C h a p t e r 5 Studies on nitric oxide dependent ...shodhganga.inflibnet.ac.in/bitstream/10603/76626/9/chapter 5.pdf · Chapter 5: Nitric oxide dependent hypersensitive reaction

Chapter 5: Nitric oxide dependent hypersensitive reaction

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5.1. Introduction

Induction of resistance expressed in the form of hypersensitive response (HR),

characterized by the formation of necrotic lesions at the infection site. HR is a form of

programmed cell death that contributes to plant resistance by restricting the invading

pathogen at the infection site and shows some regulatory and mechanistic features like

membrane dysfunction, vacuolization of the cytoplasm, chromatin condensation, and

endonucleolytic cleavage of DNA (Greenberg and Yao, 2004).

One of the earliest events in the HR is the rapid accumulation of ROS and NO

(Delledonne et al., 1998; Durner et al., 1998). Advances in the genetic, biochemical and

cytological characterization of disease resistance suggested that HR is associated with all

forms of resistance to Phytophthora and downy mildews (Kamoun et al., 1999). A peak

of NO concomitant with the oxidative burst has been detected during HR development in

soybean and Arabidopsis with an avirulent pathogen Pseudomonas syringae (Durner et

al., 2000). The simultaneous increase of NO and ROS activates hypersensitive cell

death in soybean and tobacco cell suspensions, while the independent increase of only

one component of this binary system had little effect on induction of cell death (Clarke et

al., 2000). Instead, the role of NO as an intercellular signal that triggers cell death in

adjacent cells of its generation has been reported in Arabidopsis leaves infected with two

different Pseudomonas avirulent strains. Further, the kinetics of accumulation of NO and

progression of the HR suggested NO involved in cell-to-cell spreading of HR rather than

in triggering cell death (Zhang et al., 2003).

The experimental evidence indicated that NO can induce cell death by triggering

an active process in which proteases appear to play a crucial role. Cystatin sensitive

proteases have been found to be critical regulators for HR cell death in a soybean model

system (Belenghi et al., 2003). A gene encoding the cysteine protease induced by NO in

Arabidopsis (Polverari et al., 2003) deactivation of cysteine protease regulation through

inhibitor was found to block cell death activated either by avirulent pathogens or by

nitrosative stress in Arabidopsis and tobacco plants (Belenghi et al., 2003). Finally,

caspase-specific protein fragmentation has been revealed during the HR in tobacco plants

infected with TMV, which is regulated by NO (Chichkova et al., 2004). In contrary,

cytological observations have shown that either administration of NO donors or alteration


84

of H2O2 levels has no effect on the elicitation of the HR in infected cells in oat plants,

although both molecules are required for the onset of cell death in adjacent cells (Tada et

al., 2004). Transgenic plants containing a bacterial nitric oxide dioxygenase transgene

found to have no NO failed to express the HR upon pathogen inoculation with

Pseudomonas syringae (Zeier et al., 2004). NO triggered by the infection of Blumeria

graminis powdery mildew fungus in barley contributed for formation of HR,

consequently resisted pathogn (Prats et al., 2005). Involvement of NO production during

HR formation in response to Phytophthora infestans derived protein INF1-treated

Nicotiana benthamiana plants has been reported (Kato et al 2006). A requirement for NO

and H2O2 in plant cell death has also been provided by experiments using transgenic

tobacco lines (Zago et al., 2006). INF1 induced HR and Pseudomonas cichorii elicited

HR in Nicotiana benthamiana shown to depend on mitogen-activated protein kinase

kinase which activated via NO signalling (Takahashi et al., 2007).

Visual necrosis on seedling tissues upon pathogen infection and resistant elicitor

treatment has been noticed in pearl millet downy mildew interaction and this response

has been recognized as HR, which is more rapid in resistant interaction compared to

susceptible interaction (Kumudini et al., 2001).

In pearl millet, though much work has been carried out in analysis of HR and

mechanism underneath, it was postulated that hydrogen peroxide (H2O2) is the main

radical involved in execution of HR and less was discussed about NO. Further,

involvement of NO in effecting HR during obligate parasitism of oomycetes is less

understood in any economically important crop plants; thus a detailed histochemical

analysis on NO and its interaction with H2O2 during HR formation is explored for

establishing link between HR and resistance. Further, it is aimed to elucidate the

dynamics of NO on H2O2 and it’s in turn effect on HR expression.

5.2. Materials and Methods

5.2.1. Host, pathogen and inoculation

As described in Chapter 1

5.2.2. Treatments

Treatments consisted of three different sets. The first set includes seedlings raised

from untreated resistant cultivar IP18292. The second set includes, susceptible seedlings


85

raised from the seeds treated with SNP (referred to as induced resistant seedlings). The

third set includes resistant and induced resistant seedlings that were treated with 10mM

C-PTIO separately for 1h prior to challenge inoculation. Effect of different dose of SNP,

0.5, 1, 1.5, 2, 2.5 and 5 mM treatment was also studied on HR expression. For all set of

seedlings the challenge inoculation was made for two-day-old seedlings by whorl

inoculation method.

5.2.3. Sampling for in vitro evaluation of HR and histochemical responses

Two-day-old pearl millet seedlings raised on sterile blotters in 9 cm diameter

petriplate at 25± 2oC were inoculated with zoospore suspension of S. graminicola at the

concentration of 1x104 zoospores/ ml for 24 h. The seedlings of the same age dipped in

sterile distilled water served as control. The inoculated and control seedlings were

observed for visual expression of HR and also processed for histochemical study at

hourly interval up to 24 h. For the enzymatic assay, seedlings were harvested at hourly

intervals and stored at -80 0 C wrapped in aluminum foil till further process.

5.2.4. Examination of visual expression of HR

The seedlings were observed at hourly interval during post-inoculation

incubation period for their reaction to S. graminicola. The number of seedlings showing

brown necrotic lesions/ streaks considered as HR symptoms at coleoptile and root region

were counted for each observation and percentage of seedlings showing HR was

calculated.

5.2.5. Assessment of cell death

A thin strip of epidermal peeling from the coleoptile region of pearl millet was

peeled out and immersed in a solution of 0.2% neutral red stain in 0.1 M potassium

phosphate buffer (pH 7.6) containing 0.5 M sucrose for 10 min and observed

microscopically. The cells, which took up neutral red stain and showed plasmolysis,

were considered viable and cells that remained colorless and did not show plasmolysis

were considered dead. Percentage of dead cells was calculated by averaging the 25

microscopic fields randomly in three experimental set up.

5.2.6. Hydrogen peroxide (H2O2) assay

The content of H2O2 was determined by spectrophotometric method according to

(Capaldi and Taylor, 1983). Fresh weight 0.5 g of seedlings were homogenized in 5%


86

trichloroacetic acid (TCA). The homogenate was centrifuged for 25 min at 12, 000g at

4°C. The pH of supernatant samples was adjusted to 3.6. Reaction was performed in the

mixture containing 0.2 ml of supernatant, 0.1 ml of 3.4 mM 3-methyl-2-benzothiazoline

hydrazone (MBTH) in 3.32 mM formaldehyde and 0.5 ml horseradish peroxidase

solution in 0.2 M acetate buffer pH 3.6. After 2 min of incubation, reaction was

terminated through addition of 1.4 ml of 1M HCl. Changes in the absorbance were

measured at 630 µM, 15 min after substrates mixing. The concentration of H2O2 in the

supernatant was estimated on the basis of the calibration curve.

5.2.7. H2O2 localization in tissue

H2O2 was detected in the tissues of coleoptile region of pearl millet at the

indicated time interval after inoculation with S. graminicola following the method of

Thordal- Christensen, 1997). The number of coleoptile regions showing localization of

H2O2 in 20 random microscopic fields were counted and the percentage was calculated.

5.2.8. H2O2 tissue detection

Tissue printing was performed essentially as described by Olson and Varner

(1993). To make a print of H2O2 in tested pearl millet coleoptiles, seedlings were hand-

cut with a razor blade and the cut surface was immediately pressed for 5 s to the

nitrocellulose (NC). Before tissue printing, the NC membranes were submerged in a

solution of 10% (w/v) starch and 1 M KI, subsequently dried at 30°C and stored in the

dark. Staining reaction was visible on the membrane 5–10 s after tissue print (Fig. 5.1)

5.2.9. Peroxidase enzyme assay

Peroxidase enzyme in seedlings was analyzed by extracting protein with 0.05 M

Tris buffer (pH 6.8) in pre-chilled (40C) mortar and pestle. The homogenate was

centrifuged for 15 min at a speed of 10,000 rpm at 40 C and the supernatant was used for

the study. Activity was determined using guaiacol as hydrogen donor as previously done

by Shivakumar et al. (2002). The reaction mixture (3 ml) consisted of 0.25% v/v

guaiacol in 10mM potassium phosphate buffer (pH 6.0) containing 100 mM H2O2. The

crude enzyme (5µl) was added to initiate the reaction, which was followed

spectrophotometrically at 470 uM per min.


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Coleoptile region of the seedlings

H2O2 Visualization Peroxidase

Hand cut

Drying

5.2.10. Peroxidase tissue printing

Peroxidase tissue printing was done according to the procedure of Olson and

Varner (1993). The NC membranes were conditioned in buffer (0.025 M Tris, 0.192 M

glycine, 0.1% SDS, pH 8.3) containing 20% methanol and dried at room temperature. To

detect peroxidase, the fragments of membranes with the tissue prints were incubated in

buffer pH 7.6 comprising: 25 mM Tris, 75 mM NaCl with addition of 0.015% H2O2 and

0.04% diaminobenzidine (DAB). Enzymatic reaction was carried out in the dark at room

temperature (Fig. 5.1).

Fig. 5.1. Detailed procedural diagram for tissue printing of peroxidase enzyme and hydrogen peroxide visualization 5.2.11. Catalase activity

Catalase activity was measured spectrophotometrically by monitoring the

consumption of H2O2 at 240 nm for 1 min after adding a known amount of enzyme

extract to the reaction mixture (3 ml, 10 mM hydrogen peroxide in 10 mM potassium

phosphate buffer, pH 6.9). Catalase activity was expressed in terms of the change in

absorbance at 240nm (A240/ min/mg/ protein), according to Luck (1965).

H2O2 localization (starch, KI)

Tissue print


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5.2.12. NO generation

As described in the Chapter 3

5.2.13. Statistical analysis: All the experimental results were subjected to Duncan’s

Multiple New Range Test (DMRT). Data on percentages were transformed to arcsine and

analysis of variance (ANOVA) was carried out with transformed values. The means were

compared for significance using DMRT (P=0.05).

5.3. Results

5.3.1.Observation for expression of HR

It was observed in the form of brown necrotic spots/streaks at pathogen infection

sites such as root and coleoptile regions of the seedlings. The development of HR was

very rapid in SNP treatments as well as in resistant seedlings treated seedlings. In the

SNP treatment, 69% seedlings recorded HR, which was followed by resistant cultivar

with 65.4% seedlings showing HR at 48hpi. Whereas the untreated susceptible seedlings

recorded 29.2% HR at similar point. However, prior treatment with C-PTIO affected HR

expression radically, in which resistant seedlings recorded 18% and SNP treatment

recorded 17% HR (Fig. 5.2a and 5.2b).

01020

30405060

7080

4 8 12 24 48

Time after inoculation (h)

Seed

ling

with

HR

exp

ress

ion

(%)

Resistant

SNP

Resistant+C-PTIO

SNP+C-PTIO

Susceptible

Fig. 5.2a. Hypersensitive response in pearl millet seedlings treated with NO donors to downy mildew pathogen inoculation. (Bars indicate the standard error at P=0.05).


89

Fig. 5.2b. Phenotypic expression of HR in coleoptile of pearl millet seedlings treated with 1mM SNP (A) untreated susceptible (B) at 24 h post inoculation.

5.3.2. Dose dependent action of SNP on HR expression

Effect of different dose of SNP, 0.5, 1, 1.5, 2, 2.5 and 5 mM treatment was

studied on HR expression. At the concentrations as minimal of 0.5mM, 37% seedlings

recorded HR response and at 1 mM, highest of 81% HR was recorded which was found

to be optimum level of treatment for maximum HR expression. Treatment with above to

this concentration negatively modulates HR expression. SNP treatment at the

concentration of 2mM, 40% HR was recorded and it decreased to 4% of HR at 5mM

(Fig. 5.3)

0

20

40

60

80

100

0.5 1 1.5 2 2.5 5

SNP concentrations in milli molar

Hyp

erse

nsiti

ve r

eact

ion

(%)

Fig. 5.3. Dose dependent action of NO donor on expression of hypersensitive response in pearl millet seedlings to downy mildew pathogen inoculation. (Bars indicate the standard error at P=0.05)

A B


90

5.3.3. Observation for expression of cell death

In seedlings, cell death is one of the major resistant reactions against pathogen

infection and so it was assessed in the tissues of resistant, induced resistant and

susceptible seedlings at different time intervals. Cell death was very prominent in

resistant seedlings with 65.8% seedlings showing cell death at 48hpi, similarly in the

seedlings after SNP seed treatment in which 68.5% cell death was observed at similar

time point. Prior treatment with NO scavenger reduced the cell death in resistant as well

as induced resistant seedlings in which it was 24 and 17% cell death. However, untreated

seedlings recorded 25% cell death upon pathogen inoculation (Fig 5.4a and 5.4b).

0

20

40

60

80

100

4 8 12 24 48


Cel

l dea

th a

t nec

rosis

site

(%)

Resistant

SNP

Resistant+C-PTIO

SNP+C-PTIO

Susceptible

Fig. 5.4a. Effect of NO donor treatment on cell death in coleoptile tissues of pearl millet seedlings to downy mildew pathogen inoculation. (Bars indicate the standard error at P=0.05).

Fig. 5.4b. Cell death at the necrotic region of HR formation in coleoptile of the pearl millet seedlings to downy mildew pathogen inoculation; A. Susceptible untreated, B. SNP treatment.


91

5.3.4. Hydrogen peroxide (H2O2) assay

Spectrophotometric analysis showed that H2O2 is present in resistant as well as

inducer treated seedlings. It was found that increased concentrations of SNP at 5 mM

markedly affect the level of H2O2 after pathogen inoculation. Resistant seedlings and

induced resistant seedlings recorded 9.8 µM and 6.4 µM H2O2 at 24 hpi respectively, in

similar point susceptible seedlings recorded 3.2 µM of H2O2. However, treatment of SNP

at 5 mM reduced the H2O2 accumulation in which it was recorded 0.56 and 1.2 µM of

H2O2 at 4 and 24 hpi respectively. But co-treatment with C-PTIO recorded the 2.5 µM

H2O2 at 24hpi (Fig. 5.5a).

0

2

4

6

8

10

12

0 4 8 12 16 20 24


H2O

2 um

ol 1

00 u

g/pr

otei

n

ResistantSNP (1 mM)SNP(5 mM)SNP (5mM)+C-PTIOSusceptible

Fig. 5.5a. The level of hydrogen peroxide in NO treatments of pearl millet seedling to downy mildew pathogen inoculation. (Bars indicate the standard error at P=0.05). 5.3.5. Observation of hydrogen peroxide localization

In the time-course study of hydrogen peroxide localization, it is clearly indicated

that NO donor SNP treatment to seedlings down-regulates the H2O2 localization.

Resistant seedlings recorded 69% H2O2 localization at 48hpi, but treatment with C-PTIO

enhances the rapidity and percentage of cells with higher localization. In SNP-treated

plants, mere , 24% of cells recorded H2O2 localization at 48hpi, where as in initial hours

of treatment, up to 8hpi, only 5% of cells showed its localization. While, SNP+C-PTIO

recorded relatively higher amount of H2O2 localization, in which it was recorded in 45%


92

cells. Whilst, untreated susceptible seedlings it recorded in 29% of cells (Fig. 5.5b and

5.5c).

0

20

40

60

80

100

4 8 12 24 48


Per

cent

cel

ls w

ith H

ydro

gen

pero

xide

loca

lisat

ion

Resistant

SNP

Resistant +C-PTIO

SNP+C-PTIO

Susceptible

Fig. 5.5b. Effect of NO on H2O2 localization in coleoptile of pearl millet seedlings to downy mildew pathogen inoculation. (Bars indicate the standard error at P=0.05).

Fig. 5.5c. H2O2 detection in longitudinal section of pearl millet seedlings coleoptile after SNP treatment at 5 Mm (A) and SNP at 1 mM (B) to downy mildew pathogen inoculation. 5.3.6. H2O2 tissue print detection

Analysis carried out with tissue printing method confirmed that H2O2 is present in

both resistant and susceptible of inoculated and uninoculated seedlings. However, the

intensity was higher in the inoculated resistant coleoptiles. On the other hand, seedlings

raised after treatment with NO donor SNP at 5 mM concentrations very slight printings

were seen indicating that NO treatments scavenge the H2O2 in seedlings. Conversely, at 1

mM concentration of SNP, a moderate marking of H2O2 was recorded. It was noticed that

there are quite substantial differences in the level of H2O2 among treatments of both nitric


93

oxide present and deficient situations. A marked decrease of H2O2 concentration was

observed in NO donor SNP treated seedlings compared to control and NO scavenger

treated seedlings (Fig. 5.6).

Fig. 5.6. H2O2 detection in transverse section of coleoptile of pearl millet seedlings; A. resistant, B. SNP at 1 mM C. Untreated susceptible, D. C-PTIO

5.3.7. Peroxidase activity

Different sets of treatments were subjected for assessing the peroxidase activity.

The varied level of activity was observed with the different time intervals of post

inoculation. In the resistant seedlings maximum activity was recorded after 4 hpi and it

was 3 folds higher than the control. However, C-PTIO treatment prior pathogen

inoculation slightly enhanced the peroxidase activity and maintained transiently through

out the experimental period. Very interestingly, NO donor SNP seed treatment recorded

decreased enzyme activity lesser than the susceptible seedlings. But the co-treatment with

C-PTIO did not effect peroxidase activity in the seedlings indicated the possible action of

NO on peroxidase activity, which indirectly negates the hydrogen peroxide activity.

However, susceptible seedlings recorded 2-folds lesser activity compared to resistant

seedlings (Fig. 5.7).


94

0

40

80

120

0 4 8 12 24 48


Pero

xida

se a

ctiv

ity; O

D @

470n

m/m

g/pr

otei

n/m

inResistant

Resistnat + C-PTIO

SNP

SNP +C-PTIO

Susceptible

Fig. 5.7. Effect of NO donor seed treatment on peroxidase activity in pearl millet seedlings to downy mildew pathogen inoculation. (Bars indicate the standard error at P=0.05).

5.3.8. Tissue print of peroxidase

Histo-chemical detection of peroxidase by tissue printing was studied in order to

confirm the qualitative effect of NO on peroxidase. Analyses of tissue prints revealed a

significant increase of peroxidase activity in resistant and also in NO scavenger C-PTIO

treated seedlings as compared to the SNP treated after incubation, particularly high

activity of peroxidase, visible as intense stain reaction, was found in the central part of

coleoptile markings. It has indicated that NO possibly affect the peroxidase regulate

machinery and inhibited the process of catalysis of hydrogen peroxide other than its

direct action on H2O2 to form peroxynitrate (Fig. 5.8)

Fig. 5.8. Distribution of peroxidase in transverse section of coleoptile of pearl millet seedlings to downy mildew pathogen inoculation; (A) Resistant (B) C-PTIO, (C) SNP and (D) Susceptible untreated.


95

5.3.9. Catalase activity

A significant increase in catalase activity was recorded in the seedlings raised

after the SNP treatment with 2-folds higher than the untreated control and also it was

recorded comparatively higher than the resistant cultivar. But prior treatment with C-

PTIO decreased the catalase specific activity both in resistant cultivar and SNP treatment,

which is similar to level of untreated control seedlings that indicate NO generation has

direct action on hydrogen peroxide by cleavage effect (Fig 5.9).

0

2

4

6

8

10

0 4 8 12 16 20 24


Cat

alas

e ac

tivity

24

0nM

/min

/mg/

prot

ein

Resistant

Resistant+C-PTIO

SNP

SNP+C-PTIO

Susceptible

Fig. 5.9. Time course study to assess the effect of NO on catalase activity in pearl millet seedlings to downy mildew pathogen inoculation. (Bars indicate standard error at P=0.05)

5.3.10. NO generation

Generation of NO at the time of HR expression was analyzed in the pearl millet

seedlings upon pathogen inoculation using different treatments as described earlier. The

rapidity of NO generation was high in the resistant and seedlings raised after seed

treatment with SNP in which it is recorded 6.1 and 7 µM NO generation at 4 hpi

respectively. But the treatment of C-PTIO, NO generations largely come down in which

recorded 1.5 and 1.8 µM of NO generation. Whereas untreated susceptible control

recorded 1.5 µM. When NO generation reached to minimum of 3 µM at 8hpi the

initiation of HR was observed in the susceptible seedlings also. Conversely, prior

treatment with C-PTIO negated the NO generation in both resistant as well as SNP


96

treated seedlings and affect HR expression. This indicated the possible role of NO in HR

(Fig. 5.10 and Fig. 5.11)

0

4

8

12

16

20

0 4 8 12 24 48


NO

gen

erat

ion

(µM

)Resistant

SNP

Resistant+C-PTIO

SNP+C-PTIO

Susceptible

Fig. 5.10. NO accumulation in pearl millet seedlings during HR formation after downy mildew pathogen inoculation. (Bars indicate the standard error at P=0.05).

Fig. 5.11. NO localization at the necrotic region of HR formation in coleoptile of the pearl millet seedlings to downy mildew pathogen inoculation.

5.4. Discussion

Attempted infection of plants by pathogen elicits a battery of defenses often

accompanied by the collapse of challenged host cells which is referred to as

hypersensitive cell death. This hypersensitive cell death results in a restricted lesion

delimited from surrounding healthy tissue and is thought to contribute to pathogen

restriction by limiting the nutrients supply.


97

In the present study, it was demonstrated that NO is another important molecule

required along with H2O2 for expression of HR. In an attempt to comprehend the

dynamics of NO level for expression of HR, it was understood that NO level in tissues

with 5-6.5 uM is optimum up to which maximum of HR was recorded, beyond to that

concentration, HR expression become weaken. This is attributed to the fact that NO level

more than 6 uM in tissues negatively modulates the H2O2 level, by acting on its

biosynthetic pathway as it is exemplified in peroxidase down regulation at increased

dosage of SNP. As catalytic action of peroxides activity weaken, H2O2 production

declined and HR expression was poor. On the other hand, at increased dosage of SNP it

enhanced the level of catalase, which is known to catalyze the rapid conversion of H2O2

to dioxygen and water, hence the concentration of H2O2 become deficient thus rapidity of

HR expression in higher dosage of SNP treated seedlings decreased. Thus, it was

concluded that HR response was modulated by balanced accumulation of NO and H2O2

upon pathogen inoculation and together act synergistically. Previously, Lamb et al.

(1997) demonstrated that oxidative burst is necessary but not sufficient to trigger host cell

death and NO cooperates with ROIs in the activation of hypersensitive cell death. Similar

results were reported in earlier experiments. In the Botrytis cinera and tomato

interactions, elevated NO concentration in tomato leaves strongly decreased hydrogen

peroxide concentration without affecting other studied ROS (Małolepsza and Sylwia

Rozalska 2005). In the present study, it was reported that lower NO production at the

onset of a pathogen infection reduced the rapidity and percentage of HR expression.

Further, rapidity of HR expression was also found to depend on a minimum of 3.5 uM of

NO level, which largely helps in attaining the resistance. Previously, Inhibitors of NO

synthesis treatment reduced the HR expression of Arabidopsis leaves infected with

Pseudomonas syringae pv. maculicola. Further, it was inferred that a poised production

of NO is necessary to trigger the HR and considered as an essential player in the process

of HR development (Mur et al., 2006). Pathogen-induced production of H2O2 and NO in

plant cells has been shown to regulate the HR and cell death in Arabidopsis and tobacco

plants (Delledone et al., 1998; Durner et al., 2000). Previously NO production has been

shown during the HR elicited in suspension cultures of Arabidopsis inoculated with

Psuedomonas syringae pv. maculicola (Clarke et al., 2000) and tobacco cultures


98

challenged with Psuedomonas syringae pv. tomato (Conrath et al., 2004). Activation of

the HR is part of a highly amplified and integrated signal system that also involves

salicylic acid and perturbations of cytosolic Ca2+ to trigger defense mechanisms and to

mediate the establishment of systemic immunity.

Previously, NO involvement in execution of HR using inducer ‘elicitin’ in

tobacco was also proved (Yamomoto et al., 2004). Similarly, enhancing the NO in the

system by addition of SNP at millimolar concentrations, cause cell death leading to HR in

soybean suspension cultures after inoculation with Pseudomonas syringe (Mur et al.,

2005).

NO involvement in expression of HR was further supported by the results after

using specific NO scavenger and NOS inhibitors, in which, prior treatment with these

inhibitors completely abolish the expression of HR. Although these inhibitors indirectly

allowed to amplify the accumulation of H2O2, expression of HR was not recorded, which

indicate the direct role of NO in expression of HR. Previously, a significant but transient

NO burst was observed in barley epidermal cells attacked by the powdery mildew fungus,

Blumeria graminis f. sp. hordei just prior to their HR-associated collapse (Prats et al.,

2005). As in many studies, a NO-scavenger, C-PTIO was used to suppress the fluorescent

signal and also delay cell death, suggesting a contribution of NO to the HR process. It

should be noted that the reaction product of cPTIO and NO, cPTI (2-(4-carboxyphenyl)-

4,4,5,5 tetramethylimidazole-1-oxy-3-oxide) itself suppressed cryptogein-elicited cell

death in tobacco cultures without scavenging NO (Planchet et al., 2005). Hence, although

C-PTIO remains valuable in establishing that NO generation is being detected, more than

suppression with C-PTIO may be required if seeking to correlate a reduction in NO with

a physiological effect.

Thus, the role of NO in a particular phenomenon, requires confirmation through a

multitude of approaches; actual NO measurements, the use of pharmaceutical agents

which scavenge NO or suppress NO generation, as well as mutants exhibiting reduced or

elevated NO levels. In another approach, genetic evidence of a role for NO in the HR was

provided through the expression of a nitric oxide dioxygenase (NOD), encoded by hmp

from E. coli in transgenic Arabidopsis (Zeier et al., 2004). NOD catalyzed the

dioxygenation of NO to nitrate and NOD-expressing transgenic lines challenged with


99

avirulent P. s. pv. tomato avr showed reduced NO production and, crucially, delayed cell

death. Other work shows that treating plant tissues with NO donors initiates chromatin

condensation and DNA fragmentation as reported by in situ terminal dioxynucleotide

transferase-mediated dUTP nick end labeling TUNEL (Clarke et al., 2000). Further, the

initiation of NO-mediated cell death can be suppressed with a caspase-1-inhibitor (Clarke

et al., 2000), and expression of a cysteine (cystatin-class) protease inhibitor (AtCYS1) in

transgenic tobacco suppressed cell death initiated by NO or attack by avirulent bacteria

(Belenghi et al., 2003).

In the present study, cell death during the HR is under control of a balanced

accumulation of NO and H2O2 that has pathophysiological effect by limiting the pathogen

progress. Striking evidences of NO involvement in expression of HR was demonstrated

in present study, and minimum of 4-6 µM NO is required for expression of HR at onset

of pathogen infection. At higher concentrations of NO in tissues negatively modulates the

HR expression by inhibiting the biosynthetic pathways of catalyzing H2O2 formation in

tissues.

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C h a p t e r 5 Studies on nitric oxide dependent ...shodhganga.inflibnet.ac.in/bitstream/10603/76626/9/chapter 5.pdf · Chapter 5: Nitric oxide dependent hypersensitive reaction