, aleurone cell death and senescence (Fig. 3). The function of Arabidopsis thaliana NADPH oxidases was initially thought to be ROS production during the hypersensitive response and supported with genetic data demonstrating decreased ROS accumulation after pathogen challenge in a double mutant of two NADPH oxidase homologues atrbohd and atrbohf. (19) However, the atrbohd atrbohf double mutant also has reduced ABA-mediated seed germination and root elongation inhibition. (82) Further evidence for the role of ROS in growth and development came from the studies on the atrbohc mutant which has low ROS levels in root hairs and is defective in activation of Ca 2þ channels required for formation of Ca 2þ gradient necessary for root hair growth. (18) Consistent with these findings, the BioEssays

8 Arabidopsis thaliana oxi1-null mutant has reduced root hair growth. (71) In contrast, H 2 O 2 production may have an inhibitory effect on growth, as suggested by the inhibition of auxin responses by ANP1, the MAPK kinase kinase that relays H 2 O 2 signal in Arabidopsis thaliana. (70) Indeed, many auxin-responsive genes are downregulated in response to elevated H 2 O 2 levels. (33,57) Cell death Cell death is essential for plant growth, development and proper responses to the environment. (45) At the same time, cell death can be an unwanted event during many unfavorable environmental conditions, including heat, cold, salt and xenobiotic stresses and compatible or disease-causing plant pathogen interactions. (83,84) Developmental or environmental cell death controlled by ROS occurs during the aleurone cell death, leaf senescence, a number of abiotic stresses, the hypersensitive response and allelopathic plant plant interactions. (30,85,86) Programmed cell death can be initiated by all types of ROS. (39,40,87) In addition, hydroxyl radical-initiated lipid peroxidation is a rich source of oxidized lipids that can trigger programmed cell death on their own or in concert with other ROS. (44,54) Two well-described instances of ROS-induced programmed cell death in development are organ senescence and aleurone cell death. ROS, together with ethylene, are hypothesized to regulate plant organ senescence through peroxisomal and chloroplast-derived signals. (88,89) During seed germination, monocot aleurone layer cells utilize their carbohydrate reserves by gibberellic acid-dependent synthesis of alpha-amylase, rapidly followed by cell death. (90) This cell death is dependent on glyoxysomal production of H 2 O 2 during the utilization of lipid reserves. There is evidence that glyoxysomal antioxidant enzymes catalase, ascorbate peroxidase and superoxide dismutase are downregulated by gibberellic acid to ensure sufficient accumulation of H 2 O 2 prior to the onset of cell death. (90) One of the best-studied types of cell death is the hypersensitive response to pathogens. (30) During the hypersensitive response, a biphasic burst of NADPH-dependent ROS production is an essential component for the onset of local cell death in the proximity of the infection as well as for the initiation of signal that migrates in the neighboring tissues to trigger distant micro-hypersensitive response and induce systemic acquired resistance. (91) Although, in the case of many plant pathogen interactions, programmed cell death is a welcome event for the plant host, there are examples of pathogen-triggered cell death that are detrimental for the plant. Several necrotrophic fungal pathogens produce mycotoxins that are able to induce ROS accumulation and eventually programmed cell death, using this strategy as a method to kill the plant and feed on the dead tissue. (57,92) In addition to plant pathogen interactions, a role for programmed cell death in allelopathic plant plant interactions have been described recently. (86) Centaurea maculosa roots secrete the phytotoxin catechin, which triggers ROS accumulation in root meristems of neighboring species and subsequent Ca 2þ - dependent cell death. In this way, Centaurea maculosa kills and eventually displaces other plant species from their habitat. Stress acclimation ROS have emerged as important regulators of plant stress responses. Many unfavorable environmental conditions lead to oxidative stress due to increased ROS production or/and impaired ROS detoxification. (4,10) Accumulation of ROS is observed during cold, heat, drought, high-light, heavy-metal stress and exposure to fungal toxins. (4,57) In addition, H 2 O 2 is a secondary messenger during wounding responses and various biotic interactions. (62,86) Redox changes are sensed by the plant cell as a warning message and, depending on the situation, genetic programs leading to stress acclimation or programmed cell death are switched on. (43) Transient and a moderate elevation of ROS result in protection against subsequent more severe abiotic or oxidative stress. This stress acclimation can be induced either by a direct application of ROS or ROS-generating agents, or by application of mild sublethal stresses that lead to transient ROS accumulation. One of the first studies on stress acclimation demonstrated that a H 2 O 2 pretreatment of maize seedlings protected them from chilling stress. (93) This protective effect resembled the effect of a pretreatment with ABA and low temperatures. Indeed, H 2 O 2 was found to accumulate during the ABA- and low temperature-acclimation treatments. (93) H 2 O 2 pretreatments have been shown to also induce salt, high-light, heat and oxidative stress tolerance. (43,94,95) Amazingly, H 2 O 2 -induced acclimation to high temperature is very durable, lasting more than a month after the initial H 2 O 2 treatment. (95) Applications of salicylic acid, causing elevation of H 2 O 2 levels, or low doses of the O 2 -generating herbicide paraquat protect against subsequent severe heat or oxidative stress, respectively. (39,96) In both mustard and potato, heat acclimation treatment elevates H 2 O 2 levels and results in subsequent thermotolerance against severe heat shock, which is consistent with the thermoprotective role of H 2 O 2 treatment. (95,96) Likewise, light acclimation and H 2 O 2 pretreatment can result in tolerance against high light stress. (94) Moreover, it has been proposed that H 2 O 2 can initiate acclimation not only in local leaves but also in distant non-acclimated leaves, a process referred to as systemic acquired acclimation. (94) ROS are also the signal messengers that initiate cell death-protective responses in the neighboring cells that surround the sites of the hypersensitive response to pathogens and trigger systemic acquired resistance in distant tissues. (29,91) In this context, it is not surprising to find that 1098 BioEssays 28.11

9 manipulation of ROS levels can alter the resistance to pathogen attack. (97) The stress tolerance achieved by transient elevations in ROS levels can be explained by preactivation of defense mechanisms, including kinases, transcription factors and other components of the signaling network, antioxidant enzymes, dehydrins, low-temperature-induced, heat-shock and pathogenesis-related proteins. (39,43,73,94) H 2 O 2 pretreatments of maize and tobacco that resulted in induced protection against chilling, light and oxidative stress increased the activities of the catalase, ascorbate peroxidase, guaiacol peroxidases and elevated the levels of GPX protein. (43,93) Tobacco plants with reduced catalase activity have elevated levels of pathogenesis-related proteins and enhanced resistance against pathogens. (97) A number of transcriptome surveys supported these initial observations and identified new batteries of genes highly regulated at the transcriptional level during ROS-induced stress acclimation. (39,98) The newly identified genes implicated in the acclimation process included putative components of signaling cascades (kinases, phosphatases, Ca 2þ -interacting proteins), transcription factors that presumably govern the global transcriptional re-programming during acclimation and other genes, most of them representing functions that directly or indirectly ensure stress protection. (39,98) Subsequent functional studies confirmed the role of some of those genes in stress tolerance and acclimation. (73,80,81) Both sets of genes that participate in stress acclimation induction and genes responsible for the maintenance of systemic acquired acclimation are important for durable acclimation. The heat-shock protein HSA32, for example, is required not for induction but for maintenance of acquired thermotolerance, as demonstrated by the inability of knockout hsa32 plants to survive severe heat shock followed by a recovery period longer than 24 hours. (99) Multiple stresses exist in nature and different stressors may require diverse responses and adjustment of multiple adaptation mechanisms. (100) Manipulating ROS levels provides us with an opportunity to enhance specific and common protective mechanisms against different stresses to ensure plant growth and survival under a variety of unfavorable environmental conditions. Conclusions It is becoming increasingly evident that ROS regulate a complex signal transduction network within the plant development and its response and adaptation to both biotic and abiotic stressors. We highlighted the major sources of ROS and sites of production in plant cells, together with the key antioxidant molecules and enzymes that scavenge ROS. 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