Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol159.Issue1.Oct2000:

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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 Uni6ersity,Pusan614-714,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 sati6usL.). 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. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Chilling stress; Cucumber; H2O2; Antioxidant enzymes

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1. Introduction

Various tolerance mechanisms have been sug-gested on the basis of the biochemical and physio-logical 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 accel-erates the conversion of lipids of a liquid-crys-talline 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.

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 (O2−),

hydroxyl radicals ( · OH), hydrogen peroxide

(H2O2) and singlet oxygen (1O2) [7].

Active oxygen species act both as cytotoxic compounds and as mediator on the induction of stress tolerance. In order to protect cellular mem-branes and organelles from the damaging effects of active oxygen species, complex antioxidant

sys-* Corresponding author. fax: +00-82-518901521. E-mail address:[email protected] (C.B. Lee).

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tems are very important in plants. Antioxidants can be divided into three classes as follows: (1) lipid soluble and membrane-associated tocophe-nols, (2) water soluble reductants such as ascorbic acid and glutathione, and (3) antioxidant enzymes such as superoxide dismutase (SOD), catalase, per-oxidase, ascorbate peroxidase (APX) and glu-tathione reductase (GR) [8]. SOD is a group of metalloenzymes that catalyze the

disproportiona-tion of superoxide to H2O2 and O2, and plays an

important role for protection against superoxide-derived oxidative stress in plant cells [9,10].

Detox-ification of cellular H2O2 through the activity of

the Asada-Halliwell scavenging cycle is an impor-tant step in the defense mechanisms against active oxygen species. The cycle found in the chloroplast and cytosol involves the oxidation and re-reduc-tion of ascorbate and glutathione through the activation of enzymes such as APX and GR [11,12]. APX catalyzes the reaction of ascorbic

acid with H2O2, and GR catalyzes the

regenera-tion of ascorbic acid [13]. Catalase can also reduce

H2O2 to water, but it has a very low affinity for

H2O2 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 sug-gested 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 an-tioxidant defense system have been shown to be involved in the chilling acclimation process, little is known about the responses of antioxidant en-zymes against chilling stress which induces the overproduction of active oxygen species. Analysis of isoforms of antioxidant enzymes regulated dur-ing chilldur-ing acclimation when coupled with physio-logical and biochemical analyses will also provide important new insights into chilling tolerance pro-cesses. Therefore in order to clarify the protective mechanism of antioxidant enzymes against chilling

stress, we describe the changes of H2O2contents 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.sati6usL. cv.

Pyunggang-naebyungsamchuk) 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 condi-tions in the growth chamber were 70% humidity,

25°C, and light intensity of 200 mmol 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 mmol 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.

2.2. Measurement of H2O2 content

For assay of H2O2 content, 1 g of leaves was

homogenized in 3 mL of 100 mM sodium phos-phate buffer (pH 6.8). To remove cellular debris the homogenate was filtered through four layers of

cheesecloth and then centrifuged at 18 000g for 20

min at 4°C. The supernatant was collected for

assay of H2O2 content. Measurement of H2O2

content was performed according to the modified method of Bernt and Bergmeyer [21] using perox-idase 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-diani-sidine, 40 mg 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

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and centrifuged at 5000g for 5 min. The resultant supernatant was read at 436 nm and its ab-sorbance was compared to the extinction of an

H2O2 standard.

2.3. Preparation of enzyme extracts

For determination of antioxidant enzyme activi-ties, 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 ho-mogenate was filtered through four layers of

cheesecloth and centrifuged at 18 000 gfor 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.

2.4. Enzyme assay

Catalase activity was determined by monitoring

the decomposition of H2O2 (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 H2O2. One unit of

catalase is defined as the amount of enzyme which liberates half the peroxide oxygen from 10 mM

H2O2solution in 100 s at 25°C. Peroxidase activity

was determined by monitoring the formation of guaiacol dehydrogenation product (extinction

co-efficient 6.39 mM cm−1_{) at 436 nm following the}

method of Pu¨tter [24]. 3.18 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 H2O2.

One unit of peroxidase specific to guaiacol is

defined as the oxidation ofmmol of guaiacol from

0.3 mM guaiacol and 0.1 mM H2O2 per min at

25°C at pH 7.0.

Determination of SOD activity was performed by the method of Beyer and Fridovich [25]. 30.25 mL of the reaction mixture was composed of 50

mM potassium phosphate buffer (pH 7.8), 9.9 mM

methionine, 57 mM 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-in-hibitable NBT reduction. APX activity was deter-mined 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 H2O2 and the suitable volume of enzyme

extract [26]. GR activity was determined by the oxidation of NADPH at 340 nm (extinction

coeffi-cient 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 vol-ume of enzyme extract in a 1 mL volvol-ume. The reaction was initiated by the addition of NADPH at 25°C.

2.5. Acti6ity 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 non-denaturing, nonreducing conditions essentially as described by Laemmli [28], except that SDS was omitted and the gels were supported by 10% glyc-erol. 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)

contain-ing 28 mM riboflavin and 28 mM tetramethyl

ethylene diamine (TEMED) under dark condition. Expression of SOD was achieved by light exposure for 10 – 20 min at room temperature. Identification of SOD isoforms was achieved by incubating gels in 50 mM potassium phosphate buffer (pH 7.0)

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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 H2O2 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 10 – 20 min with gentle agitation. GR activity was detected by incu-bation of gel in 50 mL of 0.25 M Tris – HCl buffer (pH 7.5) containing 10 mg of 3-(4,5-dimethylthia-zol-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 H2O2 levels was

similar to that of protein contents during chilling stress (Fig. 1A). During the poststress period,

however, the level of H2O2was 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). 3.2. Changes in the acti6ity of SOD

In comparison to the control, chilling stress induced a significant increase of total SOD activ-ity, 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 H2O2in 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 9SD (n=3).

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Fig. 2. Identification of SOD isoforms in the leaves of cucum-ber plants. Aliquats of 150 mg protein of cucumber leaves were loaded and separated on a nondenaturing polyacry-lamide 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 H2O2which inhibits both Cu/Zn- and Fe-SOD.

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 H2O2

content (Fig. 1A).

3.3. Changes in the acti6ities 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

combined action of Cu/Zn-, Mn- and Fe-SOD.

Using 3 mM KCN to inhibit Cu/Zn-SOD or 5

mM H2O2 to inactivate both Cu/Zn-SOD and

Fig. 3. Native gel stained for the activity of SOD of cucumber leaves. Equal amounts of protein (200mg) 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. Arrows indicate the isoforms whose staining intensity was preferentially enhanced by chilling stress. Arrowheads indicate the isoforms whose staining intensity was preferen-tially enhanced in 24 – 48 h of poststress period.

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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 H2O2solution in 100 s at 25°C. One unit of peroxidase specific to guaiacol is defined as the oxidation ofmmol of guaiacol from 0.3 mM guaiacol and 0.1 mM H2O2per 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 9SD (n=3).

until 8 h of poststress, the activity was gradually decreased. Peroxidases are known to utilize

differ-ent substrates to metabolize H2O2. 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.

3.4. Changes in the acti6ity of APX

With catalase deactivation in chilling stressed-plants, there is little detailed study on the metabolic role of APX together with other

antiox-idant enzymes in H2O2 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 H2O2content (Fig. 1A). The

enzyme activity results shown in Fig. 4B represent total foliar activity and not the activities of indi-vidual APX isoforms. To determine whether there were developmental or chilling-mediated differ-ences among individual APX isoforms, APX activ-ity 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 signifi-cant 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 expres-sion was significantly increased after 24 h of poststress.

3.5. Changes in the acti6ity of GR

Although APX plays an important role for the

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essen-Fig. 5. Native gel stained for the activity of APX of cucumber leaves. Equal amounts of protein (200mg) 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 24 – 48 h of poststress period.

tial catalyzer in the conversion of H2O2in order to

maintain the redox state of ascorbate and glu-tathione [31]. The potential of APX to metabolize

H2O2 depends on the redox state of such

com-pounds. 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 post-stress, 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.

4. Discussion

The application of chilling stress to cucumber plants induced the increase of protein content,

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 H2O2. On

the other hand, the level of H2O2 after 24 h of

poststress was significantly higher than the level at

chilling stress. The increase of H2O2 content

dur-ing the poststress period may be attributed to visible injury symptoms such as leaf sensescence

Fig. 6. Glutathione reductase activity in the leaves of cucum-ber plants subjected to chilling stress. Data are mean 9SD (n=3).

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Fig. 7. Native gel stained for the activity of GR of cucumber leaves. Equal amounts of protein (200mg) 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 preferen-tially enhanced by chilling stress. Arrowheads indicate the isoforms whose staining intensity was preferentially enhanced in 24 – 48 h of poststress period.

ing the cell. SOD, which is a key enzyme in the dismutation of superoxide radicals, can be distin-guished 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 O3- exposure have been shown to

pref-erentially 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 re-ported to be dramatically effective in the activity

of cytosolic Cu/Zn-SOD [35]. However, in the

present study chilling stress caused the enhance-ment of total SOD activities (Fig. 1B), and ap-peared 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 con-tribute 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 plumb-aginifolia [36]. On the other hand, the fact of the

discrepancy between H2O2 content and total SOD

activity after 12 h of poststress indicates that

overproduction of H2O2could 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 H2O2 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 chill-induced by leaf yellowing which starts after 24 h

of poststress. The metabolism of active oxygen

species such as H2O2 is dependent on various

functionally interrelated antioxidant enzymes such as catalase, peroxidase, SOD, APX and GR. Al-though chilling stress has been shown to induce one or more antioxidant enzymes [20,32], there has been little detailed study concerning the re-sponses 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 re-sponses 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 H2O2 to form highly reactive

hy-droxyl radicals that are thought to be primarily responsible for oxygen toxicity in the plant cells. Thus, the dismutation of superoxide radicals into

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protect-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

mul-tiple isozymes require H2O2 as an essential

sub-strate. 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 H2O2. Unlike catalase

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

com-pared with catalase, because H2O2 generated at

the intercellular space of the plant during environ-mental stress appears to diffuse first into the cyto-sol in which cytocyto-solic APX is localized and only then into peroxysome in which catalase is typi-cally found, and because cytosolic APX has a

higher affinity for H2O2 than catalase does [41].

And as argued for SOD isoforms, changes in the relative distribution of APX isoforms could con-tribute to stress tolerance or response in the

chill-ing stressed- or poststressed-plants. In this

experiment, the pattern of changes in the APX activity was parallel to that of changes in the

H2O2content (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 H2O2 to water through an

ascor-bate-glutathione cycle. GR activity was signifi-cantly 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 en-zyme activity was lower than the level in chilling stressed-plants (Fig. 6). The enhancement of total GR activity against chilling stress could be in-duced by the preferential induction of almost all isoforms, particularly the synthesis of new iso-forms 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 H2O2 can

in-duce the activation of a defense mechanism against chilling stress or programmed cell death

such as leaf yellowing. The accumulation of H2O2

can be induced by the increase of total SOD activity or alterations in the relative distributions of SOD isoforms. And the newly accumulated

H2O2, in turn, may trigger a protective mechanism

that increases the activity of several enzymes such as peroxidase, APX and GR or induces alter-ations in the relative distributions of several

en-zyme isoforms under catalase deactivation.

However, it is not clear whether the responses of

an antioxidant enzyme system against excess H2O2

levels induced by leaf yellowing may offer a toler-ance or cytotoxicity. Therefore, further studies are necessary to investigate antioxidant enzyme sys-tems on leaf senescence. These results on the chill-ing stress and leaf yellowchill-ing suggest that the response timing of enzyme isoforms against vari-ous environmental stresses on the antioxidant en-zyme 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. 1999-2-203-001-3 from the Interdisciplinary Research Program of the KOSEF and partially supported by grant No. 961-0507-055-2 from KOSEF.

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in 100 s at 25°C. One unit of peroxidase specific to guaiacol is defined as the oxidation ofmmol of guaiacol from 0.3 mM guaiacol

and 0.1 mM H2O2per 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 9SD (n=3).

until 8 h of poststress, the activity was gradually decreased. Peroxidases are known to utilize differ-ent substrates to metabolize H2O2. When guaiacol

3.4. Changes in the acti6ity of APX

With catalase deactivation in chilling stressed-plants, there is little detailed study on the metabolic role of APX together with other antiox-idant enzymes in H2O2 scavenging metabolism.

that of changes in the H2O2content (Fig. 1A). The

3.5. Changes in the acti6ity of GR

Although APX plays an important role for the conversion of H2O2to water, GR is also an

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essen-Fig. 5. Native gel stained for the activity of APX of cucumber leaves. Equal amounts of protein (200mg) 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 24 – 48 h of poststress period.

tial catalyzer in the conversion of H2O2in order to

maintain the redox state of ascorbate and glu-tathione [31]. The potential of APX to metabolize H2O2 depends on the redox state of such

4. Discussion

The application of chilling stress to cucumber plants induced the increase of protein content,

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 H2O2. On

the other hand, the level of H2O2 after 24 h of

poststress was significantly higher than the level at chilling stress. The increase of H2O2 content

dur-ing the poststress period may be attributed to visible injury symptoms such as leaf sensescence

Fig. 6. Glutathione reductase activity in the leaves of cucum-ber plants subjected to chilling stress. Data are mean 9SD (n=3).

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D.H.Lee,C.B.Lee/Plant Science159 (2000) 75 – 85 82

Fig. 7. Native gel stained for the activity of GR of cucumber leaves. Equal amounts of protein (200mg) 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 preferen-tially enhanced by chilling stress. Arrowheads indicate the isoforms whose staining intensity was preferentially enhanced in 24 – 48 h of poststress period.

ing the cell. SOD, which is a key enzyme in the dismutation of superoxide radicals, can be distin-guished 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 O3- exposure have been shown to

pref-erentially 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 re-ported to be dramatically effective in the activity of cytosolic Cu/Zn-SOD [35]. However, in the present study chilling stress caused the enhance-ment of total SOD activities (Fig. 1B), and ap-peared 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 con-tribute 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 plumb-aginifolia [36]. On the other hand, the fact of the discrepancy between H2O2 content and total SOD

activity after 12 h of poststress indicates that overproduction of H2O2could 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 H2O2 levels resulting from

of poststress. The metabolism of active oxygen species such as H2O2 is dependent on various

The metal ions present in the cell such as Fe+++

and Cu++ _{reduced by superoxide radicals can}

interact with H2O2 to form highly reactive

hy-droxyl radicals that are thought to be primarily responsible for oxygen toxicity in the plant cells. Thus, the dismutation of superoxide radicals into H2O2and oxygen is an important step in

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APX is also an important antioxidant enzyme in scavenging or utilizing H2O2. 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 in-crease of total APX activity after 24 h of post-stress 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 com-pared with catalase, because H2O2 generated at

And as argued for SOD isoforms, changes in the relative distribution of APX isoforms could con-tribute to stress tolerance or response in the chill-ing stressed- or poststressed-plants. In this experiment, the pattern of changes in the APX activity was parallel to that of changes in the H2O2content (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 H2O2 to water through an

ascor-bate-glutathione cycle. GR activity was signifi-cantly enhanced by chilling stress as compared

In summary, the findings in the present study suggest that high cellular levels of H2O2 can

in-duce the activation of a defense mechanism against chilling stress or programmed cell death such as leaf yellowing. The accumulation of H2O2

can be induced by the increase of total SOD activity or alterations in the relative distributions of SOD isoforms. And the newly accumulated H2O2, in turn, may trigger a protective mechanism

that increases the activity of several enzymes such as peroxidase, APX and GR or induces alter-ations in the relative distributions of several en-zyme isoforms under catalase deactivation. However, it is not clear whether the responses of an antioxidant enzyme system against excess H2O2

Acknowledgements

This work was supported by grant No. 1999-2-203-001-3 from the Interdisciplinary Research Program of the KOSEF and partially supported by grant No. 961-0507-055-2 from KOSEF.

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