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CHAPTER 5 - SECTION 2

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					                        CHAPTER 5 - SECTION 2


   Potentiation of heat-induced Hsp70/Hsc70 accumulation by

                 salicylic acid is associated with decreased

                      programmed cell death in tobacco



INTRODUCTION



The heat shock response is ubiquitous and highly conserved - in all organisms from

bacteria to plants and animals - as an essential defence mechanism for protection of cells

from a wide range of harmful conditions, including heat shock, inhibitors of energy

metabolism, heavy metal- or oxidative stress, fever or inflammation (Jolly and Morimoto,

2000). The cellular response to stress at the molecular level is characterised by the induced

synthesis of heat shock proteins (HSP). Those that function as molecular chaperones aid in

protein folding, translocation and refolding of intermediates that were either misfolded or

denatured (Pirkkala et al., 2001). A key HSP family is the 70 kDa group of proteins,

consisting of both constitutive (Hsc70) and inducible members (Hsp70). Hsp70, in both

mammalian and plant cells, is involved in thermotolerance (Frossard, 1999; Harrington et

al., 1994; Vierling, 1991) and provides protection against oxidative stress (Chong et al.,

1998; Härndahl et al., 1999). In mammalian cells, Hsp70 protects mitochondria from

oxidative injury (Polla et al., 1996) and ATP depletion (Vayssier and Polla, 1998; Mallouk

et al., 1999), while it suppresses apoptosis (Beere and Green, 2001; Jäättelä et al., 1998).




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Programmed cell death (PCD) is an innate and evolutionary conserved process by which

cells systematically inactivate and disassemble their structural and functional components

to effect their own demise. Apoptosis (a form of PCD in metazoans) is divided into three

distinct stages. During the commitment stage a cell receives a lethal apoptotic stimulus and

becomes irreversibly committed to death. The execution stage involves rapid changes

within the cell including condensation of nuclear chromatin, cytoplasmic shrinkage,

membrane blebbing, nuclear fragmentation, the formation of apoptotic bodies and

translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane.

These changes are the result of the activation of various enzymes, including the nucleases,

caspases and lipases (Cohen, 1997). The clearance phase involves phagocytosis and

degradation of apoptotic bodies by macrophages or neighbouring cells. Mitochondria are

central to the execution of cell death and include events such as release of apoptosis

inducing factor (AIF) (Susin et al., 1999) or the caspase activator(s) (i.e. cytochrome c),

disruption of electron transport and energy metabolism, loss of mitochondrial membrane

potential (∆Ψm) and regulation of mitochondrial permeability transition pores via the Bcl-2

family (Zamzami and Kroemer, 2001). Uncoupling of electron transport from ATP

production leads to a decrease in ∆ψm and subsequent production of ROS, and concomitant

alterations of cellular redox states are considered to be important signalling components of

the induction phase of apoptosis (Jabs, 1999).



The plant response to attempted infection by microbial pathogens is often accompanied by

rapid cell death in and around the initial infection site, a reaction known as the

hypersensitive response (HR). This response is associated with restricted pathogen growth

and represents a form of PCD (Lam et al., 2001). Some facets of apoptosis in animals are




                                            130
shared with PCD in plants, such as PS exposure (O’Brien et al., 1998a) and protease

activation or activity (Sun et al., 1999; del Pozo and Lam, 1998).



In plants, salicylic acid (SA) has been shown to play an important role in the activation of

various plant defence responses following a pathogen attack. It is involved in the induction

of local and systemic disease resistance (Dempsey et al., 1999), the potentiation of the

oxidative burst and host cell death - via a self-amplification loop that involves increasing

SA synthesis and H2O2 accumulation (Van Camp et al., 1998) - and the containment of

pathogen spread. Recent data suggest a complex role of SA and an involvement in the

activation of the HR at primary infection sites and in fact, appears to define the rate of cell

death in R-gene triggered HR reactions (Shirasu and Schulze-Lefert, 2000). In humans SA

or aspirin, used as non-steroidal anti-inflammatory drugs (Kataoka et al., 1997), inhibit

cyclo-oxygenase enzymes and prostaglandin synthesis that promote pain and fever, and has

been shown to modulate apoptosis through changes in mitochondria.



In Chapter 5 section 1, it was shown that SA at low concentrations during HS potentiates

the induction of Hsp70/Hsc70, while SA at high concentrations on its own induces

Hsp70/Hsc70. In plants, the effect of SA-modulated Hsp70 expression on apoptosis has not

been investigated. In this section the effects of salicylic acid-mediated increases in

Hsp70/Hsc70 expression, whether potentiation or induction, on events associated with

programmed cell death were investigated. This was approached by evaluating the effect of

SA at normal temperatures or following HS on levels of Hsp70/Hsc70 and on

morphological and physiological changes associated with apoptosis (MMP, ROS,

phosphatidylserine exposure and DNA fragmentation) in Nicotiana tabacum protoplasts.




                                             131
MATERIALS AND METHODS



Cell Cultures

All chemicals were obtained from Sigma (St. Louis, MO), unless stated otherwise.

Suspension cultures of tobacco, N. tabacum, were maintained in liquid MS medium in

conical flasks on an orbital shaker at 25 ºC in the dark and subcultured at 10-day intervals.

To obtain protoplasts, cell walls of suspension cultures in logarithmic growth phase were

digested for two hours at 25 ºC in an enzyme mixture containing 1% cellulase, 0.3%

macerozyme (Yakult Honsha, Tokyo, Japan) and 0.05% Pectolyase Y-23 (Seishen

Pharmaceuticals, Japan), supplemented with 0.5 M sorbitol and 5 mM Ca(NO3)2. After

digestion, protoplasts were pelleted (100 x g, 5 min), washed twice in PWF, containing

1/10 strength VKM (Binding and Nehls, 1977) macro-nutrients and 2.69 mM NaCl

(O’Brien and Lindsay, 1993). Protoplasts were collected by centrifugation (80 x g, 4 min)

and resuspended in VKM until further treatments. Protoplast concentration was determined

by measuring OD600 nm at 0.5.



Treatments

Heat shock. Protoplasts (∼106 ml-1) were heat shocked (HS) (32 °C, 15 min) in a circulating

water bath and allowed to recover for 2 h 45 min at 25 ºC. Salicylic acid. Protoplasts were

incubated with 70 µM, 100 µM or 140 µM SA for a total incubation time of 3 h. Heat

shock plus salicylic acid. SA was added to cells prior to a 15 min heat shock and allowed

to recover for 2 h 45 min. Control cells were maintained at 25 °C.




                                            132
Protoplast Fixation and Permeabilization

After various treatments, protoplasts were fixed in 2% paraformaldehyde-PBS (4 ºC, 30

min) washed with PWF and dehydrated overnight at -20 °C in methyl cellusolve

(PiersolveTM, Pierce Chemical Co., Rockford, Ill.) (O’Brien et al., 1997). Protoplasts were

permeabilized (4 °C, 6 min) using 0.1% Triton X-100 prepared in PBS followed by

centrifugation (200 x g, 4 min, 4 ºC) and washed with PBS before immunolabelling.



Immunolabelling with Hsp70/Hsc70

A mouse monoclonal antibody directed against both the constitutive Hsc70 (73 kDa) and

inducible Hsp70 (72 kDa) isoforms of the Hsp70 family (SPA 820, Stressgen, Victoria,

Canada) served as the primary antibody (100-fold dilution). The primary antibody was

detected with a 50-fold dilution of a goat anti-mouse IgG R-Phycoerythrin (PE)-conjugated

F(ab’)2 fragment (Beckman Coulter, Brea, CA.). Non-specific labelling due to the

secondary antibody was detected by the omission of the primary antibody during the

labelling protocol. Unstained protoplasts were used as controls for autofluorescence in

protoplasts.



Measurement of mitochondrial membrane permeability (MMP) and reactive oxygen

species (ROS)

Cytofluorometric evidence supports mitochondrial alterations as an early apoptotic event

(Hirsch et al., 1998) and include changes in mitochondrial membrane permeability and

subsequent production of reactive oxygen species (ROS). Changes in mitochondrial

membrane permeability (MMP) were analysed by using the mitochondrial selective probe,

MitoTracker Red™ (Molecular Probes, Eugene, OR, USA). After the various treatments of

protoplasts (3 h), MitoTracker was added to a final concentration of 350 nM to viable


                                           133
protoplasts and incubated for 30 min. Protoplasts were washed and resuspended in VKM.

Fluorescence was measured on a 96-well fluorescence reader (Fluoroskan II, Labsystems,

Helsinki, Finland) at excitation 584 nm and emission 612 nm. The ionophore CCCP

(carbonyl cyanide m-chlorophenylhydrazone) that dissipates the mitochondrial membrane

potential (Koll et al., 2001), was used as a positive control for MMP. Protoplasts were

incubated with CCCP at a final concentration of 5 µM for 4 h followed by washing and

resuspension in VKM. MitoTracker was added and MMP determined as described above.

The washing step was included in order to minimise the observed quenching effect of

CCCP on MitoTracker (results not shown). Untreated protoplasts were used as

autofluorescent controls. To detect the generation of ROS, H2DCFDA (2’,7’-

dichlorodihydrofluorescein diacetate) (Molecular Probes, Eugene, OR, USA) (final

concentration 1 µM) was added to unfixed protoplasts after the various treatments,

incubated in the dark at room temperature for 10 min and analysed by flow cytometry.

Untreated protoplasts served as an autofluorescent control, while protoplasts treated with

H2O2 (130 µM) served as a positive control for ROS.



Detection of phosphatidylserine externalisation by annexin V binding

One of the characteristics of cells undergoing apoptosis is the loss of phosholipid

asymmetry of their plasma membranes and externalisation of phosphatidylserine (PS) to

the outer leaflet of the membrane. A sample of 500 µl protoplasts (∼106 ml-1) was analysed

for cell death, while the rest of the sample was fixed and used for TdT-mediated dUTP

nick-end labelling (TUNEL) or Hsp70/Hsc70 analysis. The apoptotic cells were stained

with a fluorescein conjugate of annexin V for 5 min according to the manufacturer’s

protocol (Annexin V FITC Kit, Immunotech, Marseille, France) before analysis.

Propidium iodide, which stains DNA in cells after entry through damaged membranes but


                                           134
is excluded by normal or apoptotic protoplasts possessing intact membranes, was added

together with annexin V and served to identify necrotic protoplasts (positive for both PI

and annexin V binding).



Determination of DNA fragmentation by TUNEL

In order to assess nucleosomal changes associated with apoptosis, DNA fragmentation was

detected by the TUNEL assay. Protoplasts were fixed and permeabilized as described

above, except that 45 mM sodium citrate was added to the Triton X-100 solution (O'Brien

et al., 1997). Cells were washed and incubated for 1 h at 37 °C with terminal

deoxynucleotidyl transferase (TdT) and fluorescein-labelled dUTP according the in situ

Cell Death Detection Kit (Roche, Mannhein, Germany) and analysed by flow cytometry

for DNA fragmentation. Positive controls consisted of protoplasts treated with DNAse I or

camptothecin (5 µM for 4 h).



Flow cytometry

For all flow cytometric analysis, unstained protoplasts consistently prepared under similar

physiological conditions were used as autofluorescent controls. Protoplasts were analysed

using an Epics Altra ESP flow cytometer (Beckman-Coulter, Miami, Florida) and an air-

cooled argon laser emitting light at 488 nm. PE was excited at 488 nm with fluorescence

emission measured using a 575 nm ± 10 nm bandpass filter, while FITC was excited at 488

nm with fluorescence measured at 525 nm ± 10 nm. Protoplasts were gated based on their

forward scatter (FS) versus side scatter (SS) properties, with the discriminator set on the

PE log fluorescence signal instead of forward scatter, to reduce background noise from

autofluorescence. The probes H2DCFDA and PI were excited at 488 nm, while




                                           135
fluorescence was measured using a 525 nm ± 10 nm bandpass filter and a 610 nm ± 10 nm

bandpass filter respectively.



Statistical analyses

ANOVA and comparison of means were performed using CoStat Software (CoHort

Software, Berkeley, CA, 1990). Differences in mean values were considered significant if

the least significant difference (LSD), calculated from the pooled variance at p < 0.05, was

exceeded. Data in Table 1 was analysed by Duncan’s multiple range test and are expressed

as means ± SEM of triplicate values and were considered significant at confidence levels

of 95%. Non-parametric correlation (Spearman’s roh) was performed on all data (Table 2).




RESULTS



The effect of SA on Hsp70/Hsc70 expression at normal temperatures or following

heat shock

In order to confirm previous results obtained by Western blot analysis with tomato cell

suspensions, a dose-response study to evaluate the effect of SA concentration on

Hsp70/Hsc70 accumulation was undertaken in tobacco (N. tabacum) protoplasts. The

effects of SA (70 µM, 100 µM or 140 µM) on Hsp70/Hsc70 expression in protoplasts are

shown in Fig. 1. SA did not induce Hsp70/Hsc70 accumulation at 70 µM (Fig. 1 A, green

line and Fig. 1 C) or 100 µM (Fig. 1 A, orange line and Fig. 1 C), but at high doses

(140 µM; Fig. 1 A, light blue line) an increase in Hsp70/Hsc70 accumulation (p < 0.05,

Fig. 1 C) in tobacco protoplasts was observed when compared to the controls (Fig. 1 A, red

line and Fig. 1 C).


                                            136
Cell number   A                                                                                   B




                                                                                    Cell number
                  Hsp70/Hsc70 PE-fluorescence                                                           Hsp70/Hsc70 PE-fluorescence


                     C
                                              120
                       Relative Hsp70/Hsc70 accumulation




                                              100                             ##
                                                                                                      ##

                                                           80       **
                                                                                                               **
                                                                                                                             C
                                                           60
                                                                                                                             HS

                                                           40


                                                           20


                                                           0
                                                                0        70                       100            140

                                                                              SA [µM]




              FIG. 1 The effect of SA on Hsp70/Hsc70 accumulation in tobacco protoplasts at normal
              temperatures or following HS. Representative histograms illustrate flow cytometric detection of
              Hsp70/Hsc70 fluorescence in protoplasts exposed to (A) various concentrations of SA (70 µM -
              green line, 100 µM – orange line or 140 µM – light blue line) for 3 h at normal temperatures or (B)
              to 70 µM SA in combination with heat shock (HS, 32 °C, 15 min, followed by 2 h 45 min recovery
              - orange line). Dark blue line (A and B) indicates background fluorescence (omission of primary
              antibody), red line (A and B) indicates control (non-heat shock) and green line (B) indicates heat-
              shocked sample. (C) Bar chart depicts mean values (n=6) of Hsp70/Hsc70 accumulation under
              various conditions. Significant differences of samples to control (C 0) is indicated by ** (p < 0.05)
              and to each SA control by ## (p < 0.05).

                                                                                   137
A significant increase in Hsp70/Hsc70 accumulation (p < 0.05) is observed in protoplasts

exposed to a heat shock (HS) when compared to the non-HS control (Fig. 1B, green line

vs. red line, Fig. 1 C). Hsp70/Hsc70 accumulation was significantly potentiated (p < 0.05)

in protoplasts exposed to 70 µM SA and HS simultaneously (Fig. 1 B, orange line) when

compared to protoplasts that received HS only (Fig. 1 B, green line). This potentiation was

also observed at 100 µM SA (Fig. 1 C) but not at 140 µM SA.



Effect of SA on mitochondrial membrane permeability (MMP) at normal

temperatures or following heat shock

Changes in mitochondrial functioning after various treatments were investigated in tobacco

protoplasts. Protoplasts treated with CCCP (5 µM, 3 h), an ionophore known to dissipate

the mitochondrial membrane potential, showed a significant decrease in MMP when

compared to untreated protoplasts. Protoplasts exposed to the various concentrations of SA

(70, 100 and 140 µM) for 3 h at normal temperatures showed no significant changes in

MMP (Fig. 2) when compared to control protoplasts that received no SA. MMP was

significantly increased in the presence of HS at a SA concentration of 70 µM and 140 µM,

compared to protoplasts that received only SA at the same concentrations. MMP is

significantly increased in all SA plus HS treatments when compared to the mean of the

control values obtained at all SA concentrations. HS per se did not change MMP

significantly compared to similar samples at normal temperatures.



Effect of SA on reactive oxygen species (ROS) at normal temperatures or following

heat shock

To investigate alterations of cellular redox states in protoplasts after various treatments, the

fluorescent probe, H2DCFDA was used.


                                              138
                               25                                                  #
                                                     #                             *
                                                                                                         C
                               20
                                                     *                 *
                                                                                                         HS




       Relative fluorescence
                                                                                                         CCCP

                               15


                               10
                                                                                                     *
                               5


                               0
                                        0        70                100             140        5 uM
                                                         SA [uM]


FIG. 2 Effect of SA on mitochondrial membrane permeability (MMP) in protoplasts at
normal temperatures or following heat shock (HS). Protoplasts were exposed to 70, 100 or
140 µM SA (3 h) alone (C) or in combination with HS (32 °C, 15 min, followed by 2 h 45
min recovery). Mito Tracker ™ (350 nM) was added for 45 min followed by analysis of
MMP. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a selective inhibitor of
mitochondrial respiration, was used as a positive control for inhibition of MMP. Values
represent the means of four replicates. Significant differences of samples to control (C 0) is
indicated by * (p < 0.01) and to each SA control by # (p < 0.01).


                               140

                               120
       Mean log fluorescence




                                                                                                         C
                               100                                                                       HS

                                80          *

                                60
                                                                   *
                                                     *
                                40                                             *

                                20

                                    0
                                        0       70           100             140         AF      130uM
                                                                                                 H2O2
                                                     SA [µM]


FIG. 3 Effect of SA on production of reactive oxygen species (ROS) in protoplasts at normal
temperatures or following HS. Protoplasts were exposed to SA (70, 100, 140 µM) for 3 h at normal
temperatures (C) or in combination with HS (32 °C, 15 min, 2 h 45 min recovery). The fluorescent
probe H2DCFDA, used to detect oxidative activity in cells, was added 10 min before flow
cytometric analysis. Values represent means of six replicates. Significant differences of samples to
HS control by * (p < 0.01). Hydrogen peroxide (130 µM) served as positive control.
Autofluorescence controls (AF) containing no probe is also shown.



                                                                       139
Autofluorescent controls (Fig. 3) were included to exclude false positives that might occur

due to fluorescence energy transfers. Protoplasts treated with 130 µM H2O2 showed a

significant increase in ROS production and were used as positive controls (Fig. 3). The

addition of H2O2 to the probe in the absence of protoplasts had no effect on fluorescence

(results not shown). Protoplasts exposed to increasing concentrations of SA for 3 h at

normal temperatures showed no significant increase in ROS when compared to control

protoplasts that received no SA (Fig. 3). Heat shocked protoplasts showed a significant

increase in ROS when compared to protoplasts kept at normal temperatures (Fig. 3, C 0 vs.

HS 0). Protoplasts exposed to increasing concentrations of SA (70, 100 and 140 µM) plus

HS showed a significant decrease in ROS when compared to protoplasts receiving HS

only. No significant differences in ROS production was observed in protoplasts treated

with the various concentrations of SA plus HS relative to their counterparts at normal

temperatures. However, at 70 µM and 140 µM SA plus HS a tendency of decreased ROS

was observed in all replicates (n=6) when compared to their HS counterparts.



Effect of SA on phosphatidylserine (PS) externalisation at normal temperatures or

following heat shock

To discriminate between apoptosis and necrosis in the various treated protoplasts, annexin

V binding in combination with PI was used. Representative cytograms of protoplasts

treated with HS (Fig. 4 B), 70 µM SA (Fig. 4 D), or both (Fig. 4 E) followed by annexin V

labelling and propidium iodide staining showed subpopulations of normal protoplasts with

neither annexin V or PI binding (lower left quadrant), apoptotic protoplasts, positive for

annexin V binding but negative for PI (upper left quadrant) and necrotic protoplasts,

positive for both annexin V and PI (upper and lower right quadrant). Gate settings were

adjusted to exclude debris.


                                           140
A



                                                     D




B




                                                     E




C




    FIG. 4 Effect of SA on apoptosis in tobacco protoplasts at normal temperatures or following HS.
    Phosphatidylserine externalisation determined by binding of annexin V-FITC and PI exclusion
    indicated apoptotic protoplasts, while PI staining indicated necrosis. Protoplasts were treated with
    70 µM SA alone (D) for 3 h or in combination with HS (32 °C, 15 min followed by recovery for 2
    h 45 min) (E). Untreated (A), heat shocked (B) and H2O2-treated (130 µM) protoplasts (C) served
    as controls.




                                                    141
Protoplasts treated with hydrogen peroxide (130 µM) were used as a positive control for

cell death (Fig. 4 C), and showed 40.3 % apoptosis and 36.9 % necrosis respectively.

Control protoplasts showed a subpopulation of at least 88.7 % normal protoplasts, with a

small subpopulation of 1.5 % apoptotic and 9.8 % necrotic ones (Table 1). Incubation of

protoplasts with 70 µM SA increased the apoptotic population to 8.5 % (p < 0.001; Table

1, C 70) and necrosis to 14.03 % (p < 0.05; Table 1, C 70), while the normal population

was 77.47 % (p < 0.05; Table 1, C 70). At higher SA concentrations (100 and 140 µM),

necrosis increased significantly in protoplasts, while control and apoptotic protoplasts

decreased. (Table 1, C 100 and C 140). Protoplasts exposed to SA (70 µM) in combination

with HS, showed a decrease in apoptosis (2.34 %, p < 0.001; Table 1, HS 70) and in

necrosis in comparison to non heat-treated protoplasts exposed to SA only.



Table 1 The influence of SA with or without HS on protoplast survival or death.
Protoplasts were exposed to various concentrations of SA (70, 100 and 140 µM) (3 h)
alone at normal temperature (C) or in combination with heat shock (HS; 32 °C, 15 min,
followed by 2 h 45 min recovery). Normal, apoptotic or necrotic protoplasts were
identified based upon phosphatidylserine externalisation and PI staining quantified by flow
cytometry. Values represent the mean (SEM) of three replicates.

                                                  % Protoplasts
Treatment              Normal                 Apoptotic              Necrotic
C0                     88.70 ± 2.95           1.50 ± 0.83            9.80 ± 3.23
C 70                   77.47 ± 4.24**         8.50 ± 3.24***         14.03 ± 3.24
C 100                  80.26 ± 7.53           4.13 ± 1.54            15.61 ± 5.23
C 140                  76.62 ± 6.08           3.79 ± 1.82            19.59 ± 6.25**
HS 0                   81.70 ± 3.83           4.17 ± 1.13            14.13 ± 4.07
HS 70                  85.37 ± 2.99##         2.34 ±1.13###          11.79 ± 3.09
HS 100                 81.84 ± 3.98           3.23 ± 1.24            14.93 ± 3.85
HS 140                 80.80 ± 4.34           3.87 ± 0.85            15.33 ± 4.37
** (p<0.05), *** (p<0.001) relative to C0
## (p<0.05), ### (p<0.001) relative to SA treated equivalent

                                            142
Effect of SA on DNA fragmentation at normal temperatures or following heat shock

Early changes in nucleosomal DNA in the various treated protoplasts was investigated

with the TUNEL assay. Histogram overlays of DNA fragmentation (Fig. 5 A) showed

protoplasts with 78 % and 35 % DNA fragmentation that were treated with DNAse I (1000

U, 5 min, orange) or with camptothecin (1 µM, 4 h, yellow) respectively.



A significant increase (p < 0.05) of DNA fragmentation in protoplasts exposed to 70 µM

SA (Fig. 5 C, C 70) was observed in comparison to protoplasts that received no SA (Fig. 5

C, C 0), but not in protoplasts exposed to 100 µM or 140 µM SA. However, a significant

decrease in DNA fragmentation was observed in protoplasts exposed to 100 µM and 140

µM SA when compared to protoplasts that received 70 µM SA (Fig. 5 C, C 100 and C 140

vs. C 70). Protoplasts exposed to 70 µM SA in combination with HS showed a significant

decrease (p < 0.05) in DNA fragmentation (Fig. 5 B, blue; Fig 5 C, HS 70) when compared

to protoplasts exposed to only 70 µM only (Fig. 5 B, purple; Fig. 5 C, C 70). This decrease

in DNA fragmentation was not observed in protoplasts exposed to 100 µM or 140 µM SA

plus HS when compared to non-HS counterparts.



Correlation analysis of the various parameters evaluated

The relation between Hsp70/Hsc70 levels and the various parameters of cellular viability

and cell death type at different temperatures for all SA concentrations is shown in Table 2.

A significant negative relation between SA-modulated heat-induced Hsp70/Hsc70 potenti-

ation and apoptosis was observed. The induction of Hsp70/Hsc70 at high doses of SA at

normal temperatures was not accompanied by increased ROS or MMP, but correlated

positively. At 70 µM SA plus HS, a negative correlation (r = - 0.9993, p < 0.05) between

Hsp70 accumulation and DNA fragmentation was observed.


                                            143
          A                                                             B

   300-                                                       200-
Counts




                                                              Counts
                                     Log FITC                                Log FITC
         C

                                40

                                35

                                30                                      **
          % DNA fragmentation




                                                                                        C
                                                  ##
                                25                **                                    HS

                                20
                                            ##
                                15

                                10

                                 5

                                 0
                                        0        70                    100   140

                                                  S A [µM ]

 FIG. 5 The effect of SA on DNA fragmentation in tobacco protoplasts at normal temperatures or
 following HS. Histogram overlays show DNA fragmentation detected by TUNEL in (A) positive
 controls consisting of protoplasts exposed to camptothecin (yellow) or DNAse (orange) or (B)
 protoplasts consisting of the control (green), treated with 70 µM SA at normal temperature (purple)
 or 70 µM SA plus HS (blue). Bar charts depict mean % DNA fragmentation (n=3) in protoplasts
 exposed to various SA concentrations (0, 70, 100 and 140 µM) for 3 h at normal temperatures or in
 combination with HS (32 °C, 15 min, followed by 2 h 45 min recovery). Significant differences
 of samples to HS control (HS 0) is indicated by ** (p < 0.05) and relative to each SA
 control by ## (p < 0.05). DNA fragmentation increased at 70 µM SA but was significantly
 suppressed when protoplasts were exposed to SA in combination with HS.



                                                        144
Table 2 Correlation analysis of Hsp70/Hsc70 accumulation with parameters of cellular

viability and cell death type in protoplasts exposed to different SA concentrations at

normal (C) and elevated temperatures (HS).



X variable                Y variable                 Correlation     Probability      Signifi-
                                                     coefficient                       cance
Hsp70      (C)            MMP                           0.099         9.00 x 10-1        ns

Hsp70                     ROS                            0.686        7.50 x 10-1       ns

Hsp70                     normal                        -0.840        1.50 x 10-2        *

Hsp70                     apoptosis                      0.687        7.64 x 10-1       ns

Hsp70                     necrosis                       0.969        3.06 x 10-3       **

Hsp70                     DNA fragmentation              0.689        7.81 x 10-1       ns

Hsp70      (HS)           MMP                           -0.338        6.61 x 10-1       ns

Hsp70                     ROS                           -0.786        2.13 x 10-1       ns

Hsp70                     normal                         0.829        1.70 x 10-2        *

Hsp70                     apoptosis                     -0.992        7.87 x 10-4       ***

Hsp70                     necrosis                      -0.676        3.23 x 10-1       ns

Hsp70                     DNA fragmentation              0.651        3.49 x 10-1       ns

*** = significant at p < 0.001, ** at p < 0.05, * at p < 0.01, ns = not significant




DISCUSSION



This section showed that low doses of salicylic acid (70 µM) did not increase

Hsp70/Hsc70 accumulation but led to increased apoptosis in protoplasts. Increased

expression of Hsp70/Hsc70 in protoplasts at normal temperatures due to high doses of SA


                                              145
was accompanied with increased necrosis. Heat-induced Hsp70/Hsc70 potentiation at low

doses of salicylic acid correlated negatively with apoptosis.



A dose-responsive effect of SA on Hsp70/Hsc70 accumulation (Fig. 1) in tobacco (N.

tabacum) protoplasts was observed: low doses of SA did not induce Hsp70/Hsc70, while

SA doses considered to be phytotoxic induced Hsp70/Hsc70. These dose-responsive

effects agree with previous findings in N. plumbaginifolia protoplasts (Chapter 5, section

1) and tomato cell suspension cultures as detected by Western blot analysis (Cronjé and

Bornman, 1999; Chapter 3, section 2). The induction of Hsp70/Hsc70 at high doses of SA

was not accompanied by increased ROS or MMP, but correlated positively with increased

necrosis. High doses of SA in plant cells can be phytotoxic and has been associated with

increased membrane permeability in tomato cell suspensions (Cronjé and Bornman, 1999).

Protein damage caused by high doses of SA most likely mediated the induction of

Hsp70/Hsc70 observed at 140 µM SA. It has been proposed that the induction of Hsp70 is

an antioxidant strategy to protect against oxidative damage in plants (Härndahl et al., 1999;

Banzet et al., 1998) and human cells, while mitochondria appear to be the target for this

protection (Vayssier and Polla, 1998, Polla et al., 1996). This is on par with decreased ATP

levels that are associated with the induction of Hsp70 (Mallouk et al., 1999). Since SA has

been known to decrease oxidative phosphorylation and ATP synthesis in tobacco cells (Xie

and Chen, 1999), this may well be the reason for Hsp70/Hsc70 induction in tobacco

protoplasts. The absence of significant differences of both ROS and MMP between control

protoplasts (no SA) and those that received SA could suggest changes in ROS and MMP

occurring earlier than the interval studied (Jabs, 1999). Transient increases in ROS

production associated with the oxidative burst and HR during an incompatible plant-




                                             146
pathogen interaction, is usually rapid and is further influenced by the host type and

challenging factors (Wojtaszek, 1997).



The ability of low doses of SA (70 µM) to increase apoptosis (increased

phosphatidylserine externalisation (Fig. 4) and nuclear DNA fragmentation (Fig. 5)) agrees

with previous results observed in tobacco (N. plumbaginifolia) protoplasts (O’Brien et al.,

1998a). It has been reported that SA is able to induce apoptosis in mammalian cells by the

activation of caspase-3 and p38 mitogen-activated kinase (Bellosillo et al., 1998; Wong et

al., 2000; Schwenger et al., 1997) and the mitochondrial permeability transition in

mammalian cells (Al-Nasser, 1999; Trost and Lemasters, 1997). Whether SA is able to

induce apoptosis in plants via these mechanisms is unknown. It may well be that the

decline in ATP caused by SA (Xie and Chen, 1999) is the signal perceived by

mitochondria to activate apoptosis (Lam et al., 2001). The absence of changes in MMP

(Fig. 2) or ROS (Fig. 3) during the interval where increased apoptosis (PS externalisation

and DNA fragmentation) was observed, may indicate that these changes occur early,

preceding the morphological events.



Protoplasts exposed to 70 µM SA in combination with HS showed significant potentiation

of heat-induced Hsp70/Hsc70 accumulation (Fig. 1). This was also previously observed in

tomato cells (Chapter 3, section 2; Cronjé and Bornman, 1999). The partial activation of

HSF1 contributing to enhanced expression of Hsp70 in mammalian cells (Amici et al.,

1995; Fawcett et al., 1997) might also be the mechanism of potentiation in plants. The

potentiation of heat-induced Hsp70/Hsc70 was accompanied by a significant increase in

MMP (Fig. 2) relative to the control protoplasts (no HS, 70 µM SA) and a significant drop

in ROS when compared to HS control (Fig. 3). Potentiation was negatively correlated with


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phosphatidylserine externalisation and also accompanied by a significant decrease and

DNA fragmentation (Fig. 5). These results suggest that the SA-mediated potentiation of

heat-induced Hsp70/Hsc70 expression could oppose apoptosis in protoplasts. In

mammalian cells, Hsp70 has been shown to suppress apoptosis by preventing the assembly

of the apoptosome complex (Beere et al., 2000; Beere and Green, 2001; Garrido et al.,

2001). Increased expression of Hsp70 was also shown to decrease phosphatidylserine

externalisation in monocytes (Guzik et al., 1999). The formation of an apoptosome

complex in plants has not been demonstrated but a plant homologue cannot be ruled out in

the light of already identified similarities between morphological changes and components

of the mammalian and plant cell death pathways.



The apparent contradictory data reported on the effect of Hsp70 in promoting or inhibiting

apoptosis could be reconciled if one considers their effects on both necrosis and apoptosis.

In some instances, the apoptosis-enhancing effects of Hsp70 could be a logical indirect

consequence of protection against necrosis (Vayssier and Polla, 1998). Thus resistance to

apoptosis by induced expression of Hsp70 can be advantageous depending on

circumstances (Garrido et al., 2001). For instance, apoptosis induced by the pathogen

Staphylococcus aureus is abrogated as evidenced by a decrease in DNA fragmentation and

PI staining in monocytes exposed to expressing high levels of Hsp70, contributing to host

resistance (Guzik et al., 1999). As such, Hsp70/Hsc70 can act as an anti-apoptotic protein

necessary for protection against cell death. Heat-treated barley seedlings showed reduced

infection when being subsequently exposed to the powdery fungus (Schweizer et al.,

1995). If inhibition of apoptosis/PCD by high levels of Hsp70 with concomitant increased

cell survival is observed in planta, senescence or fruit-ripening could be delayed,

potentially benefiting post-harvest industries.


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Resistance of cells to apoptosis or even necrosis can have a negative impact on survival,

e.g. when pathogens use host cell apoptosis to gain access to tissues during

infection/disease (Grassme et al., 2001; Dickman et al., 2001) or when cancer cells

expressing high levels of HSP become resistant to chemo- or radiation therapy (Polla et al.,

1998). This “Jekyll and Hyde” scenario of increased Hsp70 expression has been well

documented in human diseases, also in asthma and chronic bronchitis. In plants, increased

expression of Hsp70 could be counterproductive when it has to launch a response by

programming cells to undergo cell death (HR), an essential host defence strategy to confine

the pathogen to the initial site of infection. Inhibition/reversal of PCD could promote

pathogen survival, replication and spread, leading to disease susceptibility. For example, it

was shown that exposing tobacco cells to high temperatures lead to decreased levels of SA

and concomitant susceptibility to TMV infection, although they did not refer to Hsp70

expression (Malamy et al., 1992).



DeMeester et al. (2001) coined the phrase heat shock paradox for the ability of the HS

response to induce both cytoprotection and -toxicity. Whereas it is well known that a prior

heat shock/stress can protect cells against inflammatory stress in vitro and in vivo, it has

been shown that induction of a subsequent heat stress in cells ‘primed’ by inflammation

can precipitate cell death by apoptosis. Since this study showed that HS abrogated the

induction of apoptosis by SA in protoplasts, it is tempting to speculate that heat-related

disease susceptibility may be due to the suppression of apoptosis by increased expression

of Hsp70. In future studies, logistic regression may provide a model for the prediction of

the occurrence of apoptosis in plants under heat stress using Hsp70 as independent

variable. However, evidence is required showing increased expression of Hsp70 providing

protection against PCD in plants during a pathogen attack.


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