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 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 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.
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 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 9 SD n = 3.
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 CuZn-SOD, or in the presence of 5 mM H
2
O
2
which inhibits both CuZn- 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
CuZn-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 CuZn-SOD-2 were little
detected in native gel. Chilling stress caused a significant increase in the activation of all SOD
isoforms,
particularly CuZn-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.
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 CuZn-, Mn- and Fe-SOD. Using 3 mM KCN to inhibit CuZn-SOD or 5
mM H
2
O
2
to inactivate both CuZn-SOD and
Fig. 3. Native gel stained for the activity of SOD of cucumber leaves. Equal amounts of protein 200 mg 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.
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 mmol 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 9 SD n = 3.
until 8 h of poststress, the activity was gradually decreased. Peroxidases are known to utilize differ-
ent 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.
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 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 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
conversion of H
2
O
2
to water, GR is also an essen-
Fig. 5. Native gel stained for the activity of APX of cucumber leaves. Equal amounts of protein 200 mg 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 H
2
O
2
in order to maintain the redox state of ascorbate and glu-
tathione [31]. The potential of APX to metabolize H
2
O
2
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
