Journal of Experimental Botany, Vol. 53, No. 372, pp. 1305-1319,
May 15, 2002
© 2002 Oxford University Press
Original Papers |
Regulation and function of ascorbate peroxidase isoenzymes
1 Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan
2 Faculty of Life and Environmental Science, Shimane University, Nishikawatsu, Matsue, Shimane 690-8504, Japan
Received 15 November 2001; Accepted 7 January 2002
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Abstract |
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 Top Abstract IntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS). Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level. Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle. Ascorbate peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H2O2 in higher plants. APX is also found in eukaryotic algae. The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes. Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons. Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses. Transgenic plants expressing E. coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions. It is clear that a high level of endogenous ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.
Key words: Ascorbate peroxidase, gene regulation, oxidative stress.
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Introduction |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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In agreement with the fact that ascorbate (AsA) is a reductant of H2O2 in chloroplasts (Foyer and Halliwell, 1977
), the occurrence of APX in plants (Groden and Beck, 1979; Nakano and Asada, 1981) and Euglena (Shigeoka et al., 1980a) shows that AsA is a very important reducing substrate for H2O2 detoxification in photosynthetic organisms. The code number (EC 1.11.1.11) for APX was first given on the basis of detailed information that was available about the enzymological properties of the Euglena enzyme (Shigeoka et al., 1980a, b).
APX utilizes AsA as its specific electron donor to reduce H2O2 to water with the concomitant generation of monodehydroascorbate (MDAsA), a univalent oxidant of AsA (Fig. 1). MDAsA is spontaneously disproportionated to AsA and dehydroascorbate (DAsA). MDAsA is also directly reduced to AsA by the action of NAD(P)H-dependent MDAsA reductase. DAsA reductase utilizes glutathione (GSH) to reduce DAsA and thereby regenerate AsA. The oxidized GSH is then regenerated by GSH reductase, utilizing reducing equivalents from NAD(P)H. Thus, APX in combination with the effective AsA–GSH cycle functions to prevent the accumulation of toxic levels of H2O2 in photosynthetic organisms (Asada, 1992, 1997). In the chloroplasts of higher plants, in addition to the AsA–GSH cycle located in the stroma the water–water cycle, which is the photoreduction of oxygen to water in PSI by the electrons derived from water in PSII, participates in the detoxification of AOS and the dissipation of the energy of excess photons (Asada, 1999).
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To date, many reports on the purification, molecular cloning, and physiological function of APX isoenzymes based on enzymological and molecular approaches have been published; these studies indicate that APX isoenzymes are critical components that prevent oxidative stress in photosynthetic organisms. Additionally, recent studies on the response of APX expression to some stress conditions and pathogen attack indicate the importance of APX activity in controlling the H2O2 concentration in intracellular signalling.
In this review, the focus will be on recent molecular and physiological findings about APX isoenzymes in photosynthetic organisms such as higher plants and eukaryotic and prokaryotic algal cells. In fact, elucidation of the regulation mechanisms of APX isoenzymes in response to environmental stress is a subject of great interest with respect to APX research and will help to clarify the oxidative stress-tolerance responses of these organisms.
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H2O2/hydroperoxide-scavenging enzymes in higher plants |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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APX has been identified in many higher plants and comprises a family of isoenzymes with different characteristics. Many isoenzymes of guaiacol peroxidase (GP) in plant tissues are localized in vacuoles, the cell wall, and the cytosol, but not in organelles (Asada, 1992
). By contrast, APX isoenzymes are distributed in at least four distinct cellular compartments: stromal APX (sAPX) and thylakoid membrane-bound APX (tAPX) in chloroplasts, microbody (including glyoxysome and peroxisome) membrane-bound APX (mAPX), and cytosolic APX (cAPX) (Chen and Asada, 1989; Miyake et al., 1993; Yamaguchi et al., 1995a, b; Bunkelmann and Trelease, 1996; Ishikawa et al., 1996a, c, 1998). A fifth APX isoenzyme (mitAPX) occurs in a mitochondrial membrane-bound form (Jiménez et al., 1997; Leonardis et al., 2000). Recently, the cDNA encoding a putative APX in the chloroplast thylakoid lumen of Arabidopsis was identified (Kieselbach et al., 2000). APX has also been found in the protozoan Trypanosoma cruzi (Boveris et al., 1980) and the bovine eye (Wada et al., 1998). The finding of APX in the bovine eye suggests that APX may also contribute, together with glutathione peroxidase (GPX: EC 1.11.1.9), to the detoxification of AOS in AsA-rich tissues of animals.
To date, cDNAs homologous to those of mammalian GPXs, especially phospholipid hydroperoxide GPX (PHGPX), have been found for higher plants, including Citrus sinensis (Holland et al., 1993), Nicotiana sylvestris (Criqui et al., 1992), and yeast (Tran et al., 1993). However, the corresponding genes carry a codon for a Cys residue at the putative catalytic site instead of the TGA codon for the selenocysteine (Sec) of mammalian GPXs. Furthermore, Arabidopsis has a homologue of thioredoxin peroxidase, which is known to be an anti-oxidative enzyme in animals and bacteria (Baier and Dietz, 1999).
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Enzymatic properties of APX isoenzymes |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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The molecular and enzymatic properties of APX isoenzymes are different from those of other haem-peroxidases such as GP. APX isoenzymes have high specificity for AsA as the electron donor, which is especially the case for the chloroplastic APX (chlAPX) and mitAPX isoenzymes (Yoshimura et al., 1998
; Asada, 1999; Leonardis et al., 2000). In addition to oxidizing AsA, cAPX and mAPX of higher plants and algal APXs can also oxidize artificial electron donors such as pyrogallol or guaiacol at appreciable rates (Chen and Asada, 1989; Ishikawa et al., 1995, 1996b; Yoshimura et al., 1998; Asada, 1999). Plant APX isoenzymes cannot reduce lipid hydroperoxides. APX, a haem-containing enzyme whose prosthetic group is protoporphyrin, is inhibited by cyanide and azide (Shigeoka et al., 1980b; Chen and Asada, 1989). APX is also inhibited by thiol-modifying reagents such as p-chloromercuribenzoate and suicide inhibitors such as hydroxylamine, p-aminophenol, and hydroxyurea (Chen and Asada, 1990). Since these suicide inhibitors do not affect GP, these reagents are useful for assays that differentiate between APX and GP (Amako et al., 1994). One of the most characteristic properties of APX is its instability in the absence of AsA. Under conditions where the concentration of AsA is lower than 20 µM, APX activity is rapidly lost. The half-inactivation times of chlAPX and mitAPX are less than 30 s, while those of cAPX and mAPX are about 1 h or more (Chen and Asada, 1989; Miyake et al., 1993; Ishikawa et al., 1998; Yoshimura et al., 1998; Leonardis et al., 2000). The instability of APX seems to be one reason that APX was not found for a long time in photosynthetic organisms. ChlAPX isoenzymes exist in a monomeric form, but cAPX is a dimer consisting of identical subunits with a molecular mass of 28 kDa (Mittler and Zilinskas, 1991a; Miyake et al., 1993). As for chlAPX isoenzymes, the molecular mass of tAPX is about 4.5 kDa larger than that of sAPX (33.2 kDa); the difference in molecular mass between tAPX and sAPX is related to the requirement for membrane binding (Chen and Asada, 1989; Miyake et al., 1993; Ishikawa et al., 1996a). The molecular masses (31 kDa) of mAPX and mitAPX are similar (Yamaguchi et al., 1995a; Ishikawa et al., 1998; Leonardis et al., 2000). |
H2O2/hydroperoxide-scavenging systems in algae |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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Both eukaryotic and prokaryotic algae produce H2O2 via O2-, as do higher plants (Patterson and Myers, 1973
; Miyake et al., 1991; Ishikawa et al., 1993; Collén et al., 1995). Algal APX isoenzymes have enzymological and immunological properties similar to those of APX isoenzymes from higher plants except for the amino acid sequence of the N-terminus, the affinity for substrates, and/or their stability. Euglena gracilis lacks catalase and contains the enzymes involved in the AsA–GSH cycle, including APX, solely in the cytosol (Shigeoka et al., 1980a; Ishikawa et al., 1996b). Chlorella vulgaris contains APX as well as catalase (Takeda et al., 1998). Galdieria partita, a unicellular red alga, has at least two APX isoenzymes (Sano et al., 2001). Fresh water Chlamydomonas reinhardtii C9 (C. C9) contains catalase, APX, and selenium-induced GPX (Yokota et al., 1988; Takeda et al., 1992). Halotolerant Chlamydomonas sp. W80 (C. W80) contains catalase, APX, and a selenium-independent GPX-like protein (Takeda et al., 2000).
In C. C9 cells grown in the absence of selenium, APX functions mainly to scavenge H2O2 in chloroplasts (Shigeoka et al., 1991; Takeda et al., 1992). When the C. C9 cells are transferred to medium containing sodium selenite, APX is completely lost after 6 h, and the GPX activity appears and gradually increases until 24 h (Takeda et al., 1993). Recently, a cDNA encoding a GPX-like protein was isolated from C. reinhardtii cw15arg7mt-(CC-325) (Leisinger et al., 1999). The GPX-like protein contained a normal Cys residue, instead of a selenocysteine, which is critical for the activity of the reactive centre in cytosolic GPX from mammals. Accordingly, it seems likely that Chlamydomonas cells contain both a selenium-dependent GPX and a selenium-independent GPX-like protein.
Cyanobacteria contain catalase-peroxidase (CPX) and thioredoxin peroxidase (Mutsuda et al., 1996; Baier and Dietz, 1999; Yamamoto et al., 1999). APX activity has been detected in some prokaryotic algae (Miyake et al., 1991). However, the corresponding protein and gene for APX have not yet been identified. The absence or low level of AsA may account for the lack of APX in these organisms. Synechocystis PCC 6803 cells have two gpx (GPX-1 and GPX-2) homologue genes (slr1171 and slr1992 according to CyanoBase) with significant similarity to the PHGPX gene in mammals. Interestingly, both recombinant proteins in E. coli are able to utilize NADPH, but not GSH, as an electron donor and alkyl hydroperoxide or unsaturated fatty acid hydroperoxides like 
-linolenic acid hydroperoxide as an acceptor (Gaber et al., 2001). It seems accurate to refer to GPX-1 and GPX-2 as NADPH-dependent GPX-like proteins that serve as a novel defence system for the reduction of unsaturated fatty acid hydroperoxides.
The photosynthesis of Euglena, Chlamydomonas, and some cyanobacteria, including Synechococcus PCC 7942 and S. PCC 6803, is not susceptible to H2O2 up to 1 mM, as a result of the resistance of fructose-1,6-/sedoheptulose-1, 7-bisphosphatase, NADP+-glyceraldehyde-3-phosphate dehydrogenase, and phosphoribulokinase in the Calvin cycle to H2O2 (Takeda et al., 1995; Tamoi et al., 1996a, b, 1998a, b, 2001). H2O2 formed in chloroplasts and mitochondria from algal cells diffuses from each organelle to the cytosol, where it is decomposed by H2O2-scavenging enzymes (Ishikawa et al., 1993). A similar finding has also been obtained in several cyanobacteria as well as Ulva rigida C. Ag (Collén et al., 1995). It is concluded that the H2O2-scavenging, H2O2-diffusion, and H2O2-tolerance systems in algal cells function co-operatively to protect cellular components against oxidative stress caused by AOS.
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Characterization of cDNAs encoding APX isoenzymes |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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The cDNAs encoding APX isoenzymes from plant species have been isolated and characterized by several research groups (Mittler and Zilinskas, 1991
b; Ishikawa et al., 1995, 1996a; Bunkelmann and Trelease, 1996). The cDNA encoding mitAPX has not been isolated yet. Higher plants contain two or more putative cAPXs, indicating that cAPX isoenzymes are encoded by a multigene family (Santos et al., 1996; Jespersen et al., 1997). In spinach, cAPX2 (SAP1), which appears to belong to a second family of cAPX-related enzymes, has been cloned and characterized (Ishikawa et al., 1995); however, neither the protein nor the activity of cAPX2 has been detected in crude homogenates of spinach leaves, although its mRNA is constitutively expressed (Yoshimura et al., 2000). Thus, the expression of cAPX2 seems to be suppressed by translational regulation. In Mesembryanthemum crystalinum, cDNAs encoding three types of putative cAPXs have been isolated; however, their corresponding proteins have not been identified yet. cDNA clones encoding APXs localized in the stroma of C. C9 and C. W80 were recently isolated (Takeda et al., 2000).
Interestingly, the N-terminal 364 amino acids of spinach sAPX are completely identical with those of tAPX, whereas the C-terminal 50 amino acids differ (Ishikawa et al., 1996a). A similar finding was also made in pumpkin (Mano et al., 1997), M. crystalinum, and tobacco. These chlAPX isoenzymes are encoded by only one gene (APXII), and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons as described below. By contrast, in Arabidopsis thaliana, the nucleotide sequences of the cDNAs for sAPX and tAPX share only 66.1% identity and these two isoenzymes are encoded by different genes (Jespersen et al., 1997).
chlAPX isoenzymes have a transit peptide consisting of approximately 70 residues in their N-terminus. This peptide contains few acidic residues and is rich in Ser and Thr, thus resulting in a net positive charge (Ishikawa et al., 1996a; Yamaguchi et al., 1996; Jespersen et al., 1997). In all sequences of transit peptides for import across the chloroplast envelopes, position–2 relative to the cleavage site is occupied by a Lys residue; however the functional significance of this is unknown (Jespersen et al., 1997). tAPX has one major hydrophobic domain which is responsible for spanning to the stroma-exposed thylakoid membranes in chloroplasts. A similar membrane-spanning region in the C-terminal region is present in mAPX and is bound to the external side of the membrane of glyoxysomes (Yamaguchi et al., 1995b; Bunkelmann and Trelease, 1996; Ishikawa et al., 1998). This fact indicates that the physiological function of mAPX is to scavenge H2O2 generated both in microbodies and the cytosol in combination with catalase and cAPX (Yamaguchi et al., 1995b; del Río et al., 1998; Ishikawa et al., 1998). The transmembrane domain within mAPX functions as an overlapping peroxisomal endoplasmic reticulum-sorting signal and a peroxisomal membrane-targeting signal type 2 (Mullen and Trelease, 2000).
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Evolution of APX isoenzymes |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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On the basis of the amino acid sequences, plant peroxidases can be classified into three well-separated classes (Welinder, 1992
). Class I includes the intracellular peroxidases of prokaryotic origin, Class II the fungal peroxidases, and Class III the classical plant peroxidases. APX, yeast cytochrome c peroxidase (CCP) and cyanobacterium CPX belong to the Class I family (Welinder, 1992; Mutsuda et al., 1996; Zámock'y et al., 2000). On the other hand, GP belongs to the Class III family, which constitutes a separate lineage from APX. A novel Class III peroxidase in tea leaves showed high specificity for AsA as an electron donor (Kvaratskhelia et al., 1997).
A phylogenetic tree constructed using the deduced amino acid sequences of the catalytic active domains of APX isoenzymes, not including the transit peptide and the membrane-spanning sequences, shows that APX isoenzymes in higher plants and algae can be divided into four groups (cAPX I, cAPX II, chlAPX, and mAPX) (Fig. 2). The earliest event in APX evolution resulted in the appearance of separate groups: a cAPX I group, a chlAPX group, and mAPX and cAPX II groups. It is likely that cAPX I, chlAPX, and mAPX share common features conserved among plant species, whereas the cAPX II group may have evolved from cAPX in a species-specific manner. chlAPX isoenzymes are divided into two types that arose by alternative splicing from a single gene and by different genes at a very recent stage.
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APX isoenzymes in higher plants show high homology (70–90%) within each group. Furthermore, the four groups of APX isoenzymes show 50–70% homology with each other. The Arg-38, Asn-71, Glu-65, and Asp-208 residues around the distal His-42 and proximal His-163 residues of pea cAPX are conserved in all APX groups as well as the entire plant peroxidase family. These residues are essential for binding of the ligand haem (Welinder, 1992
). Trp-179 is conserved in most APX groups and is the third participant in a hydrogen-bonding network together with the proximal His-163 and Asp-208 residues; however, the residue in the cAPX II group is changed to Phe, which is the predominant residue at this position in the Class II and Class III peroxidases of higher plants (Jespersen et al., 1997). The change from Trp-41 to Phe is also found in the cAPX II group as well as in Class II and III peroxidases. Phe-175 is conserved in all APX groups except for a Trp in the chlAPX group, suggesting that the Trp residue may be the major determinant of the greater specificity toward AsA of chlAPX isoenzymes. Four additional regions in the internal sequences and seven residues in the C-terminal extension of the catalytic domain are observed in chlAPX isoenzymes, but not in the other APX isoenzymes. Therefore, chlAPX isoenzymes generally have larger molecular weights than cAPX and mAPX, suggesting that these additional sequences may be associated with the greater instability of chlAPX isoenzymes.
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Structural investigations of the catalytic domain |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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The instability of APX isoenzymes seems to be one of the reasons for the difficulty in obtaining large amounts of highly purified APX isoenzymes. Therefore, advances in the expression of the recombinant APX isoenzymes utilizing cDNA clones have provided new approaches for characterizing the structure and function of each APX isoenzyme (Raven, 2000
). Some functional amino acid residues which are related to the binding of haem and AsA have been identified by mutation analysis using recombinant pea cAPX. Arg-172 of pea cAPX has an important role in AsA utilization to form Compound II (Bursey and Poulos, 2000). Glu-112 is located at the dimer interface, and is related to an alteration in solvent structure (Mandelman et al., 1998a). Arg-38 has a functional role in the control of substrate binding and orientation (Celik et al., 2001). The active site structures, including the hydrogen-bonding interactions between the proximal His-163, buried Asp-208, and Trp-179, are nearly identical to those of CCP. On the other hand, there are many structural differences between CCP and APX, including the cation binding site and orientations of the vinyl substituents on the haem chromophore (Patterson and Poulos, 1994, 1995; Nissum et al., 1998). AsA oxidation does not occur at the exposed haem edge but at an alternate binding site in the vicinity of Cys-32 and the haem propionates (Mandelman et al., 1998b).
Antibodies raised against APX isoenzymes
Monoclonal antibodies have been developed against spinach sAPX (chl-mAb6) and cAPX (cyt-mAb1) isoenzymes and Euglena cAPX (EAP1 and EAP2) (Ishikawa et al., 1996b; Yoshimura et al., 2001). Each spinach APX antibody reacts specifically with the respective isoenzyme from higher plants, but none cross-react with the others. On the other hand, EAP1 reacts with both chlAPX isoenzymes and cAPX from higher plants, although EAP2 shows much higher cross-reactivity with cAPX than with sAPX (Ishikawa et al., 1996b). EAP1 recognizes the common epitope at the site around the proximal His residue in APX isoenzymes, while EAP2 recognizes a relatively homologous region downstream of the distal His residue in the APX isoenzymes. Cyt-mAb1 and chl-mAb6 recognize the distal His region of cAPX and the inherent region of chlAPX isoenzymes, respectively.
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The potential of the defence system involving APX isoenzymes to oxidative stress |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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The rate of O2- and H2O2 formation within intact chloroplasts under optimal conditions is estimated to be 240 and 120 µM s-1, respectively (Asada and Takahashi, 1987
). SOD isoenzymes and H2O2-sacavenging enzymes are preferentially located near the site of O2- production (Miyake and Asada, 1992; Ogawa et al., 1995). As a consequence, it is estimated that the H2O2 concentration is less than 8x10-7 M under normal conditions (Asada, 1999). Under photo-oxidative conditions, the actual level of H2O2 must increase several orders of magnitude with respect to that estimated for non-stressed chloroplasts, especially within the sites of localization of antioxidant enzymes. When the AOS production far exceeds the endogenous AOS-scavenging capacity, the regulated balance between the generation system and the scavenging system of AOS may be upset, leading to the inactivation of defence enzymes. Thus, O2- and H2O2 must be completely scavenged to preserve photosynthetic activity.
A characteristic property of APX isoforms is their lability in the absence of their electron donor, AsA (Nakano and Asada, 1981; Chen and Asada, 1989). In the presence of AsA, the reaction intermediate of APX Compound I successively oxidizes AsA to produce two molecules of MDAsA in the catalytic cycle. However, unless Compound I of APX is reduced by AsA, it is oxidized and degraded to the inactivated form by 10 nM levels of H2O2 within 2 min, levels which are generated via auto-oxidation of AsA at micromolar levels. Cu/Zn-SOD is inactivated by H2O2 with an apparent rate constant of 0.8 M-1 s-1; the half-time for the inactivation is about 30 min in 0.4 mM H2O2 at neutral pH (Asada et al., 1975; Casano et al., 1997). Stromal Fe-SOD is also inactivated by H2O2 at a similar rate of 0.6 M-1 s-1. H2O2 reduces the Cu(II) and then reacts with the Cu(I) to give a ·OH at the reaction centre of the enzyme. The ·OH oxidizes His-118 of the active site of the enzyme to 2-oxo-His, resulting in the inactivation and fragmentation of the radical (Casano et al., 1997). DAsA reductase is labile in the absence of thiols and inactivated by 0.5 mM H2O2 (Hossain and Asada, 1984). GSH reductase is inactivated by NADPH in the absence of GSSG (Lascano et al., 1998). Thus, based on the data reported so far, it seems likely that the AOS-scavenging enzymes themselves show sensitivity to oxidative stress.
Manipulation of the expression of enzymes involved in the AOS-scavenging systems has focused on SOD, APX, and GR isoenzymes that are targeted into the cytosol or plastids (Sen Gupta et al., 1993; Pitcher et al., 1994; Aono et al., 1995; Foyer et al., 1995; Slooten et al., 1995; Webb and Allen, 1996). The modification of AOS-scavenging systems can lead to considerable changes in oxidative stress tolerance (Allen, 1997). To evaluate the potential of the AOS-scavenging system of chloroplasts to respond to photo-oxidative stress imposed by drought at high light intensity or paraquat treatment under illumination, E. coli catalase encoded by katE was introduced into tobacco chloroplasts (Shikanai et al., 1998; Miyagawa et al., 2000). Under high light and drought conditions (1600 µmol m-2 s-1 without watering), the degradation of chlorophyll in the leaves of wild-type plants was detectable after 24 h, and severe chlorosis occurred at 72 h. However, transgenic plants did not show any chlorosis for at least 96 h under the stress conditions. Furthermore, when sprayed with 50 µM paraquat and exposed to high light intensity (300 µmol m-2 s-1 or 1600 µmol m-2 s-1), wild-type plants developed visible severe leaf injury after 24 h, while the transgenic plants did not exhibit any signs of chlorosis. These results demonstrate that transgenic tobacco plants have increased tolerance to photo-oxidative damage imposed by AOS. Interestingly, the chlAPX isoenzymes in the wild-type and transgenic plants were completely inactivated under the stress conditions, while PRK remained active. The total SOD activity in the control plants dramatically decreased, while, in the transgenic plants, the initial activity of SOD was retained. These data suggest that AOS-scavenging enzymes, especially chlAPX isoenzymes, are much more strongly inactivated by oxidative stress than PRK, which is believed to be one of the thiol-modulated enzymes that is most sensitive to H2O2. chlAPX isoenzymes have been found to be the primary targets among the H2O2-sensitive enzymes under paraquat-induced photo-oxidative stress in spinach leaves (Mano et al., 2001).
Why are chlAPX isoenzymes sensitive to photo-oxidative stress in both plant lines? Concentrations of H2O2 as low as 2 µM inactivate chlAPX isoenzymes within several seconds when the level of AsA is too low for the catalytic cycle of the APX isoenzymes to function (Miyake and Asada, 1996). A significant decrease in the total AsA level is in agreement with the decreases in the activities of chlAPX isoenzymes in the wild-type and transgenic plants with stress treatments (Shikanai et al., 1998; Miyagawa et al., 2000). When tobacco leaves suffer from photo-oxidative stress, the level of AsA in the vicinity of chlAPX isoenzymes may decrease due to the excess of AOS generated by paraquat treatment, which causes irreversible damage of chlAPX isoenzymes. Total APX activity and AsA content show identical reductions in pea plants under stress conditions induced by severe water deficit or paraquat (Iturbe-Ormaetxe et al., 1998). The Arabidopsis mutant vtc1 is deficient in AsA, accumulating approximately 30% of wild-type levels, and is more sensitive than the corresponding wild-type to stress conditions such as ozone, UV-B light, and SO2, which generate AOS (Conklin et al., 1997). In spinach leaves, the activities of the chlAPX isoenzymes decrease as the intensity of light increases, despite the fact that the transcript and protein levels of these isoforms are not altered (Yoshimura et al., 2000). A reduction in APX activity is observed in Lupinus arbustus seedlings, in which AsA deficiency is induced by lycorine treatment (Arrigoni et al., 1997). Application of the AsA precursor L-galactono-
-lactone increases the AsA level of water-stressed leaves of wheat, enhances photochemical and non-photochemical quenching of chlorophyll fluorescence, and thus reduces oxidative damage to their photosynthetic apparatus (Tambussi et al., 2000). Based on the data accumulated thus far, it is clear that a high level of endogenous AsA is essential for effectively maintaining the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.
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Regulation of APX expression |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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Change in APX activity
Recent studies have focused on the changes in activities of APX isoenzymes in higher plants subjected to several environmental stresses such as ozone, high light, extremes of temperature, salt, and paraquat (Tanaka et al, 1985
; Mittler and Zilinskas, 1994; Prasad et al., 1994; Conklin and Last, 1995; Rao et al., 1996; Dat et al., 1998; López et al., 1996; Donahue et al., 1997; Yoshimura et al., 2000). Interestingly, APX activities generally increase along with activities of other antioxidant enzymes like catalase, SOD, and GSH reductase in response to various environmental stress factors, suggesting that the components of AOS-scavenging systems are co-regulated. Drought-resistant maize shows greater induction of APX activity than sensitive plants, in addition to a significant increase in GSH reductase activity (Pastori and Trippi, 1992). An Arabidopsis mutant (pst1) isolated based on its ability to grow photoautotrophically on a NaCl-containing medium showed significantly enhanced APX and SOD activities (Tsugane et al., 1999). The pst1 also showed cross-resistance to other stress conditions, suggesting that enhanced enzyme activities might be conferred by the mutation of a recessive nuclear gene and that regulation of these enzyme levels provides plants with additional stress-defence capabilities. Transgenic tobacco plants expressing antisense RNA for cAPX show 45–55% less activity than the non-transgenic plants, and increased susceptibility to ozone (Övar and Ellis, 1997).
Change in APX transcript level
Arabidopsis fumigated by ozone shows a large increase in the steady-state transcript level of cAPX (Conklin and Last, 1995; Övar et al., 1997). The transcript level of pea cAPX also increases 4-fold in response to drought stress, but is even more dramatically enhanced (15-fold) following recovery from stress (Mittler and Zilinskas, 1994). The analysis of the protein level and activity of APX indicates that, during recovery from drought stress, cAPX expression in pea is regulated at the post-transcriptional level, at least in part at the level of protein synthesis. Furthermore, many environmental factors such as high light, salt, wounding, pathogen infection, fruit ripening, and paraquat affect the steady-state transcription level of cAPX (Mittler and Zilinskas, 1992; Pastori and Trippi, 1992; Schantz et al, 1995; Karpinski et al., 1997, 1999; Övar et al., 1997; Biemelt et al., 1998; Mittler et al., 1998, 1999; Morita et al., 1999; Yoshimura et al., 2000). A simultaneous analysis of the response of each APX isoenzyme in spinach leaves to several stress conditions has been carried out (Yoshimura et al., 2000). Among the APX isoenzymes (chlAPX, two cAPXs, and mAPX) tested, the steady-state transcript level of the typical cAPX isoenzyme markedly increased in response to high light stress and paraquat treatment, but not in response to other stress treatments such as drought and salt. The transcript levels of the total chlAPX and mAPX did not change in response to any of the stress treatments. These results indicate that the genes for chlAPX and mAPX are constitutively expressed for the immediate and efficient detoxification of H2O2 under normal and stress conditions, while the gene expression for cAPX is responsive to environmental changes, resulting in the protection of important cellular compartments from oxidative stress and in strict control of the level of H2O2 in intracellular signalling.
Signal for APX expression
Recently, many researchers have focused on other functional aspects of H2O2 generation. The increases in H2O2 levels are rapid and transient and are thought to constitute a general signal indicating cellular stress (Foyer et al., 1997). H2O2 levels increase in plant cells after exposure to various environmental stress conditions (Okuda et al., 1991; O'Kane et al., 1996; Foyer et al., 1997; Karpinski et al., 1997; Dat et al., 1998; Yoshimura et al., 2000). Plants treated with low concentrations of H2O2 can develop stress tolerance (Prasad et al., 1994; Matsuda et al., 1994). More recent reports demonstrated a link between H2O2 and a MAP kinase pathway in plants, supporting the idea that H2O2 acts as a signal during stress (Desikan et al., 1999; Kovtun et al., 2000; Samuel et al., 2000).
Interesting observations regarding regulation of APX gene expression by H2O2 have been reported in the last few years. Treatment of cultured soybean cells with exogenous H2O2 resulted in the alteration of cAPX transcription levels (Lee et al., 1999). Furthermore, the treatment of cultured rice cells with hydroxyurea, a suicide inhibitor of APX, or aminotriazole, an effective inhibitor of catalase, led to increased cellular H2O2 content and a large increase in the cAPX transcript level (Morita et al., 1999). These results support the idea that cAPX gene expression is up-regulated in response to cellular H2O2 levels. The accumulation of H2O2 via redox signal transduction may play an important role in cAPX gene expression regulation under some stress conditions. In addition to H2O2 accumulation, during high light stress, redox changes in electron transport through plastoquinone may be essential for the induction of APX2, a member of the cAPX gene family, in Arabidopsis leaves (Karpinski et al., 1999; Karpinska et al., 2000).
Upon pathogen attack, AOS including O2-, H2O2, and nitric oxide can help to induce cell death in infected cells or serve as a signal to activate defence responses in distant uninfected cells. cAPX expression in TMV-infected tobacco leaves is suppressed by inhibition of protein synthesis in the polysomes (Mittler et al., 1998). Transgenic antisense tobacco with reduced APX suggests that the suppression of APX plays a key role in elevating cellular H2O2 levels and results in enhanced cell death in response to pathogen attack (Mittler et al., 1999). A recent report that nitric oxide and peroxynitrite derived from nitric oxide inhibit APX and catalase activities strongly supports the possibility that these enzymes contribute to controlling cellular H2O2 levels (Clark et al., 2000). Prolonged accumulation of toxic amounts of H2O2 within plant cells is eventually lethal. It is likely that the regulation of APX expression is part of a system for regulating the balance between the detrimental and beneficial roles of H2O2 in plant cells.
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The gene encoding APX isoenzymes in higher plants |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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The cAPX genes from pea, Arabidopsis, tomato, and strawberry have been isolated and characterized (Mittler and Zilinskas, 1992
; Kubo et al., 1993; Santos et al., 1996; Kim and Chung, 1998; Gadea et al., 1999). The pea cAPX gene (APXI) contains nine introns, the first of which is located within the 5'-untranslated region. Similar observations have also been made in those of other plant species, except for the Arabidopsis APX1b gene encoding a second family of cAPX. The expression of cAPX is regulated in response to several oxidative stress factors, indicating the presence of some functional cis-regulatory elements in the promoter. A putative heat-shock cis-element is present in the cAPX genes from all plant species and is associated with the in vivo induction of the gene (APXI) from Arabidopsis (Storozhenko et al., 1998). The expression of the cAPX gene in rice is mediated by high temperature; furthermore, rice seedlings previously subjected to high temperature show increased tolerance to chilling stress (Sato et al., 2001). A functional G/C-rich element, which is essential for ethylene induction, is found in the APX1 promoter (Storozhenko et al., 1998). Furthermore, the leader intron in the 5'-untranslated region of the tomato cAPX gene is required to confer the constitutive gene expression (Gadea et al., 1999).
The APXII gene, which encodes spinach chlAPX isoenzymes and is present in only one copy, consists of 13 exons split by 12 introns. The intron–exon splice junctions conform to the consensus sequences GT at the donor site and AG at the acceptor site (Brown, 1986). Exons 1–11 encode the common amino acid sequence for sAPX and tAPX isoenzymes. The important point to note regards the two 3'-terminal exons. Two splice acceptor sites occur in exons 12 (penultimate) and 13 (final) separated by 14 bp nucleotides. The penultimate exon 12 consists of one codon for Asp-365 before the TAA termination codon and the entire 3'-untranslated region including a potential polyadenylation signal (AATAAA) of the sAPX mRNA. The final exon 13 contains the corresponding coding sequence of the hydrophobic C-terminal region, the TGA termination codon, and the entire 3'-untranslated region, including a potential polyadenylation signal (AATATA) of the tAPX mRNA. These facts show that the mRNAs for the chlAPX isoenzymes arise from only one gene by alternative use of the two final exons. The same mechanism has also been observed in the cases of pumpkin, M. crystallinum, and tobacco chlAPX isoenzymes, indicating that the alternative splicing of chlAPX isoenzymes is a common regulation mechanism in higher plants (Mano et al., 1997; K Yoshimura and S Shigeoka, unpublished data).
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Generation of chloroplastic APX isoenzymes by alternative mRNA splicing |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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Alternative splicing pattern
By alternative processing of the 3'-terminal region as a result of alternative polyadenylation and splicing, four types of mature mRNA variants, one form encoding tAPX (tAPX-I) and three forms (sAPX-I, sAPX-II, sAPX-III) encoding sAPX, are produced as mature and functional forms, transported from the nucleus to the cytoplasm, and then incorporated into polysomes (Yoshimura et al., 1999
). When the AAUAAA signal in exon 12 is selected, the only resulting product after the splicing of intron 11 should be sAPX-I mRNA. In contrast, the selection of the AAUAUA signal in exon 13 causes more complexity and, as a result, three mRNA variants (sAPX-II, -III, and tAPX-I) are produced by the alternative excision of intron 11 or intron 12 (Fig. 3).
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Alternative splicing is a common mechanism of gene regulation at the post-transcriptional stage in eukaryotic organisms (McKeown, 1992
; Simpson and Filipowicz, 1996). The production of heterogeneity in the amino acid sequences by various regulation systems such as alternative splicing, 3'-end cleavage, and polyadenylation of mRNA precursors is well documented in animal systems. In higher plants, production of heterogeneous C-terminals by alternative splicing which induces an in-frame shift of the open reading frame has been shown for spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco) activase and pumpkin hydroxypyruvate reductase (Werneke et al., 1989; Hayashi et al., 1996).
Regulatory mechanism of alternative splicing of chlAPX isoenzymes
Some alternative splicing events are constitutive, with similar ratios of mRNA variants in different cells, whereas others are subject to tissue-specific or developmental regulation (McKeown, 1992). In higher plants, a number of cases of alternative splicing have been reported, although the biological significance of the isoforms produced is unknown (Simpson and Filipowicz, 1996). It is likely that a few alternative splicing events are regulated by unique exogenous factors specific to plants. In the case of Rubisco activase, although two isoforms produced by alternative splicing can activate Rubisco, only the larger one is redox-regulated (Rundle and Zielinski, 1991). In the case of pumpkin hydroxypyruvate reductase, which produces the microbody and cytosol isoenzymes by alternative splicing, the production of the cytosol type is greatly enhanced by light (Mano et al., 1999).
In the case of chlAPX isoenzymes, it seems likely that the expression ratio of tAPX to sAPX changes according to the plant species (Miyagawa et al., 2000; Yoshimura et al., 2000). However, known cis-elements that might regulate the transcription level have not been found in the promoter of the APXII gene (Ishikawa et al., 1997), and the amount of the chlAPX transcript is not changed in mature spinach leaves under various stress conditions (Yoshimura et al., 2000). Furthermore, even in non-photosynthetic tissues such as root, the chlAPX gene is expressed, and almost equal amounts of chlAPX transcripts are detected in the leaf, stem, and root of mature plants (K Yoshimura and S Shigeoka, unpublished data). However, the expression ratio of sAPX mRNAs to tAPX mRNA variants is extremely elevated in stem and root as a result of an increase in sAPX-III mRNA and a decrease in tAPX-I mRNA (K Yoshimura and S Shigeoka, unpublished data), suggesting that the efficiency of the polyadenylation site selection in the alternative splicing event of the 3'-terminal region in chlAPX pre-mRNA is not modulated in a tissue-specific manner. Therefore, it is likely that the alternative splice-site selection in the alternative splicing event of chlAPX pre-mRNA is a major regulatory mechanism for changing the expression ratio of chlAPX isoenzymes in each tissue, because sAPX-III and tAPX-I mRNAs are produced by the alternative excision of intron 11 or intron 12. In addition to this observation, the patterns of accumulation of sAPX and tAPX isoenzymes in pumpkin cotyledons were different during germination versus subsequent greening, indicating that light regulates the alternative splicing of chloroplastic APX isoenzymes (Mano et al., 1997).
What cis-elements and trans-acting regulators are involved in alternative splicing to determine the level of each mRNA variant for chlAPX isoenzymes? In higher plants, not much is known about the mechanisms underlying alternative splicing events. Judging from the data reported so far, the regulatory factors for alternative splicing in higher plants may also have unique features because the recognition of introns in higher plants differs from that in yeast and vertebrates (Lorkovi'c et al., 2000; Reed, 2000). The 3'-terminal regions of the chlAPX gene involved in the alternative splicing event show high homology; in addition, a putative splicing regulatory cis-element (SRE) in the region immediately upstream of exon 13 is highly conserved among spinach (Ishikawa et al., 1997), pumpkin (Mano et al., 1997), and tobacco (K Yoshimura and S Shigeoka, unpublished data). It is likely that the binding of a splicing-enhancing trans-acting factor to the SRE region in chlAPX pre-mRNA may induce an increase in the splicing efficiency of intron 12, leading to the enhancement of the production of the tAPX-I mRNA. The identification of a trans-acting factor is currently being attempted in the authors’ laboratory.
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Future prospects |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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Photosynthetic organisms including higher plants and eukaryotic algae have developed AOS-scavenging systems, including APX isoenzymes. Cloning of cDNAs and genes encoding APX isoenzymes has facilitated a diverse rage of molecular and physiological studies on these isoenzymes. Prokaryotic cyanobacteria have an AOS-scavenging system as well as an H2O2-tolerance system of the Calvin cycle and a H2O2-diffusion system. APX isoenzymes are expressed by distinct regulatory mechanisms in response to various environmental stresses or cell conditions, and play a co-operative role to protect each organelle and minimize tissue injury. The regulatory mechanism of the expression of chlAPX isoenzymes via alternative splicing will provide new outlooks on plant gene expression because little is known about the mechanisms of post-transcriptional regulation in plant cells (Lorkovi'c et al., 2000
).
At the present stage, there is no definite information on the signal transduction pathway that regulates the expression of APX isoenzymes, although it is clear that increased reduction-state of quinone B or plastoquinone in chloroplasts may be essential for the cAPX induction under high light intensity (Karpinski et al., 1999).
H2O2 functions as a second messenger to regulate the gene expression of some antioxidative enzymes in plant cells, and a transient increase of H2O2 is observed during the early stages under oxidative stress conditions (Foyer et al., 1997). The balance between the detrimental and beneficial roles of H2O2 could be determined by the local concentration of H2O2 related to the function of each organelle and the state of the cellular scavengers, including the levels of various antioxidants such as APX, catalase, AsA, and GSH. The cytosol fraction is a very important cellular compartment for communicating the information from each organelle to the nucleus. Taking into account these facts and the occurrence of two or more putative cAPX variants in plants, it is likely that cAPX variants have a critical function in controlling the H2O2 concentration in signalling, and that their major subcellular localization in the cytosol has strong relevance to gene regulation and thus to the mechanism of tolerance against various oxidative stress factors.
APX isoenzymes, especially chlAPX, are labile in the absence of AsA as the electron donor; therefore, they are inactivated with decreased AsA content under photo-oxidative stress conditions. It is clear that a high level of endogenous AsA is essential effectively to maintain the antioxidant system that protects plants from oxidative damage. Therefore, the biosynthesis, intracellular translocation, subcellular distribution, and the regeneration system of AsA strongly affect the final activity of each APX isoenzyme. At the present time, a major unanswered question is whether the instability of APX has a physiological role in vivo. The fact that the H2O2 level is controlled by cAPX suppression during pathogen infection suggests the possibility that the instability of APX might have functions related to stress signalling. Crystallographic studies of APX isoenzymes will provide new information about the inactivation mechanism in relation to the catalytic mechanisms. In addition to recent molecular analyses of APX isoenzymes described in this review, knowledge about AsA metabolism, including the AsA synthetic pathway and the AsA regeneratory cycle in plant cells, has greatly increased within the past few years (Smirnoff, 2000). The combined results of studies of APX isoenzymes together with advances in knowledge of AsA metabolism will provide a fuller understanding of the physiological function of APX in plant stress responses.
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Notes |
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To whom correspondence should be addressed. Fax: +81 74243 2252. E-mail: shigeoka{at}nara.kindai.ac.jp
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References |
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TopAbstractIntroductionH2O2/hydroperoxide-scavenging...Enzymatic properties of APX...H2O2/hydroperoxide-scavenging...Characterization of cDNAs...Evolution of APX isoenzymesStructural investigations of the...The potential of the...Regulation of APX expressionThe gene encoding APX...Generation of chloroplastic APX...Future prospectsReferences |
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O. Chew, J. Whelan, and A. H. Millar Molecular Definition of the Ascorbate-Glutathione Cycle in Arabidopsis Mitochondria Reveals Dual Targeting of Antioxidant Defenses in Plants J. Biol. Chem., November 21, 2003; 278(47): 46869 - 46877. [Abstract] [Full Text] [PDF]
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F. Nishikawa, M. Kato, H. Hyodo, Y. Ikoma, M. Sugiura, and M. Yano Ascorbate metabolism in harvested broccoli J. Exp. Bot., November 1, 2003; 54(392): 2439 - 2448. [Abstract] [Full Text] [PDF]
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H. H. Ratnayaka, W. T. Molin, and T. M. Sterling Physiological and antioxidant responses of cotton and spurred anoda under interference and mild drought J. Exp. Bot., October 1, 2003; 54(391): 2293 - 2305. [Abstract] [Full Text] [PDF]
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M. A. Matamoros, D. A. Dalton, J. Ramos, M. R. Clemente, M. C. Rubio, and M. Becana Biochemistry and Molecular Biology of Antioxidants in the Rhizobia-Legume Symbiosis Plant Physiology, October 1, 2003; 133(2): 499 - 509. [Full Text] [PDF]
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M. C. de Pinto, P. Lavermicocca, A. Evidente, M. M. Corsaro, S. Lazzaroni, and L. De Gara Exopolysaccharides Produced by Plant Pathogenic Bacteria Affect Ascorbate Metabolism in Nicotiana tabacum Plant Cell Physiol., August 15, 2003; 44(8): 803 - 810. [Abstract] [Full Text] [PDF]
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 K. Wada, T. Tada, Y. Nakamura, T. Ishikawa, Y. Yabuta, K. Yoshimura, S. Shigeoka, and K. Nishimura Crystal Structure of Chloroplastic Ascorbate Peroxidase from Tobacco Plants and Structural Insights into its Instability J. Biochem., August 1, 2003; 134(2): 239 - 244. [Abstract] [Full Text] [PDF]
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A. Mika and S. Luthje Properties of Guaiacol Peroxidase Activities Isolated from Corn Root Plasma Membranes Plant Physiology, July 1, 2003; 132(3): 1489 - 1498. [Abstract] [Full Text] [PDF]
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C. S. Lisenbee, M. Heinze, and R. N. Trelease Peroxisomal Ascorbate Peroxidase Resides within a Subdomain of Rough Endoplasmic Reticulum in Wild-Type Arabidopsis Cells Plant Physiology, June 1, 2003; 132(2): 870 - 882. [Abstract] [Full Text] [PDF]
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J. S. Kim, Y. O. Kim, H. J. Ryu, Y. S. Kwak, J. Y. Lee, and H. Kang Isolation of Stress-Related Genes of Rubber Particles and Latex in Fig Tree (Ficus carica) and their Expressions by Abiotic Stress or Plant Hormone Treatments Plant Cell Physiol., April 15, 2003; 44(4): 412 - 414. [Abstract] [Full Text] [PDF]
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O. V. Karpova, E. V. Kuzmin, T. E. Elthon, and K. J. Newton Differential Expression of Alternative Oxidase Genes in Maize Mitochondrial Mutants PLANT CELL, December 1, 2002; 14(12): 3271 - 3284. [Abstract] [Full Text] [PDF]
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K. Yoshimura, Y. Yabuta, T. Ishikawa, and S. Shigeoka Identification of a cis Element for Tissue-specific Alternative Splicing of Chloroplast Ascorbate Peroxidase Pre-mRNA in Higher Plants J. Biol. Chem., October 18, 2002; 277(43): 40623 - 40632. [Abstract] [Full Text] [PDF]
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