Plant Science 159 2000 75 – 85
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, Pusan
614
-
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 sati6us 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 CuZn-SOD. Fe-SOD isoform was not observed in this plant. Expression of CuZn-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 SODascorbate-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; H
2
O
2
; Antioxidant enzymes www.elsevier.comlocateplantsci
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 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 mem-
branes and organelles from the damaging effects of active oxygen species, complex antioxidant sys-
Corresponding author. fax: + 00-82-518901521. E-mail address
:
cbleehyomin.dongeui.ac.kr C.B. Lee. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0168-94520000326-5
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 H
2
O
2
and O
2
, and plays an important role for protection against superoxide-
derived oxidative stress in plant cells [9,10]. Detox- ification of cellular H
2
O
2
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 H
2
O
2
, and GR catalyzes the regenera- tion 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 CuZn-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 chilling 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 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
