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CHAPTER 5 - SECTION 1 Powered By Docstoc
					                       CHAPTER 5 - SECTION 1

         Pilot studies to investigate the relationship between
    Hsp70/Hsc70 accumulation and apoptosis as influenced by
                                    salicylic acid


Apoptosis, the best-characterised form of programmed cell death (PCD) in mammals, 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 and the formation of

apoptotic bodies. The clearance stage involves phagocytosis and degradation of apoptotic

bodies by macrophages or neighbouring cells. Some facets of apoptosis in animals are

shared with PCD in plants. The presence of a cell wall in plants, however, prevents the

formation and removal of apoptotic bodies. Recently it has been shown that Hsp70, a heat

shock protein considered to be involved in thermotolerance and protection against stress

(Frossard, 1999; Vayssier et al., 1998), was capable of negatively regulating apoptosis

(Beere and Green, 2001; Garrido et al., 2001).

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

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

to the outer leaflet of the membrane where it is exposed. This occurs in the early stages of

apoptotic cell death during which the cell membrane is still intact. Surface exposure of PS

is used as a common, early hallmark for the detection of cells dying by PCD/apoptosis, and

has been reported to occur in plants (O’Brien et al., 1997; 1998a). Annexin V, a member of

the annexin family of proteins, preferentially binds to negatively charged phospholipids

like PS in the presence of calcium and has proved to be a useful tool in detecting apoptotic

cells. By conjugating a fluorochrome (in this study, phycoerythrin, PE) to annexin V it is

possible to identify and quantify apoptotic cells on a single-cell basis by flow cytometry.

Mitochondria have been proposed to play a critical role in the process of cell death. They

are involved in a number of key events leading to cell death such as release of caspase

activators (such as cytochrome c), disruption of electron transport and energy metabolism,

loss of mitochondrial membrane permeability (MMP) and up- or down regulation of pro-

and anti-apoptotic proteins of the Bcl-2 family (Lee and Wei, 2000). Indeed,

cytofluorometric evidence supports mitochondrial alterations as an early apoptotic event

(Hirsch et al., 1998) and is seems plausible that mitochondrial permeability transition (PT)

is the central co-ordinator of apoptosis. Apoptosis-inducing agents can trigger the

uncoupling of electron transport from ATP production, leading to a decrease of

mitochondrial membrane potential (∆ψm) and subsequent production of reactive oxygen

species (ROS). The production of ROS and concomitant alterations of cellular redox states

are important signalling components of the induction phase of apoptosis (Jabs, 1999).

Events such as phosphatidylserine exposure, generation of ROS and ∆ψm were chosen as

parameters of apoptosis in tobacco protoplasts. The results shown in this section reflect the

developmental work done in conjunction with Dr. Iona Weir at the Horticulture Research

Institute in Auckland, New Zealand (see Acknowledgements). The protoplasts were

prepared from established Nicotiana plumbaginifolia cultures. Since the import/export of

any plant material from New Zealand is strictly prohibited, and due to the fact that N.

tabacum cultures were already established in our laboratories, further studies using these

techniques were done on N. tabacum and are presented in Section 2. However, due to

technical difficulties with the flow cytometer, dual labelling of protoplasts measuring ROS

and ∆ψm simultaneously has not been duplicated in our laboratories yet.


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

otherwise. Suspension cultures of tobacco, N. plumbaginifolia, were maintained in liquid

CSV medium (O' Brien et al., 1997) in conical flasks on an orbital shaker at 25 ºC in the

dark and subcultured at 7-day intervals. To obtain protoplasts, cell walls of suspension

cultures in logarithmic growth phase were digested without shaking 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 for

5 min), washed twice in protoplast wash fluid (PWF), containing 1/10 strength VK-

medium (VKM) macronutrients and 2.69 mM NaCl (O’Brien and Lindsay, 1993) and

collected by centrifugation (80 x g for 4 min). Protoplasts were finally resuspended in

either PWF or VKM (Binding and Nehls, 1977) until further use.

Treatments Heat shock. Protoplasts (∼106 ml-1) were heat shocked (HS) (40 °C, 30 min)

in a circulating water bath and allowed to recover for 2 h 30 min at 25 ºC. Salicylic acid

(SA). Protoplasts were incubated with 70 µM, 100 µM or 140 µM salicylic acid (SA) for a

total incubation time of 3 h. Heat shock plus salicylic acid. SA was added to cells prior to

the 30 min heat shock and allowed to recover for 2 h 30 min. Control cells were

maintained at 25 °C. Protoplasts treated with 130 µM or 260 µM H2O2 known to induce

apoptosis or necrosis (O’Brien et al., 1998a) were used as positive controls for annexin V

binding, the decrease of ∆ψm and the increase in ROS.

Accumulation of Hsp70/Hsc70 After the 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 in PBS

followed by centrifugation (200 x g, 4 min, 4 ºC) and washed with PBS. 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.). Labeling with the primary and secondary

antibodies was done consecutively for 1 h each at 37 ºC. Non-specific labeling due to the

secondary antibody was detected by the omission of the primary antibody. Unstained

protoplasts were used as controls for autofluorescence.

Detection of apoptotic protoplasts through Annexin V binding A sample volume of

500 µl treated protoplasts was analysed for cell death (or ∆ψm/ROS, while the remaining

sample was fixed and used to measure Hsp70/Hsc70 accumulation). The apoptotic cells

were stained with a fluorescein conjugate of annexin V for 5 min according to the

manufacturer’s recommendations (Annexin V FITC Kit, Immunotech, Marseille, France)

before analysis. Propidium iodide (PI) was added simultaneously and used to identify

necrotic protoplasts.

Measurement of mitochondrial membrane potential (∆ψm) and reactive oxygen

species (ROS)     The indo-carbocyanine probe, DiIC1(5) (Molecular Probes Inc, Eugene,

OR, USA), exhibits fluorescent changes based on potential-dependent changes in the

mitochondria and was used to determine ∆ψm. Dichlorodihydrofluorescein diacetate

(H2DCFDA, Molecular Probes Inc, Eugene, OR, USA) was used to detect ROS. The

“dihydro” derivative is readily oxidized back to the parent dye (FDA) by ROS, and serves

as a fluorogenic probe for detecting oxidative activity in cells. The probes were added

simultaneously (final concentration 40 nM and 5 µM respectively) to sample volumes (500

µl) of viable treated protoplasts after various time intervals (30 min, 1 h, 2 h and 3 h),

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

Flow cytometry      For all flow cytometric analysis, unstained protoplasts prepared under

the same physiological conditions were used as autofluorescent controls. Protoplasts were

analysed using an Epics Elite ESP flow cytometer (Beckman-Coulter, Florida). Two air-

cooled lasers emitting light at 488 (Argon) and 633 (HeNe) nm were used. Excitation of

PE was at 488 nm with fluorescence emission measured using a 575 nm ± 10 nm bandpass

filter. Protoplasts were gated based on their forward versus side scatter properties, with the

discriminator set on the PE log fluorescence signal instead of forward scatter, to reduce

background noise from autofluorescence. Excitation of the probes H2DCFDA and PI was

at 488 nm with fluorescence measured using a 525 nm ± 10 nm bandpass filter, and a 610

nm ± 10 nm bandpass filter, respectively. Excitation of DiIC1(5) was at 633 nm and

fluorescence measured using a 675 nm ± 20 nm bandpass filter. In order to analyse these

probes simultaneously within the same cell and to avoid spectral overlap the gated

amplifier on the Elite was used with a 40 µs delay between the Argon and HeNe lasers.

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, based on p values 0.05 (**)

or 0.01 (*)) calculated from the pooled variance, was exceeded.


Effect of SA on Hsp70/Hsc70 expression at normal temperature or following heat

shock as detected by flow cytometry

The effect of SA alone or in combination with HS on Hsp70/Hsc70 accumulation in

protoplasts was analysed at different concentrations of SA (70, 100 or 140 µM) after 3 h.

Hsp70/Hsc70 accumulation is shown as a line graph (Fig. 1 B) of the mean of log PE-

fluorescence. SA at low concentrations (70 µM or 100 µM) did not significantly influence

the expression of Hsp70/Hsc70 at normal temperatures, but at a concentration of 140 µM

induced Hsp70/Hsc70 (significantly at p < 0.01). Heat-induced Hsp70/Hsc70 accumulation

(Fig. 1 C, line graph) was significantly increased (p < 0.01) at all SA concentrations when

compared to non-HS controls indicated in Fig. 1 B, while 70 and 100 µM SA significantly

potentiated Hsp70/Hsc70 accumulation (p < 0.05) compared to the HS control containing

no SA.

 A                                        1 00 %
                                                                                                                                        n e c ro sis
                                           80 %                                                                                         pcd
                                                                                                                                        n o rm a l

                                % Cells
                                           60 %

                                           40 %

                                           20 %

                                                           c on trol        1 30 µ M         260 µM

                                                                            h y d ro g e n p e ro x id e

 B                           100%
                                                                                                           140                                                             necrosis

                                                                                                                     Hsc70/Hsp70 accumulation
                              80%                                                                          120                                                             pcd
                   % Cells

                                                                                              *            80

                              40%                                                                          60


                               0%                                                                          0
                                                       0               70       100          140

                                                            Salicylic acid [µM]

 C                           100%
                                                                                                                                                Hsc70/Hsp70 accumulation

                             80%                                                                                120                                                         pcd
                                                                   **             **                            100                                                         normal
                  % Cells

                             60%                                                                                                                                            Hsp70

                             40%                                                                                60


                              0%                                                                                0
                                                   0                   70         100           140

                                                           Salicylic acid [µM] + HS

FIG. 1 The effect of SA on Hsp70/Hsc70 expression at normal temperature or following heat
shock as detected by flow cytometry. A. Protoplasts were exposed to hydrogen peroxide to obtain
positive controls for apoptosis (130 µM) and necrosis (260 µM). Bar charts show the effect of
salicylic acid (SA, 70 µM, 100 µM or 140 µM for 3 h) on apoptosis (red fraction) and necrosis
(yellow fraction) in protoplasts, while line graphs show intracellular Hsp70/Hsc70 levels at normal
temperatures (B) or following heat shock (C; 40ºC 30 min HS, 2 h 30 min recovery). All values
represent the mean of six replicates. Significant differences of samples relative to each control is
indicated by * (p < 0.01) or ** (p < 0.05).

Effect of SA on cellular integrity estimated by PS externalisation and PI staining of

DNA at normal temperature and following heat shock

Increasing concentrations of H2O2 treatment were used as control for apoptosis indicated

by externalisation of PS and PI staining. H2O2 at low concentrations has been reported to

induce apoptosis (O’Brien et al., 1998a), while high concentrations cause necrotic cell

death. For this reason, two different concentrations of H2O2 (130 µM and 260 µM) were

used as positive controls for apoptosis and necrosis respectively (Fig. 1 A (n=6); Fig. 2 a

and f). H2O2 at a concentration of 130 µM resulted in 48% of a protoplast sub-population

undergoing PS externalisation without PI staining, 37% were necrotic indicated by PI

staining and 15% were normal (Fig. 1 A). Concentrations of 260 µM on average led to

necrosis in 66% of the population, 18% apoptotic and 16% normal protoplasts (Fig. 1 A).

Although a slight decrease in annexin V binding was observed in protoplasts exposed to

100 µM SA and an increase at 140 µM, these differences were not significant at p < 0.01

when compared to control samples. Representative cytograms (Fig. 2) illustrate apoptosis

(positive for annexin V binding, negative for PI staining, upper left quadrant) and necrosis

(positive for PI staining, upper and lower right quadrants) of protoplasts exposed to

increasing concentrations of salicylic acid (0, 70, 100 or 140 µM) for 3 h (Fig. 2, b-e).

No significant increases in apoptosis accompanied the SA-mediated increase in heat-

induced Hsp70/Hsc70 expression (Fig. 1 C). Representative cytograms (Fig. 2 g-j) showed

a significant increase of necrosis (p < 0.05) in heat-treated protoplasts (as indicated by

propidium iodide (PI) staining, upper and lower right quadrants), when compared to the

control protoplasts, irrespective of exposure to different concentrations of SA.

Fig 2 (see word doc Chapt 5-1 figures)

Effect of SA on intracellular ROS generation and mitochondrial integrity (estimated

by MMP) at normal temperature and following heat shock

A time- and dose-responsive increase in ROS and decrease in ∆ψm was observed in

protoplasts exposed to the two different concentrations of H2O2 (Fig. 3 I: b,d,f,h (130 µM

H2O2) and c,e,g,i (260 µM H2O2)). The amorphous region drawn around the population

indicated in (a) (of Fig. 3 I) indicates the ROS and ∆ψm levels in untreated protoplasts at

time zero. In protoplasts exposed to 130 µM H2O2 a time-dependent upward shift of the

population and shift to the left indicates an increase in ROS and depolarization of the

mitochondrial membrane. Protoplasts exposed to 260 µM H2O2 show a sub-population

migrating far left and a sub-population undergoing an upward shift over time.

Representative bivariate histogram plots are shown to illustrate changes in mitochondrial

membrane potential (∆Ψm) (X-axis) and ROS production (Y-axis) of SA-treated

protoplasts at normal temperatures over time (Fig. 3 II, a-p). A slight decrease in ROS

levels is observed in control samples (c and d) after 2 h. In protoplasts treated with 70 µM

SA a decrease in ROS is observed and after 2 h (g, h) a prominent sub-population shows a

shift to the left. After 2 h treatment with 100 µM SA (k, l) and 140 µM SA (p) a sub-

population showed an upward shift of the protoplasts as well as a sub-population

remaining to the left as was observed at with 70 µM SA. Representative bivariate

histogram plots are shown to illustrate changes in mitochondrial membrane potential

(∆Ψm) (X-axis) and ROS production (Y-axis) of SA-treated protoplasts following HS

(40°C, 30 min) (Fig. 3 III, a-p). In the absence of SA in heat-treated protoplasts a decrease

in both ROS and ∆Ψm is evident at all time intervals (a-d). At all SA concentrations (70-

140 µM) of heat-treated protoplasts, a sub-population that shifted to the left is evident at 2

h (g, k, o) and 3 h (h, l, p). However, there is also a sub-population that is shifting upwards

into the amorphous region defining normal levels of both ∆Ψm and ROS.

FIG. 3 Representative bivariate histogram plots illustrating the effect of salicylic acid

(SA), alone or in combination with heat shock (HS), on mitochondrial membrane potential

(∆ψm) and ROS production in protoplasts over time using flow cytometric detection of

DiIC1(5) and H2DCFDA fluorescence respectively. The amorphous region indicated

around the fluorescent control population (I: a) was used as reference levels of ∆ψm. and

ROS in untreated protoplasts at time zero.

I: Protoplasts exposed to hydrogen peroxide served as positive controls for increase in

ROS and alterations in ∆Ψm (130 µM: b, d, f, h) and necrosis (260 µM: c, e, g, i). Samples

were analysed at 30 min (b and c), 1 h (d and e), 2 h (f and g) and 3 h (h and i).

II: Changes in ∆ψm and ROS in protoplasts over time treated with various doses of

salicylic acid at normal temperatures. Protoplasts treated without SA (a-d), 70 µM SA (e-

h), 100 µM SA (i-l) or 140 µM SA (m-p) were analysed after 30 min (a, e, i, m), 1 h (b,

f, j, n), 2 h (c, g, k, o) or 3 h (d, h, l, p).

III: Changes in ∆ψm and ROS in protoplasts over time treated with various doses of

salicylic acid in combination with heat shock (40 ºC for 30 min). Heat-treated protoplasts

without SA (a-d) or treated with 70 µM SA (e-h), 100 µM SA (i-l) or 140 µM SA (m-p)

were analysed after 30 min (immediately following HS) (a, e, i, m), 1 h (b, f, j, n), 2 h (c,

g, k, o) or 3 h (d, h, l, p).

Fig 3: I (see word doc chapt 5-1 figures)

Fig 3: II

Fig. 3: III


The objective of the pilot studies described in this section was to determine at which

concentrations SA-mediated Hsp70/Hsc70 induction occurred at normal temperatures or

following heat shock in tobacco protoplasts and to investigate whether these events were

associated with cell death (apoptosis/necrosis) or with changes in ROS or membrane


It was found that low doses of SA (70 and 100 µM) does not induce Hsp70/Hsc70, but

potentiates heat-induced Hsp70/Hsc70 accumulation, while high SA doses (140 µM) are

considered to be phytotoxic and induced Hsp70/Hsc70.

In protoplasts exposed to 70 µM SA for 3 h, a prominent decrease in ∆Ψm was observed in

a sub-population of protoplasts as well as a sub-population that showed a decrease in ROS.

This concentration of SA has been shown to induce apoptosis in tobacco protoplasts

(O’Brien et al., 1998b), and therefore these events might be associated with the onset of

apoptosis. However, the ability of SA to activate the alternative oxidase pathway in plants

that lowers mitochondrial ROS (Maxwell et al., 1999) could also be responsible for the

decreased levels of ROS observed. The addition of 70 µM SA was not accompanied by an

induction of Hsp70/Hsc70. However, in protoplasts exposed to higher SA concentrations,

an increase in Hsp70/Hsc70 expression, albeit slight, is associated with a sub-population of

protoplasts that are returning to normal levels of oxidative activity (Fig. 3 II l-p).

In protoplasts exposed to heat shock, a sub-population shifting to the left indicating a

decrease in ∆Ψm became apparent after 3 h (Fig. 3 III, d). This population was more

prominent at all SA treatments. It was also noted that necrosis increased (Fig. 1 C, yellow

fraction) in all heat shocked protoplasts in comparison to protoplasts treated at normal

temperatures. It is proposed that this was due to the heat shock treatment being too severe.

It is known that HS per se can induce an early increase in cells with depolarized

mitochondria returning to normal after 18 h (Polla et al., 1996).

In protoplasts exposed to SA in combination with HS, there is a sub-population shifting

upwards indicative of oxidative activity returning to normal (Fig. 3 III l, p). This shift is

most prominent in protoplasts accompanied by significant potentiation of heat-induced

Hsp70/Hsc70 levels (Fig. 1 C, line graph).

Thus these results appear to suggest that there is a relationship between increased levels of

Hsp70/Hsc70 accumulation and apoptosis, although there is no direct evidence of this.

Investigation of changes in mitochondrial permeability have previously been reported to be

fraught with potential artifacts (Weir, 2001). These include autoquenching of fluorescent

probes when the intramitochondrial concentrations are too high, oxidative inactivation of

their fluorescence and alterations in uptake caused by changes in mitochondrial volume

rather than ∆Ψm (Green and Reed, 1998). Furthermore, although changes in PS exposure

as determined by annexin V binding is used as an indicator of apoptosis, analysing

nucleosomal DNA fragmentation appears to be a better marker for apoptosis, as the latter

event is considered to be unique to apoptosis (Weir, 2001). Thus using the flow cytometer

to analyse early events associated with apoptosis in plants can be an exciting area of

research, although care should be taken to consider all the possible problems that are

unique to plants when analysing apoptosis.


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