Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: in gel enzyme activity assays

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1 Plant Science 159 (2000) Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: in gel enzyme activity assays Dong Hee Lee, Chin Bum Lee * Department of Biology, Dong-Eui Uni ersity, Pusan , Korea Received 20 January 2000; received in revised form 15 June 2000; accepted 27 June 2000 Abstract To investigate the antioxidant defense system, chilling stress-induced changes of antioxidant enzymes were examined in the leaves of cucumber (Cucumis sati us L.). Chilling stress preferentially enhanced the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. In order to analyze the changes of antioxidant enzyme isoforms against chilling stress, foliar extracts were subjected to native PAGE. Leaves of cucumber had four isoforms of Mn-SOD and two isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in this plant. Expression of Cu/Zn-SOD and Mn-SOD was preferentially enhanced by chilling stress. Expression of Mn-SOD-2 and -4 was enhanced after 48 h of the poststress period. Five APX isoforms were presented in the leaves of cucumber. The intensities of APX-4 and -5 were enhanced by chilling stress, whereas that of APX-3 was significantly increased in the poststress periods after chilling stress. Gel stained for GR activity revealed six isoforms in the plant. Activation levels for most of GR isoforms were higher in the stressed-plants than the control and poststressed-plants, but that of GR-1 isoform was significantly higher in the poststressed-plants than chilling stressed-plants. These results collectively suggest that chilling stress activates the enzymes of an SOD/ascorbate-glutathione cycle under catalase deactivation in the leaves of cucumber, but the response timing of enzyme isoforms against various environmental stresses is not the same for all isoforms of antioxidant enzymes Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chilling stress; Cucumber; H 2 O 2 ; Antioxidant enzymes 1. Introduction Various tolerance mechanisms have been suggested on the basis of the biochemical and physiological changes related to chilling injury [1,2]. Levitt has suggested that a major target of chilling injury is cell membranes [3]. As temperature is reduced, a specific temperature determined by the ratio of saturated to unsaturated fatty acids accelerates the conversion of lipids of a liquid-crystalline condition into that of a solid condition in plant cell membranes [4]. The conversion of fatty acid may give rise to chilling resistance at lower temperatures in the plant cells. * Corresponding author. fax: address: cblee@hyomin.dongeui.ac.kr (C.B. Lee). However some plants, which show a similar fatty acid ratio under chilling conditions, are very sensitive to chilling injury compared to others; thus other mechanisms may also be necessary for chilling injury. In previous studies it has been suggested that oxidative stress induced by chilling stress may play a pivotal role for chilling injury in plant cells [5,6]. The oxidative stress at lower temperatures has been thought to be mediated by active oxygen species composed of superoxide (O 2 ), hydroxyl radicals ( OH), hydrogen peroxide (H 2 O 2 ) and singlet oxygen ( 1 O 2 ) [7]. Active oxygen species act both as cytotoxic compounds and as mediator on the induction of stress tolerance. In order to protect cellular membranes and organelles from the damaging effects of active oxygen species, complex antioxidant sys /00/$ - see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S (00)

2 76 D.H. Lee, C.B. Lee / Plant Science 159 (2000) tems are very important in plants. Antioxidants can be divided into three classes as follows: (1) lipid soluble and membrane-associated tocophenols, (2) water soluble reductants such as ascorbic acid and glutathione, and (3) antioxidant enzymes such as superoxide dismutase (SOD), catalase, peroxidase, ascorbate peroxidase (APX) and glutathione reductase (GR) [8]. SOD is a group of metalloenzymes that catalyze the disproportionation of superoxide to H 2 O 2 and O 2, and plays an important role for protection against superoxidederived oxidative stress in plant cells [9,10]. Detoxification of cellular H 2 O 2 through the activity of the Asada-Halliwell scavenging cycle is an important step in the defense mechanisms against active oxygen species. The cycle found in the chloroplast and cytosol involves the oxidation and re-reduction of ascorbate and glutathione through the activation of enzymes such as APX and GR [11,12]. APX catalyzes the reaction of ascorbic acid with H 2 O 2, and GR catalyzes the regeneration of ascorbic acid [13]. Catalase can also reduce H 2 O 2 to water, but it has a very low affinity for H 2 O 2 as compared with APX [14]. It has been proposed that SOD and APX isoforms are specific to the chloroplast and cytosol [15,16], whereas GR isoforms are specific to the chloroplast, cytosol and mitochondria [17]. In the previous studies, over-expression of Mn- SOD or chloroplastic Cu/Zn-SOD has offered a defense against light-mediated paraquat damage [18] and against light-associated chilling stress in tobacco transformants [19]. It has been also suggested that chilling stress causes the elevation of tissue activation of APX and GR of Arabidopsis [6], whereas it gives rise to the inhibition in the activation of catalase of rice [20]. Although several biochemical and physiological changes on the antioxidant defense system have been shown to be involved in the chilling acclimation process, little is known about the responses of antioxidant enzymes against chilling stress which induces the overproduction of active oxygen species. Analysis of isoforms of antioxidant enzymes regulated during chilling acclimation when coupled with physiological and biochemical analyses will also provide important new insights into chilling tolerance processes. Therefore in order to clarify the protective mechanism of antioxidant enzymes against chilling stress, we describe the changes of H 2 O 2 contents as well as the changes in the activation and induction of antioxidant enzymes in the leaves of cucumber plants subjected to chilling stress. 2. Materials and methods 2.1. Plant material and growth and stress condition Seeds of cucumber (C. sati us L. cv. Pyunggangnaebyungsamchuk) were allowed to germinate on filter paper (Whatmann No. 2) in a petri dish containing distilled water for 5 days under dark condition at 25 C and planted in a pot containing commercial soil, and then grown in a growth chamber for 20 days. The environmental conditions in the growth chamber were 70% humidity, 25 C, and light intensity of 200 mol m 2 s 1 with a 14 h photoperiod. For the exposure to chilling, the 25 day-old plants were transferred to a cold chamber at 4 C under the illumination of continuous light (light intensity of 50 mol m 2 s 1 ) for 12 h, and then the plants were incubated in the growth chamber at 25 C for 2 days. The second leaves of cucumber plants subjected to chilling or poststress were used as the experimental materials. Control is defined as the second leaves of 25 day-old plants without chilling stress or poststress. The leaves were collected at 6 and 12 h of chilling stress and after 4, 8, 12, 24 and 48 h of the poststress period. All of the experiments were repeated at least three times Measurement of H 2 O 2 content For assay of H 2 O 2 content, 1gofleaves was homogenized in 3 ml of 100 mm sodium phosphate buffer (ph 6.8). To remove cellular debris the homogenate was filtered through four layers of cheesecloth and then centrifuged at g for 20 min at 4 C. The supernatant was collected for assay of H 2 O 2 content. Measurement of H 2 O 2 content was performed according to the modified method of Bernt and Bergmeyer [21] using peroxidase enzyme. To initiate the enzyme reaction an aliquot of 0.5 ml of supernatant was mixed with 2.5 ml of peroxide reagent, consisting of 83 mm sodium phosphate, ph 7.0, 0.005% (w/v) o-dianisidine, 40 g peroxidase/ml and incubated for 10 min at 30 C in a waterbath. The reaction was stopped by adding 0.5 ml of 1 N perchloric acid

3 D.H. Lee, C.B. Lee / Plant Science 159 (2000) and centrifuged at 5000 g for 5 min. The resultant supernatant was read at 436 nm and its absorbance was compared to the extinction of an H 2 O 2 standard Preparation of enzyme extracts For determination of antioxidant enzyme activities, leaves (1 g) were homogenized in 100 mm potassium phosphate buffer (ph 7.8) containing 0.1 mm ethylenediamine- tetraacetic acid (EDTA), 1% (w/v) polyvinyl-pyrrolidone (PVP) and 0.5% (v/v) Triton X-100 at 4 C, except that in the case of APX activity leaves were homogenized in 100 mm sodium phosphate buffer (ph 7.0) containing 5 mm ascorbate and 1 mm EDTA. The homogenate was filtered through four layers of cheesecloth and centrifuged at g for 20 min at 4 C. The resultant supernatant was collected for determination of antioxidant enzyme activities, and stored at 80 C for further analyses. Protein content was measured according to the method of Lowry et al. [22] with bovine serum albumin (BSA) as a standard Enzyme assay Catalase activity was determined by monitoring the decomposition of H 2 O 2 (extinction coefficient 39.4 mm cm 1 ) at 240 nm following the method of Aebi [23]. The reaction mixture contained 50 mm potassium phosphate buffer (ph 7.0) and plant extract in a 3 ml volume. The reaction was initiated by adding 10 mm H 2 O 2. One unit of catalase is defined as the amount of enzyme which liberates half the peroxide oxygen from 10 mm H 2 O 2 solution in 100 s at 25 C. Peroxidase activity was determined by monitoring the formation of guaiacol dehydrogenation product (extinction coefficient 6.39 mm cm 1 ) at 436 nm following the method of Pütter [24] ml of reaction mixture contained 100 mm potassium phosphate buffer (ph 7.0), 0.3 mm guaiacol and plant extract. The reaction was initiated by adding 0.1 mm H 2 O 2. One unit of peroxidase specific to guaiacol is defined as the oxidation of mol of guaiacol from 0.3 mm guaiacol and 0.1 mm H 2 O 2 per min at 25 C at ph 7.0. Determination of SOD activity was performed by the method of Beyer and Fridovich [25] ml of the reaction mixture was composed of 50 mm potassium phosphate buffer (ph 7.8), 9.9 mm methionine, 57 M nitroblue tetrazolium (NBT) and the appropriate volume of plant extract. The reaction was initiated by light illumination. One unit of SOD is defined as the amount of enzyme which causes a 50% decrease of the SOD-inhibitable NBT reduction. APX activity was determined by following the decrease of absorbance at 290 nm (extinction coefficient 2.8 mm cm 1 ). The reaction mixture contained 50 mm potassium phosphate buffer (ph 7.0), 0.5 mm ascorbate, 0.2 mm H 2 O 2 and the suitable volume of enzyme extract [26]. GR activity was determined by the oxidation of NADPH at 340 nm (extinction coefficient 6.2 mm cm 1 ) as described by Rao et al., [27]. The reaction mixture was composed of 100 mm potassium phosphate buffer (ph 7.8), 2 mm EDTA, 0.2 mm NADPH, 0.5 mm glutathione (oxidized form, GSSG) and the appropriate volume of enzyme extract in a 1 ml volume. The reaction was initiated by the addition of NADPH at 25 C Acti ity gel analysis Plant extracts containing equal amounts of protein, with the addition of bromophenol blue and glycerol to a final concentration of 12.5%, were subjected to discontinuous PAGE under nondenaturing, nonreducing conditions essentially as described by Laemmli [28], except that SDS was omitted and the gels were supported by 10% glycerol. Electrophoretic separation was performed at 4 C for 4 h with a constant current of 30 ma per gel. For the analysis of APX activity, 2 mm ascorbate was added to the electrode buffer and the gel was pre-run for 30 min before the samples were loaded [29]. SOD activity was detected by following the modified method of Beauchamp and Fridovich [30]. After completion of electrophoresis the gel was incubated in a solution containing 2.45 mm NBT for 25 min, followed by incubation in 50 mm potassium phosphate buffer (ph 7.8) containing 28 M riboflavin and 28 mm tetramethyl ethylene diamine (TEMED) under dark condition. Expression of SOD was achieved by light exposure for min at room temperature. Identification of SOD isoforms was achieved by incubating gels in 50 mm potassium phosphate buffer (ph 7.0) containing 3 mm KCN or 5 mm H 2 O 2 for 30 min

4 78 D.H. Lee, C.B. Lee / Plant Science 159 (2000) before staining for SOD activity. APX activity was detected by the procedure described by Mittler and Zilinskas [29]. The gel equilibrated with 50 mm sodium phosphate buffer (ph 7.0) containing 2 mm ascorbate for 30 min was incubated in a solution composed of 50 mm sodium phosphate (ph 7.0), 4 mm ascorbate and 2 mm H 2 O 2 for 20 min. The gel was washed in the buffer for 1 min and submerged in a solution of 50 mm sodium phosphate buffer (ph 7.8) containing 28 mm TEMED and 2.45 mm NBT for min with gentle agitation. GR activity was detected by incubation of gel in 50 ml of 0.25 M Tris HCl buffer (ph 7.5) containing 10 mg of 3-(4,5-dimethylthiazol-2-4)-2,5- diphenyl tetrazolium bromide, 10 mg of 2,6-dichlorophenolindophenol, 3.4 mm GSSG and 0.5 mm NADPH [27]. 3. Results 3.1. Growth responses The changes of protein content in the leaves of cucumber plants treated with chilling stress are shown in Fig. 1A. A significant increase in the protein content was detected during the period of chilling stress. After 24 h of poststress, protein content reached almost the same value as that in control plants. The significant increase of protein content appeared to be due to the decrease of relative water content of chilling stressed-plants (data not shown). The pattern of H 2 O 2 levels was similar to that of protein contents during chilling stress (Fig. 1A). During the poststress period, however, the level of H 2 O 2 was significantly higher than the level at chilling stress. On the other hand, after 24 h of poststress, the leaves showed visible injury symptoms, such as leaf yellowing, starting at the tip of the leaf. Leaf yellowing was due to the breakdown of chlorophylls (data not shown) Changes in the acti ity of SOD In comparison to the control, chilling stress induced a significant increase of total SOD activity, whereas after 12 h of poststress, the plants reached almost the same activity as control plants did (Fig. 1B). Total SOD activity represents the Fig. 1. Changes in the contents of protein and H 2 O 2 in the leaves of cucumber plants subjected to chilling stress (A). Superoxide dismutase activity in the leaves of cucumber plants subjected to chilling stress (B). One unit of SOD is defined as the amount of enzyme which causes a 50% decrease of the SOD-inhibitable NBT reduction. Data are mean SD (n=3).

5 D.H. Lee, C.B. Lee / Plant Science 159 (2000) Fig. 2. Identification of SOD isoforms in the leaves of cucumber plants. Aliquats of 150 g protein of cucumber leaves were loaded and separated on a nondenaturing polyacrylamide gel. Arrows indicate different isoforms in the leaves of cucumber plants. Staining for activity was performed without any inhibitor (control), in the presence of 3 mm KCN which inhibits Cu/Zn-SOD, or in the presence of 5 mm H 2 O 2 which inhibits both Cu/Zn- and Fe-SOD. combined action of Cu/Zn-, Mn- and Fe-SOD. Using 3 mm KCN to inhibit Cu/Zn-SOD or 5 mm H 2 O 2 to inactivate both Cu/Zn-SOD and Fe-SOD [9], SOD isoforms were identified. As shown in Fig. 2, four isoforms of SOD in the cucumber leaves were identified as Mn-SOD, and the other two isoforms were identified as Cu/Zn-SOD. Fe-SOD isoform was not observed in the native gels. In order to analyze the changes in the expression of SOD isoforms against chilling stress, foliar extracts were subjected to native PAGE (Fig. 3). In control plants, activities of Mn-SOD-2, -3, -4, and Cu/Zn-SOD-2 were little detected in native gel. Chilling stress caused a significant increase in the activation of all SOD isoforms, particularly Cu/Zn-SOD isoforms, whereas the expression of Mn-SOD-2 and -4, particularly Mn-SOD-2, were preferentially enhanced after 48 h of poststress as compared with the control. On the other hand, the pattern in the change of SOD activity after 12 h of poststress was discrepant from that in the change of H 2 O 2 content (Fig. 1A) Changes in the acti ities of catalase and peroxidase Activities of catalase and peroxidase were monitored at 6 and 12 h of chilling stress and after 4, 8, 12, 24 and 48 h of the poststress period (Fig. 4A). The foliar levels of catalase activity were decreased by chilling stress as compared with the control. After slow recovery of enzyme activity Fig. 3. Native gel stained for the activity of SOD of cucumber leaves. Equal amounts of protein (200 g) were loaded on the gel. Lane A, control; lane B, chilling stress for 6 h; lane C, chilling stress for 12 h; lane D, 4hofpoststress; lane E, 8hofpoststress; lane F, 12 h of poststress; lane G, 24 h of poststress; lane H, 48 h of poststress. Arrows indicate the isoforms whose staining intensity was preferentially enhanced by chilling stress. Arrowheads indicate the isoforms whose staining intensity was preferentially enhanced in h of poststress period.

6 80 D.H. Lee, C.B. Lee / Plant Science 159 (2000) Fig. 4. Total activities of catalase and peroxidase specific to guaiacol in the leaves of cucumber plants subjected to chilling stress (A). One unit of catalase is defined as the amount of enzyme which liberates half the peroxide oxygen from 10 mm H 2 O 2 solution in 100 s at 25 C. One unit of peroxidase specific to guaiacol is defined as the oxidation of mol of guaiacol from 0.3 mm guaiacol and 0.1 mm H 2 O 2 per min at 25 C at ph 7.0. Ascorbate peroxidase activity in the leaves of cucumber plants subjected to chilling stress (B). Data are mean SD (n=3). until 8 h of poststress, the activity was gradually decreased. Peroxidases are known to utilize different substrates to metabolize H 2 O 2. When guaiacol was used as a substrate, peroxidase activities were enhanced in chilling stressed-plants as compared with control plants. After 24 h of poststress, the level of catalase activity was significantly higher than the level at chilling stress Changes in the acti ity of APX With catalase deactivation in chilling stressedplants, there is little detailed study on the metabolic role of APX together with other antioxidant enzymes in H 2 O 2 scavenging metabolism. Thus, we examined the changes of APX activity in the leaves of cucumber plant subjected to chilling stress (Fig. 4B). APX activity was enhanced in chilling stressed-plants as compared with control plants. After 24 h of poststress, the level of APX activity was significantly higher than the level at chilling stress. In this experiment, the pattern of changes in the APX activity was very similar to that of changes in the H 2 O 2 content (Fig. 1A). The enzyme activity results shown in Fig. 4B represent total foliar activity and not the activities of individual APX isoforms. To determine whether there were developmental or chilling-mediated differences among individual APX isoforms, APX activity assays were also performed on control and chilling stressed-plants using nondenaturing gels. Five isoforms of APX were visible on the activity gels (Fig. 5). There was no detectable difference in the activity of APX-1 and APX-2 between control and stressed-leaves. Chilling stress was of significant effect in enhancing the activation of APX-4 and APX-5 as compared with the control. On the other hand, the expression of APX-3 isoform was little changed during chilling stress and the expression was significantly increased after 24 h of poststress Changes in the acti ity of GR Although APX plays an important role for the conversion of H 2 O 2 to water, GR is also an essen-

7 D.H. Lee, C.B. Lee / Plant Science 159 (2000) Fig. 5. Native gel stained for the activity of APX of cucumber leaves. Equal amounts of protein (200 g) were loaded on the gel. Lane a, control; lane b, chilling stress for 6 h; lane c, chilling stress for 12 h; lane d, 4hofpoststress; lane e, 8hofpoststress; lane f, 12 h of poststress; lane g, 24 h of poststress; lane h, 48 h of poststress. Large arrows indicate different isoforms in the leaves of cucumber plants. Small arrows indicate the isoforms whose staining intensity was preferentially enhanced by chilling stress. Arrowheads indicate the isoforms whose staining intensity was preferentially enhanced in h of poststress period. tial catalyzer in the conversion of H 2 O 2 in order to maintain the redox state of ascorbate and glutathione [31]. The potential of APX to metabolize H 2 O 2 depends on the redox state of such compounds. Thus, we studied the changes of GR activity in the leaves of cucumber plants subjected to chilling stress (Fig. 6). The foliar levels of GR activity were significantly increased by chilling stress as compared with the control. After the recovery of enzyme activity until 12 h of poststress, the enzyme activity was gradually increased thereafter, but the level of enzyme activity was lower than the level at chilling stress in cucumber leaves. As shown in Fig. 7, six isoforms of GR were visible on the activity gels. Chilling stress was effective in enhancing the activities of almost all GR isoforms. However the expression of GR-1 isoform was little changed during chilling stress whereas it was significantly increased after 48 h of poststress. which was probably attributable in part to the decrease of relative water content of chilling stressed-plants (Fig. 1A). Also, chilling stress caused a marked increase in the level of H 2 O 2.On the other hand, the level of H 2 O 2 after 24 h of poststress was significantly higher than the level at chilling stress. The increase of H 2 O 2 content during the poststress period may be attributed to visible injury symptoms such as leaf sensescence 4. Discussion The application of chilling stress to cucumber plants induced the increase of protein content, Fig. 6. Glutathione reductase activity in the leaves of cucumber plants subjected to chilling stress. Data are mean SD (n=3).

8 82 D.H. Lee, C.B. Lee / Plant Science 159 (2000) Fig. 7. Native gel stained for the activity of GR of cucumber leaves. Equal amounts of protein (200 g) were loaded on the gel. Lane a, control; lane b, chilling stress for 6 h; lane c, chilling stress for 12 h; lane d, 4 h of poststress; lane e, 8 h of poststress; lane f, 12 h of poststress; lane g, 24 h of poststress; lane h, 48 h of poststress. Large arrows indicate different isoforms in the leaves of cucumber plants. Small arrows indicate the isoforms whose staining intensity was preferentially enhanced by chilling stress. Arrowheads indicate the isoforms whose staining intensity was preferentially enhanced in h of poststress period. ing the cell. SOD, which is a key enzyme in the dismutation of superoxide radicals, can be distinguished into three classes according to their metal co-factor binding at the active site: Cu/Zn-, Mn-, or Fe-SOD [25]. Although SODs can easily be classified on the basis of in situ activity staining technique on the native gel, only a few reports have been conducted to study the changes in the relative distribution of SOD isoforms to date [27,34]. In the present study four isoforms of Mn-SOD and two isoforms of Cu/Zn-SOD were observed in the cucumber leaves (Fig. 2). Fe-SOD isoform was not detected in the activity gels. Both UV-B and O 3 - exposure have been shown to preferentially induce Cu/Zn-SOD whereas they have appeared to be of little effect on the activity of Mn-SOD [27]. Also, drought stress has been reported to be dramatically effective in the activity of cytosolic Cu/Zn-SOD [35]. However, in the present study chilling stress caused the enhancement of total SOD activities (Fig. 1B), and appeared to be due to preferential induction of all SOD isoforms, particularly Cu/Zn-SOD isoforms (Fig. 3). Although there were no changes in the total SOD activities after 48 h of poststress as compared with control, an increase in the relative distributions of Mn-SOD-2 and -4 could contribute to the response against leaf senescence. As shown in the results of the expression of SOD isoforms, the induction of different SOD isoforms may be regulated differently upon exposure to various environmental stresses. The proposal agrees with the notion using Nicotiana plumbaginifolia [36]. On the other hand, the fact of the discrepancy between H 2 O 2 content and total SOD activity after 12 h of poststress indicates that overproduction of H 2 O 2 could be due to reduction of superoxide by SOD as well as by ascorbate, thiols, feredoxin, Mn ions and self-dismutation of superoxide [14]. The enhancement of H 2 O 2 levels resulting from chilling stress would be alleviated through the combined activity of catalase and APX. Mizuno et al., [37] have also suggested that an antioxidant defense system induced by chilling stress in potato tubers may result in the combined increase in catalase and APX activities. In the present study, however, the foliar levels of catalase activity were decreased not only during chilling stress but also after 24 h of poststress in the cucumber plant (Fig. 4A). In rice shoot cultures subjected to chillinduced by leaf yellowing which starts after 24 h of poststress. The metabolism of active oxygen species such as H 2 O 2 is dependent on various functionally interrelated antioxidant enzymes such as catalase, peroxidase, SOD, APX and GR. Although chilling stress has been shown to induce one or more antioxidant enzymes [20,32], there has been little detailed study concerning the responses of various antioxidant enzymes in a single species exposed to chilling stress under similar experimental conditions to date. Furthermore, the chain of events interrelated in the induction of specific isoforms in antioxidant enzyme systems against chilling stress is not understood though Edwards et al., [33] have proposed that plants can synthesize new isoforms of antioxidant enzymes with altered kinetic properties. Hence, the responses of antioxidant enzymes against chilling stress induced the generation of active oxygen species were investigated in the cucumber plants in detail. The metal ions present in the cell such as Fe +++ and Cu ++ reduced by superoxide radicals can interact with H 2 O 2 to form highly reactive hydroxyl radicals that are thought to be primarily responsible for oxygen toxicity in the plant cells. Thus, the dismutation of superoxide radicals into H 2 O 2 and oxygen is an important step in protect-

9 D.H. Lee, C.B. Lee / Plant Science 159 (2000) ing stress, a marked decline in catalase activity has also been reported [20] and this has also been observed in the chilled cucumber seedlings [38]. On the other hand, peroxidases composed of multiple isozymes require H 2 O 2 as an essential substrate. When guaiacol was used as a substrate, the foliar levels of peroxidase activity were markedly increased not only during chilling stress but also after 24 h of poststress in the cucumber leaves (Fig. 4A). Otter and Polle, [39] have suggested that anionic peroxidases known to utilize phenolic compounds as a substrate play a central role for the synthesis of secondary metabolites such as lignin. Therefore, further studies are necessary to clarify the role of peroxidase specific to coniferyl alcohol on the lignification of the chilled plants. APX is also an important antioxidant enzyme in scavenging or utilizing H 2 O 2. Unlike catalase activity, our results indicated that chilling stress caused the enhancement of total APX activities, and appeared to be due to preferential induction of APX-4 and APX-5 isozymes, whereas the increase of total APX activity after 24 h of poststress was due to the preferential expression of APX-3 isoform (Fig. 4B and Fig. 5). This has also been investigated in Arabidopsis leaves exposed to aminotriazole [40]. Induction of APX isoforms may have an even more dramatic effect on the protection of plants against chilling stress as compared with catalase, because H 2 O 2 generated at the intercellular space of the plant during environmental stress appears to diffuse first into the cytosol in which cytosolic APX is localized and only then into peroxysome in which catalase is typically found, and because cytosolic APX has a higher affinity for H 2 O 2 than catalase does [41]. And as argued for SOD isoforms, changes in the relative distribution of APX isoforms could contribute to stress tolerance or response in the chilling stressed- or poststressed-plants. In this experiment, the pattern of changes in the APX activity was parallel to that of changes in the H 2 O 2 content (Fig. 1A and Fig. 4B). These results suggest that cytosolic APX in the cucumber leaves may be a key enzyme for the decomposition of hydrogen peroxide under catalase deactivation due to chilling stress. GR is known to act in conjunction with APX to metabolize H 2 O 2 to water through an ascorbate-glutathione cycle. GR activity was significantly enhanced by chilling stress as compared with the control. After slow recovery of enzyme activity during 4 12 h of poststress period, the activity was increased again, but the level of enzyme activity was lower than the level in chilling stressed-plants (Fig. 6). The enhancement of total GR activity against chilling stress could be induced by the preferential induction of almost all isoforms, particularly the synthesis of new isoforms such as GR-4, -5 and -6. On the other hand, after 48 h of poststress, the increase could be induced by the preferential induction of GR-1 isoform (Fig. 7). Edwards et al. [33] have also suggested that the increase of total GR activity in cold-stressed peas appears to be due to changes in the isoform population and these changes in the isoform population also exist in Arabidopsis thaliana [42]. In summary, the findings in the present study suggest that high cellular levels of H 2 O 2 can induce the activation of a defense mechanism against chilling stress or programmed cell death such as leaf yellowing. The accumulation of H 2 O 2 can be induced by the increase of total SOD activity or alterations in the relative distributions of SOD isoforms. And the newly accumulated H 2 O 2, in turn, may trigger a protective mechanism that increases the activity of several enzymes such as peroxidase, APX and GR or induces alterations in the relative distributions of several enzyme isoforms under catalase deactivation. However, it is not clear whether the responses of an antioxidant enzyme system against excess H 2 O 2 levels induced by leaf yellowing may offer a tolerance or cytotoxicity. Therefore, further studies are necessary to investigate antioxidant enzyme systems on leaf senescence. These results on the chilling stress and leaf yellowing suggest that the response timing of enzyme isoforms against various environmental stresses on the antioxidant enzyme system may not be the same for all isoforms of antioxidant enzymes. The response timings of antioxidant enzyme isoforms to different stresses are little known and merit further study. Acknowledgements This work was supported by grant No from the Interdisciplinary Research Program of the KOSEF and partially supported by grant No from KOSEF.

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