American Journal of Plant Sciences, 2011, 2, 511-519
doi:10.4236/ajps.2011.23060 Published Online September 2011 (http://www.SciRP.org/journal/ajps)
511
The Benefits of Exogenous NO: Enhancing
Arabidopsis to Resist Botrytis cinerea
Hongyu Yang1*, Xiaodan Zhao2*, Jia Wu3, Min Hu1, Shaolei Xia2
1College of Life Sciences and Technology, Kunming University, Kunming, China; 2College of Life Sciences, Yunnan Normal University,
Kunming, China; 3College of Basic Medical Sciences, Kunming Medical University, Kunming, China.
Received June 23rd, 2011; revised July 26th, 2011; accepted August 17nd, 2011.
ABSTRACT
Botrytis cinerea is a necrotrophic fungal pathogen that impacts a wide range of hosts, including Arabidopsis. Pre-
treatment with nitric oxide (NO) donor sodium nitroprusside (SNP) on Arabidopsis leaves suppressed Botrytis cinerea
infection development. Additionally, in this study the dosage levels of SNP applied to the leaves had no direct, toxic
impact on the development of the pathogen. The relationship between NO and reactive oxidant species (ROS) was stud-
ied by using both spectrophotometrical methods and staining leaf material with fluorescent dyes, nitro blue tetrazolium
(NBT), and with 3,3-diaminobenzidine (DAB). The results showed that exogenous NO restrained the generation of ROS,
especially H2O2, as the pathogen interacted with the Arabidopsis plant. And this inhibition of reactive oxidant burst
coincided with delay infection development in NO-supplied leaves. The influence of elevated level of NO on antioxidant
enzymes was investigated in this study. The activities of catalase (CAT) and guaiacol peroxidase (POD) were increased
to different degrees in both: 1) SNP treated only leaves, and 2) SNP pretreated leaves that were subsequently
inoculateted with pathogens. However, the activity of superoxide dismutase (SOD) was unchanged in the leaves studied.
The decrease in H2O2 content probably resulted from the increase in activities of POD and CAT. Our study suggests
that NO might trigger some metabolic reactions, i.e. enhanced enzyme activity that restrains H2O2 which ultimately
results in resistance to B. cinerea infection.
Keywords: Arabidopsis, Botrytis cinerea, Nitric Oxide, Antioxidant Enzyme, Disease Resistance
1. Introduction
Nitric oxide (NO) has been involved in the responses of
plants to abiotic and biotic stresses like drought, salt, heat
stresses, disease resistance, and apoptosis [1-4]. Most
studies that established the role of NO in the defense
responses of plants to disease were obtained from ana-
lyzing various plant-biotrophic pathogen systems [5].
The data obtained from these studies show that oxidative
burst, a transient and rapid accumulation of reactive
oxygen species (ROS), is a widespread defense mecha-
nism of plants against the attack of pathogens. ROS, in-
cluding superoxide (2
O) and hydrogen peroxide (H2O2),
was generated following the recognition of a variety of
pathogens and has been identified as a threshold trigger
for the hypersensitive response (HR) [6]. The death of
plant cells surrounding the pathogenic penetration site
caused by HR deprived the pathogen of a nutrient source.
This has been considered a successful defense strategy of
the plant against biotrophic pathogens [7].

In contrast to biotrophic pathogens, Botrytis cinerea, a
necrotrophic pathogen, uses different strategies to avoid
incompatibility. This kind of pathogen is able to colonize
dead tissue. The pathogen induces the death of host cells
in order to facilitate the infection. Thus HR is not an ef-
ficient weapon against the infection of necrotrophic
pathogens [8]. Although the death of host cells enables
necrotrophic pathogens to spread and develop in the host,
enhanced ROS generation in the plants was still observed
[9,10]. According to Ediich, ROS contribute to over-
coming the defense of a host plant against B. cinerea and
facilitate infection [11]. This illustrates the different
ways that biotrophic and necrotrophic phytopathogenic
fungi deal with ROS as they interact with plants. Thus it
can be concluded that an efficient response of a plant to
necrotrophs like B. cinerea should include the suppres-
sion of ROS and the stimulation of the antioxidant sys-
tem responsible for the control of the redox state in the
cell. This conclusion is supported by a study [12] that
showed that radical scavengers were able to reduce the

The Benefits of Exogenous NO: Enhancing Arabidopsis to Resist Botrytis cinerea
512
severity of diseases induced by B. cinerea.
Many studies indicate that NO is involved in modify-
ing post-infection plant metabolism, often leading to a
HR of infected cells. However, only a few of these stud-
ies examined the role played by NO in the interaction
between the plant and the necrotrophic pathogen [13-15].
Baarlen reported endogenous NO generation during the
interaction between lilies and Botrytis elliptica [13].
Floryszak-Wieczorek also found that NO plays a crucial
role in the initiation of a fast, defensive response of pe-
largonium leaves to a necrotrophic pathogen [15]. The
mechanisms of antioxidant action of NO in plant defense
reactions were viewed differently. One reckoned that the
decrease in H2O2 concentration in NO-supplied tomato
leaves was probably due to a direct NO-H2O2 interaction
[14]. Another study proposed that in the nonspecific re-
sistance of pelargonium to B. cinerea, an early NO burst
served as a signal to enhance the accumulation of ascorbic
acid and the pool of other ‘fast antioxidants’, and second-
dary NO emissions provoked a noncell-death-associated
defense, following the rule that the concentration of NO
is linked to its action [15]. This present study attempts to
elucidate whether NO is involved in the resistance reac-
tions of Arabidopsis to B. cinerea infection and to deter-
mine how NO inactivates ROS in the defense responses
of Arabidopsis plants.
2. Materials and Methods
2.1. Plant Materials and Growth Conditions
The Arabidopsis thaliana plants used in this study were
ecotype Columbia. The seeds were sterilized for 1 minute
in 95% ethanol and for 5 minutes in 1% bleach, washed 5
times in sterile water, and then pre-germinated on MS
salt agar plates at 22˚C ± 2˚C under a light cycle of 12-h
light/12-h dark for 1 week. Then the plants were trans-
planted into soil and grown at 22˚C ± 2˚C under the same
light cycle.
2.2. Pathogen Culture
Botrytis cinerea was isolated from infected tomato plants
growing in a greenhouse and was maintained in stock
culture on potato sucrose agar in the dark at 22˚C ± 2˚C.
The B. cinerea spores were cultured in a potato sucrose
agar medium without exposure to light at 22˚C ± 2˚C for
8 days. The spores were collected in sterile water and
resuspended at 105 spore·ml–1.
2.3. Effect of NO on Conidial Germination and
Mycelial Growth of B. cinerea
50 mM of NO donor (sodium nitroprusside, SNP) stock
solution was added to the potato sucrose medium con-
taining 105 ml–1 fungal spore suspension in order to pro-
duce final concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5 and
10 mM [16,17]. The spores were further cultured at 22˚C
± 2˚C in the dark for 24 hours. The medium containing
sterile water served as a control. Then the germinating
spores were counted under a light microscope [15]. A
spore was recognized as a germinating spore when a tube
was longer than the spore diameter. Each experiment was
replicated three times and the germination rate was cal-
culated as follows: rate of spore germination = the num-
ber of germinated spore/total number of spore × 100%.
In order to study the effect of SNP on the mycelial
growth of B. cinerea, we added 50 mM of the SNP stock
solution to the potato sucrose agar medium which was
cooling after having been sterilized. We added various
amounts of the SNP stock to produce final concentrations
of 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 mM. 10 ml of the me-
dium was poured into a plate. We then placed 10 ml (105
spores/ml spore suspension) of B. cinerea into the center
of the medium and the diameter (cm) of the area occu-
pied by B. cinerea mycelium was measured after being
cultured at 22˚C ± 2˚C in the dark for 3 days.
2.4. Inoculation
5 leaves were selected from one 5-week-old plant and
marked. 10 µl drops of B. cinerea conidial suspension
(105 ml–1) or sterile water were transferred onto the upper
surface of each leaf blade using a 1 ml syringe without a
needle. After inoculation, the plants were placed in a
growth chamber in the dark at 22˚C ± 2˚C and 100%
relative humidity for 24 hours. After 24 hours, the
growth chamber conditions were changed to a light cycle
of 12-h light/12-h dark and the humidity was maintained
at 70%.
2.5. Assessment of Disease Development
In order to quantify the severity of the symptoms, indi-
vidual leaves were examined each day for ten days after
inoculation with the pathogen and rated on a scale of 0 to
5 on the basis of the Murray method with modifications
[18] where 0 = no visible symptoms; 1 = very few (less
than 10% of leaf area) flecks on leaf area lamina; 2 =
chlorotic flecks covered 10% - 25% of leaf area lamina;
3 = 26% - 50% of leaf area shows symptoms; 4 = disease
spots covered 51% - 70% of leaf areas; and 5 = most of
leaf lamina (71% - 100%) are covered with chlorotic
flecks. Plants were assigned a disease index (DI) as fol-
lows:


DI 100%ijnk

in which i = infection class, j = the number of plants
scored for that infection class, n = the total number of
plants in the replicate, and k = the highest infection class.

The Benefits of Exogenous NO: Enhancing Arabidopsis to Resist Botrytis cinerea513
2.6. Assays of Hydrogen Peroxide
A concentration of hydrogen peroxide (H2O2) was meas-
ured spectrophotometrically using the titanium (Ti4+)
method [19]. H2O2 was extracted from the plant leaf tis-
sues at various time intervals after the leaves were
sprayed with SNP. Some samples were then inoculated
with B. cinerea, while some samples were not. Leaf ma-
terial (0.2 g) from the plants was ground with a mortar
and pestle in 1.5 ml of pre-chilled acetone. The homoge-
nate was centrifuged at 15,000 × g for 10 min. The su-
pernatant was collected and 1 ml of the supernatant was
mixed with 0.1 ml 5% sodium titanium and 0.2 ml
hartshorn. The reaction mixture was centrifuged for 10
min at 15,000 × g after precipitation occurred. The pre-
cipitate was collected and washed 5 times with acetone.
Then 5 ml of 2 M HCl was added to dissolve the precipi-
tate. After the precipitate was dissolved, the reaction so-
lution was made with a final volume of 10 ml. This solu-
tion was used for the assay of H2O2 at 415 nm in a spec-
trophotometer. The experiment was repeated three times
with similar results, and each experiment had three rep-
lications.
2.7. H2O2 Detection by the DAB Uptake Method
The formation of hydrogen peroxide was detected by
using a fluorescent dye, 3,3-diaminobenzidine (DAB),
following Orozco-Cardenas with minor modifications [20].
Some of the sample leaves from the pathogen inoculated
plants were pretreated with SNP and some were not.
These sample leaves were excised and immersed in 2
mg· ml –1 DAB solution (pH 3.8) at specific time intervals.
The samples were then incubated in the dark at 24˚C for
48 hours. After reacting, the leaves were placed in a
boiling, decolorizing solution for 5 - 10 minutes. The
decolorizing solution contained acetic acid, glycerol, and
ethanol at a ratio of 1:1:3. After cooling, the decolorized
leaf was mounted in 60% glycerol and examined under a
light microscope. H2O2 appeared reddish-brown in colour.
2.8. Histochemical Localization of Using
NBT 2
O
In order to detect 2
formation and accumulation in the
leaves, nitro blue tetrazolium (NBT) staining was per-
formed according to Brodersen with slight modifications
[21]. Leaves inoculated with the pathogen, some of
which had been treated with SNP, were excised from the
plants and submerged in 0.1% NBT (w·v–1) (in 10 mM
NaN3 and 10 mM potassium phosphate buffer, pH 7.8)
and shaken lightly for 24 hours. After staining, the leaves
were washed with water and photographed.
O
2.9. Antioxidant Enzyme Assays
A small amount (0.2 g) of the leaf tissue was homoge-
nized at 0˚C - 4˚C in 1.5 ml of 50 mM phosphate buffer,
pH 7.8. The homogenate was centrifuged at 15000 × g
for 15 minutes and the supernatant obtained was used as
enzyme extract.
Catalase (CAT) activity was assayed by monitoring
the consumption of H2O2 at 240 nm in a spectropho-
tometer. The 3 ml reaction mixture contained 1 ml of
0.3% H2O2, 1.9 ml of H2O and 0.1 ml of enzyme extract.
The reduction rate of OD at 240 nm was recorded [16,
22]. The activity was expressed in U g–1·FW–1 where one
unit of catalase = 0.01 reduction in OD per minute.
Guaiacol peroxidase was measured colorimetrically
with guaiacol as a substrate. The 4 ml reaction mixture
contained 1ml of 50 mM phosphate buffer (pH 7.0), 2 ml
of 0.3% H2O2 and 0.95 ml of 0.2% guaiacol. Then 0.05
ml enzyme extract was added to start the reaction. The
linear increases in absorbance at 480 nm were monitored
[14]. The activity was expressed in U g–1·FW–1 where
one unit = 0.01 increase in OD per minute.
The activity of superoxide dismutase (SOD) was as-
sayed by measuring its ability to inhibit the photochemi-
cal reduction of NBT following Beauchamp with minor
modifications [23]. A volume of 3 ml of our reaction
mixture contained 2.5 ml of 13 µM methionine, 0.15 ml
of 13 μM riboflavin, 0.25ml of 75 µM NBT and 0.05 ml
of 50 mM phosphate buffer (pH 7.8), and 0.05 ml of en-
zyme extract. Instead of NBT, the mixture contained a
phosphate buffer to serve as control. Riboflavin was
added last and the reaction was initiated by placing the
tubes under 4000 lx light for 20 minutes. The absorb-
ances at 560 nm were read. The volume of enzyme ex-
tract corresponding to 50% inhibition of the reaction was
considered to be one enzyme unit.
3. Results
3.1. The Effect of SNP on Conidia Germination
and Mycelial Growth of B. cinerea
Germination of the conidia was examined 24 hours after
having been treated with 0.01, 0.05, 0.1, 0.5, 1, 5 and 10
mM of SNP respectively. Germination of the conidia was
obviously inhibited in those samples that had been
treated with 5 or 10 mM of SNP. However, the lower
concentrations of SNP treatment did not impact the ger-
mination of the conidia in the other samples (Figure 1).
The mycelial growth of B. cinerea was not affected by
the 0.01, 0.05, 0.1, 0.5 and 1mM SNP treatments, but
was significantly restrained by the 5 and 10 mM SNP
treatments (Table 1). Therefore, we chose 0.01, 0.05, 0.1
0.5, and 1 mM of SNP, levels which did not directly af-
fect the development of B. cinerea, to optimize the fol-
lowing experiments.

The Benefits of Exogenous NO: Enhancing Arabidopsis to Resist Botrytis cinerea
514
** **
Figure 1. Effect of NO generator—sodium nitroprusside
(SNP) on conidia germination of Botrytis cinerea in vitro. A
variety of SNP concentrations were supplied to medium
containing 105 ml–1 fungal spore suspension (sterile water
served as a control) and cultured at 22˚C ± 2˚C for 24 hours.
Then the germinating spores were counted and the germi-
nation rate was calculated (desciption as method ). Values
in this figure represent the means and SE from three inde-
pendent experiments with three replicates each, n = 9. **
indicates values that differ significantly from the control
at P < 0.01.
Table 1. Effect of SNP on the mycelial growth of B. cinerea.
SNPcontent
(mmol/L)
Diameter of the fungal colony
(cm/day)
suppression
(%)
control
0.01
0.05
0.10
0.50
1.00
5.00
10.00
5.10 ± 0.31
5.03 ± 0.39
4.93 ± 0.38
4.95 ± 0.39
4.44 ± 0.42
4.19 ± 0.40
1.62 ± 0.03**
0.39 ± 0.05**
-
1.52
3.72
3.30
13.41
18.34
67.97
92.60
Values represent the means and SE from three independent experiments
with three replicates each, n = 9.  indicates values that differ significantly
from the control at P < 0.01.
3.2. Optimum Concentration and Interval of
SNP Treatment
In order to test whether SNP has a toxic effect on Arabi-
dopsis plants, we used various SNP concentrations that
did not directly affect the development of B. cinerea,
namely, 0.01, 0.05, 0.1, 0.5 and 1 mM, sprayed on the
leaves. The leaves were examined each day for ten days
after having been sprayed and the results showed that
0.01 to 0.5 mM had no significant effect on the leaves.
However, the leaves treated by 1 mM of SNP turned
yellow after 5 - 7 days.
We then sprayed 0.01, 0.05, 0.1 and 0.5 mM of SNP
on the surface of the leaves. Three days later, we inocu-
lated the leaves with B. cinerea. The symptoms of infec-
tion on the leaves were checked and compared each day
from the first day to the seventh day after their inocula-
tion (dpi). The results indicated that the optimum con-
centration of SNP was 0.5 and 0.1 mM (Figure 2).
The Arab idopsis leaves that had been pre-sprayed with
0.1 or 0.5 mM SNP (sprayed sterile water as a control)
over a four-day period were then inoculated with the co-
nidial suspension of B. cinerea. Twenty-four hours after
being inoculated, the disease index was determined. The
results showed that challenging with the pathogen 1 - 4
days after 0.5 mM SNP pretreatment and 1 day and 3 - 4
days 0.1 mM SNP pretreatment had an obvious effect in
limiting the disease symptoms (Table 2). We chose 3
days pretreatment with both 0.5 and 0.1 mM SNP fol-
lowed by inoculation with the pathogen as the optimum
interval.
3.3. Effect of NO on B. cinerea Infection
Development in Arabidopsis Leaves
Symptoms of infection in leaves inoculated with B. cine-
rea conidial suspension appeared on the surface of the
leaves 2 days post inoculation. First, the edge and/or tip
of the leaf started to curl. Then water-soaked spots ap-
peared on the inoculation site of the leaf surface. These
small spots developed into maceration or necrosis lesions
(3 dpi). The lesions expanded and became dark and
chlorotic within 5 days. On the seventh day after inocula-
tion (7 dpi), the necrotic lesions had significantly ex-
panded and covered about 90% of the leaf surface.
However, on the leaves that were sprayed with 0.1 or 0.5
mM SNP three days prior to their inoculation with the
0
10
20
30
40
50
60
70
80
90
1 23 4 5 6 7
Time after inoculation (d)
Disease index (%)
0.1
0.5
con
0.01
0.05
1
Figure 2. Effect of SNP pretreatment on the disease index of
Arabidopsis. Values represent the means and SE from three
independ ent experiments with three replicate s each, n = 9.

The Benefits of Exogenous NO: Enhancing Arabidopsis to Resist Botrytis cinerea515
Table 2. Influence of interval SNP pretreatment on the rate
of disease in Arabidopsis.
VRate of Disease
wSNP
concentration
（mmol/L） 3th day 4th day 5th day
control 30.33 ± 4.91 54.00 ± 5.51 71.67 ± 4.63
0.10 - 1 12.67 ± 1.86* 29.33 ± 3.76** 56.67 ± 5.36*
0.10 - 2 24.00 ± 5.03 40.00 ± 4.36** 65.33 ± 4.06*
0.10 - 3 19.33 ± 3.53* 37.67 ± 5.90** 58.33 ± 5.49*
0.10 - 4 27.00 ± 5.20 47.33 ± 5.78** 63.67 ± 5.81*
0.50 - 1
0.50 - 2
0.50 - 3
0.50 - 4
10.67 ± 2.19*
10.33 ± 1.76*
10.67 ± 3.84*
18.33 ± 3.53*
28.00 ± 4.04**
27.00 ± 1.53**
23.33 ± 2.60**
36.00 ± 4.73**
48.00 ± 4.36*
52.67 ± 7.31*
43.00 ± 5.86*
57.67 ± 6.33*
w0.1 - 1, 0.1 - 2, 0.1 - 3, 0.1 - 4: Leaves were sprayed with 0.1 mM of SNP
and subsequently inoculation with pathogen interval 1, 2, 3 or 4 days re-
spectively. 0.5 - 1, 0.5 - 2, 0.5 - 3, 0.5 - 4: Leaves were sprayed with 0.5 mM
of SNP, subsequently inoculation with pathogen interval 1, 2, 3 or 4 days
respectively. Vdays after inoculation with pathogen, the rate of disease of
leaves was evaluated. Values represent the means and SE from three inde-
pendent experiments with three replicates each, n = 9.  indicates values that
differ significantly from the control at P < 0.05.  indicates values that
differ significantly from the control at P < 0.01.
fungus, the development of the infection was signify-
cantly restrained. The necrotic lesion appeared one day
later than those in the control group, the lesions only af-
fected about half of the leaf surface by the seventh day
after inoculation (7 dpi), and the disease index was dra-
matically lowered from 3 to 7 dpi (Figure 2).
3.4. ROS Generation and Accumulation in SNP
Treated Leaf Tissues
In our examination of the hydrogen peroxide (H2O2)
content in Arabidopsis leaves, we compared those leaves
that had been inoculated with B. cinerea with those
leaves that were pretreated with SNP and then inoculated
with B. cinerea (Figure 3). The H2O2 content in the
pathogen-inoculated leaves was significantly increased 4 -
24 hours post inoculation (hpi) and reached its peak 24
hpi. The H2O2 content in the pathogen-inoculated leaves
decreased a little and remained largely unchanged from 2 -
7 days post inoculation (dpi). The H2O2 content in the
SNP pretreated and pathogen-inoculated leaves also in-
creased 4 - 24 hpi. However, the increase was lower than
that of the pathogen-inoculated leaves. Furthermore, the
H2O2 content in the SNP pretreated and pathogen-in-
oculated leaves reached its peak at 48 hpi, one day later
than the pathogen-inoculated leaves, and it decreased
sharply after reaching this peak (Figure 3).
The cytochemical analyses detected the presence of
H2O2 in the inoculated leaves that were pretreated with
SNP and those that were not. The reddish-brown colour-
ing of the leaves indicated a considerable accumulation
of H2O2 (Figure 4). On those leaves without the SNP
pretreatment, the reddish-brown colouring covered the
whole surface on which the conidial suspension was
transferred, starting from 2 dpi. On the leaves sprayed
with 0.1 or 0.5 mM of SNP and inoculated with the fun-
gus, the portions that were coloured were smaller and
lighter. H2O2 was present only at the site of the necrosis
formation. As the disease developed, the number and size
of the spots on the leaves marking H2O2 synthesis in-
creased. This was especially evident at the boundary be-
tween healthy and diseased tissue. In both experiments of
H2O2 content assay and cytochemical detection, we got
the same results. This suggests that NO retards the ac-
cumulation of H2O2 in leaves during the process of plant
and pathogen interaction.
Time after inoculation
4h 8h12h16h1d 2d 3d 4d 5d
H2O2 content ( umol l-1 FW )
0
200
400
600
800 con
0.1
0.5
*
**
Figure 3. Time course of changes in H2O2 accumulation in
Arabidopsis leaves pretreatment with SNP and infection with
B. cinerea. Values represent the means and SE from three
independent experiments. * indicates values that differ sig-
nificantly from the control at P < 0.05. ** indicates values
that differ significantly from the control at P < 0.01.
(a) (b) (c)
Figure 4. A comparison of H2O2 accumulation revealed by
3,3-diaminobenzidine (DAB) staining in Arabidopsis leaves.
(a) Control, sprayed with water and inoculation with B.
cinerea; (b) sprayed with 0.1 mM SNP and inoculation with
B. cinerea; (c) supplied 0.5 mM SNP and inoculation with B.
cinerea. Photos were taken 2 dpi and shown the typ ical on es.

The Benefits of Exogenous NO: Enhancing Arabidopsis to Resist Botrytis cinerea
516
The NBT staining of leaves was performed on the se-
cond day after the plants were inoculated with the B.
cinerea conidial suspension. The dark blue coloring that
appeared indicated the accumulation of 2. In the
pathogen-inoculated leaves, intensely dark blue spots
appeared and were scattered across the whole surface of
the leaves. However, in the leaves which were treated
with SNP prior to being inoculated with the pathogen,
the areas of dark blue coloring were smaller and confined
(Figure 5).
O
3.5. Effect of NO on the Antioxidant Enzymes
We tested four groups of Arabidopsis leaves: leaves
treated with SNP; leaves sprayed with sterile water as a
control; leaves inoculated with B. cinerea; and leaves
pretreated with SNP which were then inoculated with B.
cinerea. CAT and guaiacol peroxidase (POD) showed an
increase in activity during the 7 day study period. This
increase was especially significant in the leaves treated
with SNP and then inoculated with pathogen (3 dpi)
when compared with the increase in leaves without the
SNP treatment [Figures 6(a), (b)]. The CAT seemed
more sensitive to 0.1 mM of SNP, and 0.5 mM worked a
little better than the 0.1 mM in elevating POD activity.
Although SOD activities were slightly increased by the
seventh day only in those leaves treated with SNP [about
7.79% in 0.1 mM and 10% in 0.5 mM SNP treatment
was observed Figure 6(c)], no significant change in SOD
activity in all of the leaves studied was observed.
4. Discussion
The results of this study showed that NO enhanced the
resistance of Arabidopsis to B. cinerea infection. The
treatment of NO donor SNP on Arabidopsis leaves three
(a) (b) (c)
Figure 5. Superoxide anion accumulation in Arabidopsis
leaves. Shown is a comparison of typical photos of superox-
ide accumulation no treated or pretreated with SNP and
inoculation with pathogen revealed by NBT. (a) Treated
with water and inoculation w ith B. cinerea; (b) 0.1 mM SNP
pretreatment and inoculation with B. cinerea; (c) 0.5 mM
SNP pretreatment and inoculation with B. cinerea. Photos
were taken 2 dpi.
1d 2d 3d 4d 5d 6d 7d
CAT activity ( U g-
1
FW )
0
20
40
60
80
100
120 con
0.1
0.5
Time after inoculation



(a)
Time after inoculation
1d 2d 3d4d5d 6d7d
POD activity ( U g-1 FW )
0
200
400
600
800
1000
1200
1400 con
0.1
0.5


(b)
Time after inoculation
1d 2d 3d 4d 5d 6d 7d
SOD activity ( U g-1 FW )
0
10
20
30
40
50
60 con
0.1
0.5
(c)
Figure 6. CAT (a) POD (b) and SOD (c) activities in Arabi-
dopsis leaves treated with 0.1 mM or 0.5 mM SNP, sprayed
sterile water served as control (con) and inoculation with B.
cinerea. Values represent the means and SE from three
independent experiments. * indicates values that differ sig-
nificantly from the control at P < 0.05.

The Benefits of Exogenous NO: Enhancing Arabidopsis to Resist Botrytis cinerea517
days prior to inoculation with fungus significantly ham-
pered the development of symptoms of disease. The ef-
fective concentration (0.1 and 0.5 mM) of SNP did not
have any direct role in reducing pathogen viability or
inhibiting their development (Figure 1 and Table 1). This
suggests that NO induces the intrinsic resistance of
Arabidopsis plants to B. cinerea.
Production of ROS during oxidative burst is one of the
earliest and most effective defensive responses of plants
[24-28]. The process of ROS generation in plant leaves
during the interaction between Arabidopsis and B. cine-
rea has been previously elucidated [8,9,11,12,29,30]. B.
cinerea can induce an oxidative burst and hypersensitive
cell death in Arabidopsis. Additionally, levels of ROS,
especially H2O2 levels during hypersensitive response,
correlate positively with B. cinerea growth in plant tis-
sues [8,31]. In this study we have examined the genera-
tion of ROS and the actions of antioxidant enzymes in an
attempt to determine their roles in the NO treatment in-
duced resistance reactions of Arabidopsis plants to B.
cinerea infection.
The results show that the concentration of H2O2 in-
creased rapidly during the early stage of pathogen infec-
tion both in plant leaves treated with SNP three days
prior to inoculation with the pathogen and in those leaves
not treated prior to inoculation. However, the time when
the H2O2 concentration reached its peak was different
between the two groups (those leaves treated with SNP
and those not treated). The time of maximal H2O2 con-
centration was delayed one day in the leaves that were
treated with SNP (Figure 3). H2O2 production in the SNP
pretreated and pathogen-inoculated plants was lower than
in the pathogen inoculated and SNP non-treated plants
during the entire study period. Similar results were ob-
tained from the histochemical determination of H2O2 and
superoxide, which showed less accumulation of H2O2
and superoxide in SNP pretreated and pathogen-inocu-
lated leaves. In conclusion, it is evident that the lower
levels of H2O2 concentration induced by NO treatment in
Arabidopsis plants enhanced their resistance to infection
caused by B. cinerea. Thus, there appears to be a correla-
tion between the induction and accumulation of H2O2 in
host plants during compatible interactions with B. cine-
rea [8,9,11,12]. In many plant-pathogen systems, H2O2
also plays a prominent role in host cell death during in-
fection by necrotrophic pathogens, and in oxidative burst
[32]. But some studies noted that pretreatment with o-
hydroxyethylorutin enhanced H2O2 generation in tomato
leaves during the interaction of the tomato plants with B.
cinerea. This higher level of H2O2 may act as a direct
antimicrobial agent limiting the germination of pathogen
spores, and enhancing the tomato plant’s resistance [17].
A dual role for H2O2 in the process of plant-pathogen
interaction is either protective or toxic, probably depend-
ing on its concentration and the plant species.
Early studies revealed that secretion of alcohol oxi-
dases by B. cinerea can release H2O2 into the extracellular
medium [33]. Additionally, superoxide dismutase (SOD),
which is an H2O2-generating enzyme of B. cinerea, con-
tributed to pathogenesis during the interaction of the
pathogen with beans [34]. H2O2 could be produced by the
fungus in order to enable Botrytis to colonize plant tis-
sues [35]. However, Rij indicated that toxic levels of
extracellular H2O2 were likely to be more harmful to the
fungus than the plant [36]. Thus, it would be much more
advantageous for the fungus if the H2O2 generation were
to take place inside the plant cell compartment and thus
be separated from sensitive fungal hyphae. A recent
study showed that diffuse H2O2 probe signals were not
observed in lily cells in the presence of Botrytis elliptica
(a necrotrophic pathogen) mycelium, demonstrating that
intracellular H2O2 was exclusively produced by live plant
cells [13]. Although the sources of H2O2 still need to be
clarified during the process of plant-necrotrophic inter-
action, the levels of ROS, mostly H2O2, in plants are in-
creased and the accumulation of these compounds is
concomitant with disease progression and Arabidopsis
cell death. A significant reduction in H2O2 and superox-
ide content and/or the delay of the advent of the highest
H2O2 content in Arabidopsis leaves as a result of NO
donor treatment may suggest that NO can act as an anti-
oxidant that inhibits H2O2 from signaling pathways that
lead to cell death.
Data about the relationship between NO and antioxi-
dant enzymes are also a source of controversy. SOD,
CAT, ascorbate (APX) and guaiacol peroxidases (POD)
have been shown to be inhibited by NO in tobacco and
zinnia plants [16,37]. But different results also have been
observed. The activities of SOD, CAT and APX in to-
mato cells remain unchanged in NO donor SNP pre-
treated plants [17]. Our results indicated that the active-
ity of the enzyme (SOD) generating H2O2 in Arabidopsis
plant cells remained unchanged in the plants studied.
However, the activity of CAT and guaiacol peroxidase
(POD), enzymes that reduce H2O2, was significantly in-
creased in Arabidopsis leaves through the treatment of
SNP prior to inoculation with the pathogen. This sug-
gests that the decrease in H2O2 content is due to the work
of the antioxidant enzyme system. It also indicates that
NO may play a role through regulating metabolic activity
in the protection of cells against the destructive action of
ROS. In the present study, we found that in the leaves
pretreated with SNP, POD activity rapidly and signify-
cantly increased 24 hours after the leaves were inocu-
lated with B. cinerea and this activity kept increasing
during the entire study period (Figure 6(b)). Tiedemann

The Benefits of Exogenous NO: Enhancing Arabidopsis to Resist Botrytis cinerea
518
reported that B. cinerea suppressed the activity of plant
peroxidase in bean leaf discs [9]. His findings suggested
that peroxidases play a role as scavengers of harmful
H2O2 in plant resistance. POD can not only scavenge
H2O2 in the initial stage of infection, but can also be ac-
tive in further resistance reactions (e.g., the cross-linking
of cell wall proteins and the polymerization of lignin pre-
cursors), thus providing protection from pathogen inva-
sion. Our results offer evidence that NO might stimilate
antioxidant enzymes which destroy ROS in plant cells.
This triggers subsequent defense reactions in the plant
and enhances the resistance of the Arabidopsis plant to
infection by B. cinerea.
5. Conclusions
1) Low concentrations (0.1 and 0.5 mM) of nitric oxide
donor sodium nitroprusside (SNP) had neither a direct,
toxic effect on conidial germination and mycelial growth
of B. cinerea, nor a toxic impact on development of
Arabipdopsis. But high concentrations of SNP, such as 5
mM and 10 mM, were harmful to both pathogen and
plant.
2) Applying 0.1 and 0.5 mM SNP to Arabidopaia
leaves could induce disease resistance of Arabidopsis to
B. cinerea. The exogenous NO could delay ROS burst
and restrain the rapid accumlation of H2O2 and 2 O

in
plants thereby delaying progression of disease.
3) The biochemical mechanism of ROS reduction
might be related to exogenous NO stimulation of the an-
tioxidase POD and CAT activities. The changes of SOD
(generating H2O2) activities were not obvious in plants
which were pretreated with SNP and subsequently in-
oculated with a pathogen. Therefore NO might play an
important role in scavenging H2O2 and enhancing resis-
tance of Arabidopsis to B cinerea.
6. Acknowledgements
This work was funded by the National Natural Science
Foundation of China (grant no. 30860121) and the Open
Program of State Key Laboratory of Plant Physiology
and Biochemistry, China Agriculture University (PP-
B08010).
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