Botanical Studies (2006) 47: 129-136.

*

Corresponding author: E-mail: yangjiading@yahoo.com

Presoaking with nitric oxide donor SNP alleviates heat

shock damages in mung bean leaf discs

Jia-Ding YANG*, Jian-Ying YUN, Tong-Hui ZHANG, and Ha-Lin ZHAO

Department of Ecology and Agriculture, Cold and Arid Regions Environmental and Engineering Research Institute,

Chinese Academy of Sciences, Lanzhou 730000, P.R. China

(Received July 25, 2005; Accepted December 5, 2005)

ABSTRACT.

The objective of this study was to examine whether exogenously applied nitric oxide (NO)

have some protective role on mung bean (Phaseolus radiatus) leaf discs under heat shock. Leaf discs were

presoaked in distilled water and sodium nitroprusside (SNP, a NO donor) solution (150 £gM) for 60 min re -

spectively, then submitted to a heat shock at 45¢XC for 90 min in dark. Control materials were presoaked with

distilled water and laid under room temperature (25¢XC). The chlorophyll a fluorescence parameters, membrane

integrity, hydrogen peroxide (H

2

O

2

) content and activities of antioxidant enzymes [catalase (CAT), guaiacol

peroxidase (POD) and superoxide dismutase (SOD)] were assayed. Compared with heat-shocked leaf discs,

the maximal quantum yield of photosystem II (PSII) (measured as F

v

/F

m

) was significantly increased, electro-

lyte leakage due to heat shock was reduced by 48%, lipid peroxidation and H

2

O

2

content were kept at control

level by SNP presoaking. The suppressed activities of antioxidant enzymes by heat shock were all recuper-

ated by SNP presoaking. On the other hand, these role of SNP presoaking were reversed fully or partially by

bovine hemoglobin (a powerful NO scavenger), suggesting that protective effect by SNP is attributable to

released NO.

In conclusion, it appears that the exogenously applied NO donor SNP can promote higher leaf

photochemical activity and cell membrane integrity in mung bean leaf discs under heat shock. This role is pu-

tatively due to that the released NO can recuperate suppressed activities of anti-oxidant enzymes, thus elimi-

nating oxidative damage under heat shock stress.

Keywords: Antioxidant enzyme activity; Heat shock; Membrane integrity; Mung bean (Phaseolus radiatus);

Nitric oxide; Photochemical efficiency.

INTRODUCTION

Heat stress can influence many physiological processes

or factors in plants, e.g., inhibition of photosynthesis, limi-

tation of carbohydrate accumulation and destruction of

cell membranes and cytoskeleton (Liu and Huang, 2000).

Usually, the primary site of damage associated with non-

optimal temperatures was indicated to be the photosynthet-

ic apparatus (Yamane et al., 1998; Bukhov et al., 1999),

and PSII (the H

2

O-oxidising, quinine-reducing complex)

was the most heat sensitive of the chloroplast thylakoid-

membrane protein complexes involved in photosynthetic

electron transfer and ATP synthesis (Heckathorn et al.,

1998). The adverse effects of heat stress may be related

to the overproduction of reactive oxygen species (ROS)

(i.e. superoxide anion [

¡E

O

2¡V

], hydrogen peroxide [H

2

O

2

],

hydroxyl radical [

¡E

OH] and singlet oxygen [

1

O

2

]) (Pastori

and Foyer, 2002).

Plants have well-developed enzymatic and nonenzy-

matic scavenging systems to quench ROS (Vranova et

al., 2002). So the ROS is always generated at a controlled

balance under unstressed conditions. When plants are sub-

jected to adverse conditions, the scavenging system may

lose its function and the balance between producing and

quenching ROS can be disturbed, resulting in accumula-

tion of ROS, which in general, cause lipid peroxidation,

protein modification, breakage of DNA strands, chloro-

phyll decay, ion leakage and cell death (Scandalios, 1993).

Nitric oxide (NO) is a bioactive molecule involved in

many biological events, and its effects have been reported

either protective or toxic in plants (Beligni and Lamat-

tina, 2001). It can act as a signal molecule in plant defense

interactions with microorganisms (Dangl, 1998); or as a

compound with hormonal properties (Leshem and Hara-

maty, 1996) to affect photomorphogenesis (Beligni and

Lamattina, 2000), and to play a central role in determining

lateral root development (Correa-Aragunde et al., 2004).

At the same time, NO may act as an antioxidant to reduce

toxicity induced by herbicide paraquat, heavy metal or

H

2

O

2

and to delay the senescence of rice leaf induced

BIOCHEMISTRY

130

Botanical Studies, Vol. 47, 2006

by methyl jasmonate or abscisic acid (Hung et al., 2002;

Hung and Kao, 2003, 2004, 2005; Hsu and Kao, 2004;

Laspina et al., 2005). However, mechanical stresses, such

as centrifugation, induced Arabidopsis to produce NO,

which further cause DNA fragmentation (Garces et al.,

2001).

The exogenous application of NO donors conferred an

increased tolerance to severe drought stress conditions

in plants, with higher water retention, less transpiration

rate, higher proportion of stomatal closure and so (Garcia

Mata and Lamattina, 2001). Uchida et al. (2002) reported

that NO can increase both salt and heat tolerance in rice

seedlings by sodium nitroprusside (SNP, 1¡V10 £gM) in

the hydroponic solution for 2 days. Considering that the

temperature of an individual plant cell can change in the

time range of a few minutes to hours (Pastenes and Hor-

ton, 1996), being much more rapid than other factors that

cause stress (e.g. water levels or salt levels) (Larkindale

and Knight, 2002), this study aimed to further explore the

protective role of exogenously applied NO in plant tissues

submitted to a rapid heat stress.

MATERIALS AND METHODS

Plant material and experimental design

Seeds of mung bean (Phaseolus radiatus) were ger-

minated on moist paper towels and transferred in plastic

pots containing a sterile mixture of soil: vermiculite (3:1,

v/v). Plants were grown in a greenhouse exposed to direct

full sunlight, watered daily and fertilized with a complete

nutrient medium every three days. The fully-expanded

healthy leaflets (on different compound leaves) were ex-

cised during mid-morning (about two hours after sunrise),

midribs removed and cut into discs with a diameter of 1.5

cm. Leaf discs were immersed in small beakers containing

distilled water, or 150 £gM SNP (an NO donor), or SNP

(150 £gM) plus bovine hemoglobin [4 g L

-1

, a powerful NO

scavenger (Takahashi and Yamasaki, 2002)]. After submit-

ted to a pulse of 45 s of vacuum, the beakers were left 1

h in a plant incubator with a temperature of 25¢XC and a

photosynthetic photon flux density (PPFD) of about 20

£gmol m

-2

s

-1

. Referred to the method of rapid heat stress

(Law and Crafts-Brandner, 1999), presoaked leaf discs

were put on moist filter papers in Petri dishes and laid in

a plant incubator at a prearranged temperature 45¢XC with

no light illumination for 90 min. One Petri dish containing

water-pretreated leaf discs was left at 25¢XC in dark as the

control. So there were four treatments in the present ex-

periment, i.e. 25¢XC with water presoaking (Control), 45¢XC

with water presoaking (H), 45¢XC with SNP presoaking (S),

45¢XC with SNP and Hb presoaking (S+Hb).

Chlorophyll fluorescence measurement

The initial fluorescence (F

o

), maximal fluorescence

(F

m

) in leaf discs after treatments were determined using a

portable fluorometer (Handy PEA, Hansatech Instruments

Ltd., UK) in the dark room. The saturating PPFD of the

actinic light was 3000 £gmol m

-2

s

-1

(0.8 s). The ratio of F

v

/

F

m

, where the variable fluorescence yield, F

v

, is defined as

(F

m

-F

o

), is a direct measure of the maximal quantum yield

of PSII (Meyer and Santarius, 1998).

Electrolyte leakage and TBARS contents

Cell membrane permeability was measured via elec-

trolyte leakage (Meyer and Santarius, 1998). Treated leaf

discs were washed in deionised water to remove surface

ions and fully immersed in 50-ml-glass bottles containing

25 ml of distilled water of known conductivity. The bottles

were capped and shaken in plant incubator at 25¢XC with-

out light illumination for 12 h. Then the conductance was

measured using an Multiline P4 Universal Meter (WTW,

Weilheim, Germany). All electrolyte leakage data were

expressed as conductivity readings related to the material

mass after treatments, i.e., unit was £gS cm

-1

g

-1

.

The extent of lipid peroxidation in leaf discs was deter-

mined in terms of thiobarbituric acid-reacting substances

(TBARS) content by the method of Fryer et al. (1998).

H

2

O

2

Content

According to the method of Freguson et al. (1983) with

some modification, treated leaf discs (about fresh weight

1.5 g) were homogenized quickly in 3 ml of cold acetone

with a mortar and pestle in an ice bath, centrifuged at

5,000 g for 10 min at 4¢XC. The supernatant (0.2 ml) was

complement to 1.0 ml with 0.8 ml cold acetone, and fully

mixed with 0.1 ml of 20 % TiCl

4

-HCl solution and 0.2 ml

strong ammonia hydroxide, centrifuged at 3,000 g for 10

min. The resulted peroxide-Ti compound was washed with

acetone for 3~5 times and dissolved in 3 ml 2 M H

2

SO

4

and the absorbance was measured at 410 nm. The content

o f H

2

O

2

(£gmol g

-1

FW) was calculated by comparison

with a standard curve relating H

2

O

2

concentrations to

absorbance.

Enzymes activities

Treated leaf discs (about 0.3~0.4 g) were homogenized

in 2.5 ml cold extraction buffer [50 mM phosphate buffer

(pH 7.0), containing 1% (m/v) polyvinylpyrrolidone] with

a mortar and pestle in an ice bath, centrifuged at 15000

g for 20 min at 4¢XC. The supernatant was used for assays

of catalase (CAT; EC 1.11.1.6) and guaiacol peroxidase

(POD; EC 1.11.1.7) activity. The same process was con-

ducted for extracting superoxide dismutase (SOD; EC

1.15.1.1) except that the extraction buffer was 50 mM

phosphate buffer (pH 7.8) [containing 2% (m/v) poly-

vinylpyrrolidone, 0.3% (v/v) triton X-100, and 0.1 mM

EDTA].

Activities of CAT and POD were measured according

to Liu and Huang (2000) with some modification. The

CAT reaction solution (3 ml) contained 50 mM phosphate

buffer (pH 7.0), 20 mM H

2

O

2

, and 0.05 ~ 0.1 ml enzyme

extract. Reaction was initiated by adding enzyme extract.

The absorbance change at 240 nm per min was recorded

and calculated automatically. The POD reaction solution

YANG et al. ¡X Presoaking with nitric oxide donor SNP

131

(3 ml) contained 100 mM phosphate buffer (pH 6.0), 20

mM guaiacol, 40 mM H

2

O

2

, and 0.1 ml enzyme extract.

Absorbance changes at 470 nm were monitored as in CAT

activity measurement. One unit CAT or POD activity was

defined as an absorbance change of 0.01 per min.

Exactly according to the method of Cavalcanti et al.

(2004), the activity of SOD was determined by measur-

ing its ability to inhibit the photoreduction of nitro blue

tetrazolium (NBT). One unit of SOD activity was defined

as the amount of enzyme that would inhibit 50% of NBT

photoreduction. Considering that there are three SOD

isoenzymes with different sensibility to KCN and H

2

O

2

(Alscher et al., 2002), enzyme extracts were mixed respec-

tively with the equal volume of KCN (1.5 mM) or H

2

O

2

(1

mM), and incubated at 25¢XC for 10 min. The residual ac-

tivities of these treated enzyme extracts were determined

to calculate the activity of different isoenzymes.

All of the above enzyme activities were measured by

using UV-1601 UV-Visible Spectrophotometer (Shimadzu

Corporation, Japan).

RESULTS

Chlorophyll fluorescence parameter

As shown in Table 1, heat shock caused a significant

decrease about 50% to the maximal quantum yield of

PSII (F

v

/F

m

), while SNP presoaking could alleviate this

negative effect about 16.5%, both relative to control.

Presoaking with SNP + bovine hemoglobin (Hb) blocked

the action of SNP. The initial fluorescence (F

o

) in four

treatments had no statistical difference, meaning that heat

shock and SNP presoaking to influence F

v

/F

m

were mainly

through their action on the maximal fluorescence (F

m

) (Ta-

ble 1). This suggested that exogenous NO could improve

the electron flux from the primary quinine acceptor (Q

A

)

to the electron transfer chain (Ortiz and Cardemil, 2001).

Considering that besides NO, NO

2

-

and [Fe(CN)

6

]

3-

are

also products when SNP dissolved in water (Ruan et al.,

2004), NaNO

2

and K

3

[Fe(CN)

6

] as well as 2-(4-carboxy-

2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

(cPTIO, another NO scavenger) were used to test if the

role of SNP presoaking was solely due to NO released

(Table 2). After heat shock, SNP presoaking significantly

increased F

v

/F

m

compared with H

2

O presoaking, while

F

v

/ F

m

values had no statistical difference in discs

presoaked with H

2

O, SNP plus NO scavengers (Hb

or cPTIO) and byproducts of SNP dissolution [NO

2

-

,

Fe(CN)

63-

]. This result further confirmed that the role of

SNP presoaking was attributed to released NO, not other

derived compounds.

Membrane permeability, lipid peroxidation and

H

2

O

2

content

The degree of cell membrane injury induced by stress

may be easily estimated through measurements of electro-

lyte leakage from the cells (Bajji et al., 2002). As shown

Table 1. Chlorophyll a fluorescence parameters in treated leaf discs. Values in brackets are percentage of control. Leaf discs were

treated as described in "Materials and Methods".

Parameters

Treatments

Control

H

S

S + Hb

F

o

658 ¡Ó 6 a

656 ¡Ó 6 a

(99.7%)

652 ¡Ó 2 a

(99.1%)

653 ¡Ó 5 a

(99.2%)

F

m

4071 ¡Ó 15 a

1138 ¡Ó 53 b

(28.0%)

1486 ¡Ó 67 c

(36.5%)

1159 ¡Ó 47 b

(28.5%)

F

v

/F

m

0.838 ¡Ó 0.001 a

0.421 ¡Ó 0.030 b

(50.2%)

0.559 ¡Ó 0.018 c

(66.7%)

0.433 ¡Ó 0.026 b

(51.7%)

Control, 25¢XC incubation with water presoaking; H, 45¢XC heat shock with water presoaking; S, 45¢XC heat shock with 150 £gM SNP

presoaking; S + Hb, 45¢XC heat shock with presoaking of 150 £gM SNP plus 4 g L

-1

bovine hemoglobin. In each row values fol-

lowed by the same letter are not significantly different at P<0.05 (n = 8).

Table 2. Values of F

v

/F

m

in heat shocked leaf discs presoaked with different solutions. Leaf discs were treated as described in "Ma -

terials and Methods".

Treatments

Presoaked respectively in following solutions before 45¢XC heat shock

H

2

O

SNP

SNP + Hb SNP + cPTIO

NaNO

2

K

3

[Fe(CN)

6

]

F

v

/F

m

0.423 ¡Ó 0.034 a 0.561 ¡Ó 0.022 b 0.431 ¡Ó 0.031 a 0.429 ¡Ó 0.017 a 0.426 ¡Ó 0.013 a 0.418 ¡Ó 0.024 a

These different solutions were H

2

O, SNP (150 £gM), SNP (150 £gM) + Hb (4 g L

-1

), SNP (150 £gM) + cPTIO (150 £gM), NaNO

2

(150

£gM), K

3

[Fe(CN)

6

] (150 £gM). Values of F

v

/F

m

followed by the same letter are not significantly different at P<0.05 (n = 6).

132

Botanical Studies, Vol. 47, 2006

in Figure 1, heat shock increased the electrolyte leakage

by 105% in water-presoaked leaf discs, by 57% in SNP-

presoaked and by 81% in SNP + Hb presoaked ones. This

meant that SNP presoaking would decrease the electrolyte

leakage by 48%, while NO scavenger Hb could reverse

the effect of SNP presoaking by 24% (Figure 1).

TBARS are the product of lipid peroxidation, and

higher levels of these substances are found in plants that

are subject to higher levels of oxidative stress (Larkindale

and Knight, 2002). As shown in Figure 1, after heat shock,

TBARS contents in leaf discs with water presoaking or

SNP+Hb presoaking were significantly higher than in

control, while there was no statistical difference between

SNP-presoaked leaf discs and control. This suggested that

SNP presoaking would reduce the lipid peroxidation de-

gree in heat-shocked tissue to the non-heat shocked level

and this role was due to released NO.

A previously suggested role of NO as antioxidants to

scavenge ROS was tested by directly measuring the H

2

O

2

content in present work (Figure 1). Compared with control

discs, H

2

O

2

content in SNP-presoaked leaf discs had no

statistical difference after heat shock, while H

2

O

2

content

in both water presoaked and SNP+Hb presoaked discs

were significantly higher than control. This suggested that

heat shock would result in H

2

O

2

production while NO

would significantly decrease heat shock-induced H

2

O

2

to

the control level.

Activities of antioxidant enzymes

The activities of radical scavenger antioxidant enzymes

(CAT, POD and SOD) were declined significantly in

water presoaked leaf discs by heat shock. SNP presoaking

would increase POD activity by 16% and recuperate the

suppressed activities of CAT and SOD to the control

level. However, NO scavenger Hb could fully or partially

reverse these influences (Figure 2).

Activities of SOD isoenzymes

The three isoforms of SOD [i.e. iron SOD (Fe SOD),

manganese SOD (Mn SOD) and copper-zinc SOD (Cu-Zn

SOD)] are located in different compartments of the cell,

and exhibit different responses to the same kind stress (see

Kliebenstein et al., 1998; Alscher et al., 2002). In the pres-

ent research, Mn SOD activity remained approximately

constant in four treatments. Activities of Cu-Zn SOD and

Fe SOD were significantly decreased by heat shock in leaf

discs with water presoaking or SNP+Hb presoaking, while

were recuperated by SNP presoaking (Figure 3).

DISCUSSION

PSII photochemical activity

Environmental stresses that affect PSII efficiency lead

to a characteristic decrease in the maximal quantum yield

of PSII (measured as F

v

/F

m

) (Krause and Weis, 1991). So

F

v

/F

m

was employed to compare the heat stress tolerance

between different plants (Ortiz and Cardemil, 2001; Liu

and Huang, 2000; Law and Crafts-Brandner, 1999), and

to estimate the role of exogenously applied chemicals on

plant tissue submitted to heat stress (Logan and Monson,

1999), referring to that a higher F

v

/F

m

after stress stands

for a higher thermotolerance, or higher protective role of

the applied chemical. In the present work, the significantly

increased F

v

/F

m

due to exogenous NO, relative to water

Figure 1. Electrolyte leakage (EL), lipid peroxidation level

(TBARS contents) and H

2

O

2

content in four treatments. Control,

25¢XC incubation with water presoaking; H, 45¢XC heat s hock

with water presoaking; S, 45¢XC heat shock with 150 £gM SNP

presoaking; S + Hb, 45¢XC heat shock with presoaking of 150

£gM SNP plus 4 g L

-1

bovine hemoglobin. The unit for electro-

lyte leakage (EL) is £gS cm

-1

g

-1

FW; for TBARS content is nmol

g

-1

FW; for H

2

O

2

content is £gmol g

-1

FW. Bars, SE (n = 6).

Figure 2. The activities of catalase (CAT), guaiacol peroxidase

(POD) and s uperoxide dismutas e (S OD) in four treatments .

Control, 25¢XC incubation with water presoaking; H, 45¢XC heat

shock with water presoaking; S, 45¢XC heat shock with 150 £gM

SNP presoaking; S + Hb, 45¢XC heat shock with presoaking of

150 £gM SNP plus 4 g L

-1

bovine hemoglobin. Calculation of

enzyme activity was described in "Materials and methods". The

activity unit for CAT and POD is 10

3

U min

-1

g

-1

FW; for SOD is

U min

-1

g

-1

FW. Bars, SE (n = 6).

YANG et al. ¡X Presoaking with nitric oxide donor SNP

133

presoaking or SNP+Hb presoking, meant an evident pro-

tective role on leaf discs under heat stress (Table 1). Con-

sidering that decrease in F

o

was suggested to be associated

with an increased capacity for energy dissipation within

light-harvesting complexes (Demmig-Adams, 1990), and

increase in F

o

to be related to partly reversible inactiva-

tion or irreversible damage in the reaction centers of PSII

(Yamane et al., 1997), the statistically similar F

o

in four

treatments (Table 1) meant that PSII reaction centers was

not influenced by heat shock or SNP presoaking in dark.

Cell membrane integrity

Cell membranes are one target of many plant stresses

and it is generally accepted that the maintenance of their

integrity and stability under stress conditions is a major

component of stress tolerance in plants (Bajji et al., 2002).

Lipid peroxidation is commonly taken as an indicator of

oxidative stress and is quantified by the thiobarbituric acid

(TBA) test, which is easy to perform and allows the results

to be conveniently expressed as TBARS (Iturbe-Ormaetxe

et al., 1998). However, results from the TBA test need

to be compared with more specific assays, because the

hydroxyl radical (

¡E

OH) and other highly reactive radical

species can oxidize proteins in addition to lipids (Iturbe-

Ormaetxe et al., 1998). In the present study, besides

TBARS contents, membrane integrity was also assayed by

directly measuring the electrolyte leakage from plant tis-

sue because electrolyte leakage from the cell is regarded as

a consequence of an oxidative burst leading to membrane

peroxidation (Bajji et al., 2002). Although the TBARS

content was alleviated to the control level by exogenous

NO, the electrolyte leakage was still significantly higher

than that in control despite of NO action (Figure 1). This

meant exogenous NO could reduce the lipid peroxida-

tion degree in heat-shocked tissue to the control level, but

could not recuperate the membrane integrity fully. The

similar result was reported in wheat plants submitted to

drought and UV-B irradiation in combination (Alexieva et

al., 2001). It was suggested that heat shock may destroy

the cell membrane integrity through some way besides

lipid peroxidation. The possible one was the oxidative

denaturation of membrane protein, and exogenous NO

could not prevent this process.

Antioxidant enzyme activity

After heat shock, activities of antioxidant enzymes

(CAT, POD and SOD) decreased in water presoaked

leaf discs and partially or fully recuperated due to SNP

presoaking (Figure 2). Because the physiological role

of CAT and POD is to break down H

2

O

2

in the cell, de-

creases in activities of these two enzymes would result in

H

2

O

2

accumulation (Figure 1). SOD is the key enzyme

to catalyze the conversion of

¡E

O

2

¡V

into H

2

O

2

and O

2

, and

reduction in SOD activity would be related to accumula-

tion of

¡E

O

2

¡V

. Through Herbert-Weiss reaction, H

2

O

2

and

¡E

O

2

¡V

react and form the most reactive hydroxyl radical

(

¡E

OH), which can directly attack unsaturated fatty acids of

lipid to induce lipid peroxidation in the cell (Bowler et al.,

1992). The decreases in activities of CAT, POD and SOD

suggested that the ROS-scavenging ability was partially

destroyed by heat shock. SNP presoaking recuperated the

antioxidant enzyme activities in heat-shocked leaf discs,

which may eliminate the possible accumulation of ROS

and reduce the oxidative damage induced by heat shock

(Figure 1). Therefore, it was suggested putatively that the

role of exogenous NO may act partially through its capac-

ity to increase the antioxidant enzyme activities.

SOD is unique in that its activity controls the con-

centrations of

¡E

O

2

¡V

and H

2

O

2

and therefore likely to be

central in the defense mechanism (Bowler et al., 1992).

In this study, after heat shock, activities of Cu-Zn SOD

and Fe SOD decreased in water presoaked leaf discs and

was recuperated by exogenous NO (Figure 3). Since Cu-

Zn SOD and Fe SOD both were thermostable in present

experimental conditions (see Asada, 1999), the decrease

in their activity was not caused by enzyme protein de-

naturation in heat shock. However, the inactivation of

H

2

O

2

scavenging enzymes (CAT and POD) by heat shock

would result in H

2

O

2

accumulation in leaf discs with

water presoaking or SNP+Hb presoaking (Figure 1), thus

decreasing the activities of Cu-Zn SOD and Fe SOD due

to their susceptibility to H

2

O

2

(Asada, 1999). Therefore,

it is indicated that the recuperation of Cu-Zn SOD and Fe

SOD activities should be related to the improved activi-

ties of CAT and POD by exogenous NO (Figure 2). On the

other hand, the activity of SOD can be induced by diverse

stress conditions and the effect of a particular stress on

SOD gene expression is likely to be governed by the sub-

cellular sites at which oxidative stress is generated (Bowler

et al., 1992). For example, in N. plumbginifolia, mitochon-

drial Mn SOD responds to increased oxyradical formation

in the mitochondria while chloroplastic Fe SOD responds

to such an event occurring in the chloroplasts (Tsang et

Figure 3. The activity of superoxide dismutase (SOD) isoen-

zymes (i.e. Cu-Zn-SOD, Fe-SOD, and Mn-SOD) in four differ-

ent treatments. Control, 25¢XC incubation with water presoaking;

H, 45¢XC heat shock with water presoaking; S, 45¢XC heat shock

with 150 £gM SNP presoaking; S + Hb, 45¢XC heat shock with

presoaking of 150 £gM SNP plus 4 g L

-1

bovine hemoglobin.

Bars, SE (n = 3).

134

Botanical Studies, Vol. 47, 2006

al., 1991). Cytosolic Cu-Zn SOD probably responds to

cytosol-localized reactions in a similar fashion (Bowler et

al., 1992). So, that Mn SOD activity remained unchanged

in the four treatments (Figure 3) may putatively indicate

that chloroplast and cytosol were the primary targets of

heat shock under present experimental conditions.

In general, the presented data here show that exogenous

NO could significantly alleviate the negative effects of a

heat shock at 45¢XC for 90 min on photochemical activity

of PSII, cell membrane integrity, and antioxidant enzyme

activities, thus improving the performance of mung bean

leaf discs under heat shock.

Acknowledgments. This work was financially supported

by the National Natural Science Foundation of China (No.

40471004).

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