Programmed Cell Death and Adaptation: Two Different Types of Abiotic Stress Response in A Unicellular Chlorophyte

Programmed Cell Death and Adaptation: Two Different Types
of Abiotic Stress Response in a Unicellular Chlorophyte

Anna Zuppini, Caterina Gerotto and Barbara Baldan
Eukaryotic microalgae are highly suitable biological indicators of environmental changes because they are exposed
to extreme seasonal uctuations. The biochemical and
molecular targets and regulators of key proteins involved in
the stress response in microalgae have yet to be elucidated.
This study presents morphological and biochemical evidence
of programmed cell death (PCD) in a low temperature
strain of Chlorella saccharophila induced by exposure to
NaCl stress. Morphological characteristics of PCD, including
cell shrinkage, detachment of the plasma membrane from
the cell wall, nuclear condensation and DNA fragmentation,
were observed. Additionally, a signicant production of H2O2
and increase in caspase 3-like activity were detected. We
demonstrated that singly applied environmental stresses
such as warming or salt stress trigger a pathway of PCD.
Intriguingly, the prior application of salt stress seems to
reduce heat shock-induced cell death signicantly, suggesting
a combined effect which activates a defense mechanism in
algal cells. These results suggest that C. saccharophila can
undergo PCD under stress conditions, and that this PCD
shares several features with metazoan PCD. Moreover, the
simultaneous exposure of this unicellular chlorophyte to
different abiotic stresses results in a tolerance mechanism.
Keywords: Chlorella saccharophila Heat shock Programmed
cell death Salt stress Tolerance.
Abbreviations: DCF, 2,7-dichlorouorescein; H2DCFDA, 2,7dichlorouorescein diacetate; HO, Hoechst 33342; HS, heat
shock; PBS, phosphate-buffered saline; PCD, programmed cell
death; rbcL, ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco) large subunit; ROS, reactive oxygen species; TdT,
terminal deoxynucleotidyl transferase; TEM, transmission
electron microscopy; TUNEL, terminal deoxynucleotidyl
tranferase-mediated dUTP nick end labeling.
Introduction
Salt stress is one of the major abiotic stresses affecting plant
growth and crop production. A high salt content of the environment of terrestrial or aquatic plants hampers water as well as
nutrient uptake (Huh et al. 2002, Mahajan and Tuteja 2005),

leading to plant growth inhibition and even death (Tuteja
2007). In response to high salinity stress, plants generate
second messengers including Ca2+ and reactive oxygen species
(ROS) (Witzel et al. 2009, Miller et al. 2010). These key players
initiate a phosphorylation cascade which in turn activates the
major stress-responsive genes leading, in most cases, to plant
adaptation and helping the plant to cope with unfavorable
conditions (Mahajan and Tuteja 2005). The accumulation of
many metabolites, i.e. fructose, sucrose and trehalose, which
possess an osmolyte function, or are charged, such as proline
and glycinebetaine A, is one of the plant responses to prevent
water loss from the cells and to facilitate osmotic adjustment
(Shabala and Cuin 2007, Banu et al. 2009). Moreover, the
detoxication of ROS produced following salt application
by antioxidative enzyme activation is used by plant cells to
limit salt-induced damage (Sekmen et al. 2007, Tuteja 2007,
Kavitha et al. 2008). The regulation of K+ and H+ ux patterns
across the plasma membrane by K+-permeable channels, the
maintenance of a correct K+/Na+ ratio in the cytosol and
the compartmentation in the vacuole of excessive Na+ by the
tonoplast Na+/H+ exchanger have been demonstrated to be
key determinants of plant salt tolerance and result in the ability
of plants to survive severe saline environments (Shabala and
Cuin 2007). Furthermore, programmed cell death (PCD) has
been proposed amongst the salt adaptation strategies in higher
plants (Huh et al. 2002).
PCD is a primary process during plant growth and development and it can also be triggered by several environmental
stresses including salt stress. High salt-induced PCD in rice root
tip cells, in tobacco protoplasts and in cultured tobacco cells
occurred with the well described morphological and biochemical PCD hallmarks such as ROS accumulation, nuclear alteration and degradation, chromatin condensation, mitochondrial
involvement, endonuclease activity and DNA fragmentation
(Lin et al. 2006, Jiang et al. 2008, Banu et al. 2009).
Despite an increasing literature on PCD events in bacteria
and unicellular eukaryotes in response to numerous triggers,
little is known about salt-induced PCD in these organisms
which play an important role in the equilibrium of aquatic ecosystems and represent highly suitable biological indicators of
Plant Cell Physiol. 51(6): 884895 (2010) doi:10.1093/pcp/pcq069, available online at www.pcp.oxfordjournals.org
The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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884
Plant Cell Physiol. 51(6): 884895 (2010) doi:10.1093/pcp/pcq069 The Author 2010.
Downloaded from http://pcp.oxfordjournals.org/ at UFZ Umweltforschungszentrum Leipzig-Halle GmbH on November 12, 2012
Regular Paper
Dipartimento di Biologia, Universit di Padova, via U. Bassi 58/B, 35131 Padova, Italy
Corresponding author: E-mail, zuppini@bio.unipd.it; Fax, +39-049-8276280
(Received April 1, 2010; Accepted May 2, 2010)
C. saccharophila abiotic stress responses
Results
Salt treatment reduces cell growth and induces cell
death in Chlorella saccharophila
Chlorella saccharophila vegetative cells are ellipsoids of
5 m 2 m. After this stage cells increase their dimension to
reach a size of about 5 m 9 m, with a parallel extension of
the cell wall thickness (data not shown), corresponding to
the autospore mother cell (Fig. 1A). The number of daughter
cells formed depends upon cell size at initiation of division
(Donnan et al. 1985). Chlorella vulgaris is believed to replicate

producing 2n daughter cells per division burst (Donnan et al.
1985); a mature cell can produce two, four, eight or 16 autospores
(Bold and Wynnie 1978). Chlorella saccharophila show a similar
behavior, and a mature cell can produce 28 autospores of
2 m 3 m (Fig. 1A). In order to investigate the effects of
NaCl on the growth of C. saccharophila, we applied an NaCl
concentration range (from 0.05 to 0.3 M) to C. saccharophila
cell culture medium and incubated the algal cultures (in the
exponential growth phase) under the new salt conditions
at 4C and a 16 h photoperiod. Cells grown in the medium with
0.05 and 0.1 M NaCl, corresponding to about 120 and 240 times
higher than the usual medium, did not show signicant differences compared with untreated control (data not shown).
However, NaCl at a concentration of 0.2 and 0.3 M caused an
increase in the percentage of dead cells in Chlorella cell culture
(Fig. 1B). However, after the initial inhibition of the cell growth,
the cell population seems to recover, and for times longer than
48 h the percentage of dead cells in the treated cell population
is comparable with that in the untreated control (Fig. 1B). The
viability of C. saccharophila cells was determined by Evans blue
staining to quantify the rates of cell death. The dye enters cells
with injured membranes, but not intact cells. Evans blue can be
detected by absorbance measurements and it serves to assess
the integrity of cells. About 21.9 1.8% (0.2 M NaCl) and
33.1 2.3% (0.3 M NaCl) of cells in the treated populations
died within the rst 24 h (Fig. 1B). These results suggest that
exogenously added NaCl plays a certain physiological role in
C. saccharophila, probably perturbing the cell cycle, but at times
longer that 48 h the cell population seems to be able to acclimate to the new high salinity growth conditions. The proportion in the number of vegetative cells and autosporangia in
control and salt-treated cell populations did not show relevant
differences (Fig. 1C, D).
environmental changes because of their exposure to seasonal

uctuations. In different Anabaena strains, exposure to a moderate salt concentration causes cell death with features similar
to those of metazoan PCD (Ning et al. 2002). In yeast, salt causes
lysigenous PCD which is mediated by ion disequilibrium and
involves enzymes released from the vacuoles (Huh et al. 2002).
In Micrasterias denticulata, a unicellular freshwater green alga,
high salinity induces morphological and ultrastructuctural cell
changes with the occurrence of organelle degradation by
autophagy, a recognized form of plant PCD (Affenzeller et al.
2009a, Affenzeller et al. 2009b).
In this work we have investigated the effects of high salinity in
the unicellular green alga Chlorella saccharophila. Biochemical
and morphological hallmarks of PCD, i.e. H2O2 production, caspase 3-like activity, chromatin condensation and DNA fragmentation, are analyzed after the application of NaCl at different concentrations. We used a snow strain of C. saccharophila
as model system. Psychrophilic communities include organisms
that are exposed to a highly variable environment, concerning
UV irradiation, temperature, pH and osmolarity, and are generally subjected to more than one stress simultaneously. Another
purpose of this work was to attempt to analyze the response of
C. saccharophila to a combination of different environmental
conditions. The simultaneous presence of two different stress
conditions could trigger the activation of overlapping stress
response pathways that culminate in either PCD, adaptation
or the so-called cross-tolerance in which a particular stress
can induce resistance to a subsequent stress that is different
from the initial one (Mittler 2006). This unicellular chlorophyte
has been previously demonstrated to undergo PCD following
a heat shock (HS) treatment (Zuppini et al. 2007, Zuppini
et al. 2009). Based on published (Zuppini et al. 2007, Zuppini
et al. 2009) and preliminary results, we have used salt stress
and HS as a choice entry point for exploring the response of
C. saccharophila to environmental changes. Our results
demonstrate that singly applied environmental stresses set off
a PCD pathway, whereas the occurrence of the heat-induced
PCD-like process is completely inhibited by a preliminary
administration of the salt stress. These data suggest the existence of a probable overlap between the two signaling pathways leading to the activation of a cross-tolerance mechanism,
a combined effect which strengthens the defense strategies
in algal cells.
Salt stress-induced PCD markers

Cells treated with 0.2 and 0.3 M NaCl exhibited some biochemical and cytological hallmarks of PCD. The time-dependent
effects of salt stress were analyzed evaluating H2O2 production,
the presence of caspase 3-like activity, chromatin condensation
and DNA fragmentation, which are all well-known PCD markers.
ROS are either directly or indirectly involved in cell death.
We used a ROS-sensitive dye, H2DCFDA (2,7-dichlorouorescein
diacetate), and a xylenol orange colorimetric assay to monitor
ROS production during salt stress. H2DCFDA, a non-uorescent
dye, is rapidly oxidized to highly uorescent 2,7-dichlorouorescein (DCF) by intracellular ROS (Hoffmann et al. 2005),
whereas xylenol orange detects H2O2 released from the cells
in the culture medium. The evaluation of intracellular ROS
with H2DCFDA showed a signicant increase in ROS content
(1.8-fold) after 10 min of 0.2 and 0.3 M NaCl treatment, compared with the untreated control (Fig. 2A). For time periods
longer than 10 min (up to 90 min) the H2DCFDA uorescence
was comparable with the level in untreated control cells
885
A. Zuppini et al.
Vegetative cells
A
Autospore
mother cell
5 m
B
5 m
10 m
30
25
20
15
b
b
b b
b a,b
36
48
10
5
Co
24
Vegetative cells
Control
Autosporangia
Control
0.2 M
5.0
0.2 M
0.7
0.3 M
4.5
4.0
0.3 M
0.6
Cell number 106
Cell number 106
72
Time of the treatment (h)
Autosporangium
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
a a
5 m
0.2 M
0.3 M
35
0.5
0.4
0.3
0.2
0.1
24
48
72
96
24
48
72
96
Fig. 1 (A) Chlorella saccharophila life cycle (A, vegetative cells; B, autospore mother cell; C, autosporangium; D, autospores). (B) Cell viability of
the untreated cell population (Co) and NaCl-treated cells (0.2 M, 0.2 M NaCl-treated cells; 0.3 M, 0.3 M NaCl-treated cells); the 100% value
corresponds to cells treated for 10 min at 100C. Bars labeled with a different letter differ signicantly (P < 0.05) by Students t test. (C, D) Number
of vegetative cells (C) and autosporangia (D) in control (white bars), 0.2 M NaCl-treated (grey bars) and 0.3 M NaCl-treated (black bars) cell
populations. Values are the mean SD of three independent experiments.
(Fig. 2A). It was not possible to detect any differences between

control and treated cell populations by monitoring extracellular H2O2 within the rst 75 min of treatment (Fig. 2B). These
ndings are probably due to the presence of intracellular scavenging antioxidant systems that rapidly remove the excess ROS
(Shao et al. 2007).
Caspase 3 protease is an important regulator of apoptotic
cell death. In order to identify the proteases responsible for the
PCD response, caspase 3-like activity was analyzed in untreated
and salt-treated cells. We employed the synthetic colorimetric
substrate for animal caspase 3 (DEVD-pNA) to examine the
caspase 3-like activity in extracts from treated and control cells.
In cytosolic protein extract of cell populations treated with 0.2
and 0.3 M NaCl, an increase in caspase 3-like activity was
detected after 4, 8, 24 and 36 h of salt stress (Fig. 2C). DEVDase
proteolytic activities signicantly increased about 1.8 0.1- and
2.2 0.2-fold after 4 h treatment with 0.2 and 0.3 M NaCl,
respectively, compared with the control, to reach a maximum
886
after 8 h treatment (4.3 0.2-fold compared with the control)

in the 0.3 M NaCl-treated cell population (Fig. 2C). To identify
the specic proteolytic enzyme activity in NaCl-treated cells,
we pre-incubated the cytosolic extracts with the specic caspase 3 inhibitor Ac-DEVD-CHO (Fig. 2C). Ac-DEVD-CHO at
20 M signicantly inhibited NaCl-induced DEVDase activity
by about 50% (P < 0.005) (Fig. 2C). We also used an antibody
raised against human caspase 3 in an attempt to provide further information on the NaCl-induced PCD process. Western
blot analysis of protein extracts using a human caspase 3 antibody highlighted the presence of a 34 kDa cross-reacting protein band in protein extracts from control and NaCl-treated
cells, corresponding to inactive procaspase 3-like (Fig. 2D).
An additional protein band of approximately 12 kDa, corresponding to active caspase 3-like, was detectable only in
NaCl-treated cells (Fig. 2D). Banding is consistent with the
expected 34 kDa for procaspase 3 and 12 kDa for caspase 3
according to the supplier. The data reported in Fig. 2D refer to
Dead cells (relative %)
Autospores
40
Co
0.2 M
0.3 M
Light
500
450
350
Control
H2O2 (mM)
50
0.2 M
40
30
0.3 M
400
60
0.3 M
a
aa
aa
aa
aa
aa
aa
300
250
Co
200
Fluorescence (arbitrary units)
H2DCFDA
0.2 M
150
20
100
10
50
0
Co
10
15
30
45
60
75
90
10
20
30
40
Time of the treatment (min)
caspase 3-like activity (relative %)
500
Proc3
Control
50
60
70
80
Time (min)
kDa
34
Co 0.2 0.3
0.2 M
400
0.3 M
h,d
Cas3
300
h,d,c
d,c
200
f
100
0
Ac-DEVD
12
a a
a,f
a,f
a,f
a
g
a,g
a,g
a,g
-+
- + - +
- + - +
- + - +
- + - +
- + - +
- + - +
Co
24
36
48
72
Fig. 2 Reactive oxygen species (ROS) production and caspase 3-like activity upon salt stress. (A) Time course of H2O2 production; insert, light
and uorescence (H2DCFDA) representative images of control cells (Co) and cells incubated for 10 min with NaCl at a concentration of 0.2 and
0.3 M, stained with 2,7-dichlorouorescein diacetate (H2DCFDA). The histogram represents the uorescence analysis of images with ImageJ
software; data are the mean SD of three independent experiments. White bar, control; gray bars, 0.2 M NaCl-treated cells (0.2 M); black bars,
0.3 M NaCl-treated cells (0.3 M). Scale bar: 5 m. (B) H2O2 release in the culture medium in untreated (Co) and NaCl-treated cells (0.2 and 0.3 M
NaCl); values are normalized per g of fresh weight. (C) Time course of caspase 3-like activity in control cells (Co, white bars) and cells treated with
0.2 M NaCl (0.2 M, gray bars) and 0.3 M NaCl (0.3 M, black bars) assayed at different time points, in the absence () and presence (+) of the specic
caspase 3 inhibitor Ac-DEVD-CHO (20 M). Data are means SD of three independent experiments. Bars labeled with a different letter differ
signicantly (P < 0.05) by Students t-test. (D) Western blot analysis of total protein extracts (30 g) from control cells (Co) and cells treated for
8 h with 0.2 M (0.2) and 0.3 M (0.3) NaCl using an antibody raised against human caspase 3. Proc3, procaspase 3; Cas3, active caspase 3.
8 h treatment; for time treatments of 4, 24 and 36 h we found

similar results (data not shown).
To conrm further the effects of salt stress, the prole of
chromosomal DNA in treated cells was analyzed by Hoechst
(HO) staining and TUNEL [terminal deoxynucleotidyl tranferase (TdT)-mediated dUTP nick end labeling] assay. Intense
uorescence staining nuclei (HO positive) show chromatin
condensation (Fig. 3B); an increased number of HO-positive
887
A. Zuppini et al.
Hoechst negative
120
Light
Hoechst
Light
Hoechst
Co
Hoechst positive
80
24h
60
24h
40
20
0
Co
24 36 48 72
48h
24 36 48 72
0.2 M NaCl
48h
Vegetative cells
0.3 M NaCl
60
Co
Autosporangia
Treated cells
0.2M
0.3M
Light
50
b
30
b
b
b
20
TUNEL
AI (%)
40
Co
24 36 48 72
24 36 48 72
0.2 M NaCl
0.3 M NaCl
DAPI
10
Fig. 3 Chromatin condensation (A, B) and DNA fragmentation (C, D) in C. saccharophila cells. Untreated and NaCl-treated cells were stained with
Hoechst 33342 (HO) and observed under a uorescence microscope. (A, C) Time course of the percentage of cells displaying chromatin
condensation (A, HO positive) and DNA strand breaks (C). (B, D) Examples of typical light and uorescence (UV) images of the same cells stained
with HO (B) and 4,6 diamidino-2-phenylindole (DAPI) (D) and assayed for TUNEL positivity (D). Co, untreated cells; 24 h, cells treated for 24 h
with 0.2 M NaCl; 48h, cells treated for 48 h with 0.2 M NaCl. Bar: 5 m (B), 20 m (D).
nuclei was observed in cells treated with 0.2 and 0.3 M NaCl
for 24, 36 and 48 h, compared with the control (Fig. 3A).
The number of stained nuclei increased from 10% of the
control to 2040% in cells treated with 0.2 and 0.3 M NaCl,
respectively (Fig. 3A). DNA fragmentation is a specic morphological feature of PCD, and it can be identied in situ by TUNEL
analysis. This assay was employed to establish whether DNA
fragmentation occurs in salt-treated cell populations. To avoid
false-positive/negative staining, positive and negative labeling
control reactions were performed (data not shown). The
obtained results highlighted a signicant increase in DNA
fragmentation in cells treated for 24, 36 and 48 h with 0.2 and
0.3 M NaCl (Fig. 3C, D).
To acquire greater cellular detail on the effects of salt stress
treatment, transmission electron microscopy (TEM) analysis
888
was conducted, showing normal subcellular morphology in

untreated cells (Fig. 4A). Characteristics of PCD were observed
in some of the treated cells within the rst 24 h of treatment.
There was clumping of nuclear chromatin into more densely
packed material (Fig. 4DG) that becomes marginalized against
the nuclear membrane (Fig. 4EG). This was accompanied
by nuclear condensation (Fig. 4D, E) and detachment of the
plasma membrane from the cell wall (Fig. 4E). However, within
this 24 h period the cytoplasmic membrane remained intact
(Fig. 4B, E, F). These ultrastructural features indicated that PCD
appeared to be the dominant mechanism of NaCl-induced cell
death in C. saccharophila.
Loss of photosynthetic pigments is a common response of
plants to stress. Symptoms of changes in the photosynthetic
apparatus were analyzed in salt-treated C. saccharophila cells.
Treated cells
Cell nuclei (%)
100
pi
py
pi
chl
py
chl
nu
nu
nu
chl
nu
cc
E
cc
pm
de
nu
de
cw
nu
cc
cc
cc
Fig. 4 Effect of salt stress on C. saccharophila cell ultrastructure. (A) Control cell; (BD) cells treated with 0.2 M NaCl for 24 h; (EG) cells treated
with 0.2 M NaCl for 48 h. Arrows indicate the nuclear shrinkage. cc, condensed chromatin; chl, chloroplast; cw, cell wall; de, detachment of the
plasma membrane from the cell wall; nu, nucleus; pi, pyrenoid; pm, plasma membrane; py, pyrenoglobuli. Bar: 1 m.
Photosynthetic pigments (Chla, Chlb and carotenoids) exhibited a similar trend in variation upon 0.2 and 0.3 M NaCl
treatment (Fig. 5A, B). The total Chl content (Chl a + b)
decreased at 4, 8, 24 and 36 h of NaCl treatment, compared
with the control (Fig. 5A, B). However, the decrease in Chl
(a + b) content is signicantly different from that of the
control only in 0.3 M NaCl-treated cells from 4 to 36 h (Fig. 5B).
The highest drop in Chl concentration occurred after 24 h of
exposure to 0.3 M NaCl (Fig. 5B). The carotenoid contents
in 0.2 M NaCl-treated cells was comparable with those of the
control cell population (Fig. 5A), whereas these pigments
decreased signicantly after 8 h of 0.3 M NaCl treatment,
dropping from a value of about 0.4 0.06 mg g FW1 in the
untreated cells to 0.2 0.05 mg g FW1 in the cells treated
for 8 h (Fig. 5B).
Salt treatment abolishes HS-induced PCD

In a previous work by Zuppini et al. (2007) it was demonstrated
that C. saccharophila cells undergo PCD following an HS treatment of 2 h at 44C. In this study we demonstrate that salt
stress also induces PCD in some of the C. saccharophila cell
population, whereas a fraction of the treated cells seems to
tolerate the high salt content applied. Different stress conditions can result in the activation of similar response pathways
in plant cells, leading to the so-called cross-tolerance, in which
a particular stress in plants can induce resistance to a subsequent stress that is different from the initial one.
In this work we applied a 24 h 0.2 or 0.3 M NaCl salt stress
to a C. saccharophila cell population and a subsequent HS of
44C for 2 h. Monitoring cell death immediately after the HS
and at 4, 24 and 48 h from the HS, the percentage of dead cells
889
A. Zuppini et al.
1.8
mg g.f.w.-1
1.6
1.4
a
a
1.2
1.0
0.8
0.6
0.4
0.2
0
Chl (a+b)
carotenoids
24
36
Discussion
48
72

0.2 M NaCl
1.8
1.6
Chl (a+b)
carotenoids
mg g.f.w.-1
1.4
1.2
1.0
0.8
0.6
0.4
b
c
b
d
b
c
0.2
0
Co
24
36
48
72

0.3 M NaCl
Fig. 5 Time course of the pigment contents in untreated cells (Co)

and cells treated (for 472 h) with 0.2 M NaCl (A) and 0.3 M NaCl (B).
Chl (a + b), total chlorophyll content; data are means SD of ve
independent replicates. Bars labeled with a different letter differ
signicantly (P < 0.05) by Students t test.
in the population was comparable with that in the untreated

control (Fig. 6A), whereas application of HS without an NaCl
pre-treatment caused an increase of the cell death percentage
of >50% at 24 h after the HS, compared with the untreated
control (Fig. 6A). Moreover, Western blot analyses showed the
absence of the 12 kDa protein band corresponding to active
caspase 3-like protein in cells treated with the combined stresses
(Fig. 6B), and in these cells it was also not possible to detect any
increase in caspase 3-like activity (data not shown). Salt stress
somehow seems to prevent the processing of procaspase 3-like
protein. In C. saccharophila cells treated with HS alone, there is
a time-progressive alteration of chloroplast structure and function: the amount of ribulose-1,5-bisphosphate carboxylase/
oxygenase (Rubisco) large subunit (rbcL) decreases with time
after the HS (Zuppini et al. 2007). In C. saccharophila cells
treated with the combination of salt and heat stresses, we did
not detect variation in the amount of rbcL protein (Fig. 6B).
Moreover, the salt pre-treatment also prevented the decrease
in photosynthetic pigments (Fig. 6C) normally occurring in
HS-treated cells (Zuppini et al. 2007). Intriguingly, an NaCl
890
Cold ecosystems occupy >70% of the earth and are often dominated by microorganisms which represent the most abundant
cold-adapted life forms at the level of species diversity and biomass (Feller and Gerday 2003, Hoham et al. 2007). Snow is the
most conspicuous part of this system and provides a habitat for
microbial communities, typically dominated by green algae
(Hoham and Ling 2000, Remias and Ltz, 2007, Leya et al. 2009)
or bacteria (Carpenter et al. 2000). The microorganisms that
thrive in these extreme habitats are still poorly understood:
this makes low-temperature environments an interesting unexplored frontier. We utilized a snow strain of C. saccharophila,
a unicellular chlorophyte collected in the Antelao glacier
(Venetian Dolomites, Italy), as a model system for biochemical
and morphological studies on the stress response. Eukaryotic
microalgae are highly suitable biological indicators of environmental changes because they are exposed to extreme seasonal
uctuations. The primary goal of this study was to investigate
the activation and execution of PCD in C. saccharophila and to
clarify the responses of this psychrophilic alga to a combination
of environmental conditions. NaCl has been shown to induce
PCD in higher plants (Lin et al. 2006, Shabala et al. 2007, Jiang
et al. 2008) and more recently in the unicellular green alga
M. denticulata (Affenzeller et al. 2009a, Affenzeller et al. 2009b).
Our results show that no signicant alterations in cell growth
and cell death can be induced with 0.05 and 0.1 M NaCl treatments, about 120 and 240 times higher than normal growth
conditions. Whereas treatments of cell cultures with 0.2 and
0.3 M NaCl triggered a signicant increase in cell death within
the rst 24 h of treatment, longer treatments seem to lead to
acclimation to the new growth conditions. Enhanced salinity is
experienced as a stress condition in this microalga since it
induces changes in the growth rate and cell death. A certain
portion of the C. saccharophila cell population showed PCD
features such as chromatin condensation, DNA fragmentation,
nuclear shrinkage, caspase 3-like activity, decrease in pigment
contents and detachment of the plasma membrane from the
cell wall. These data support our previous ndings on the occurrence of PCD in the unicellular C. saccharophila, which belongs
to one of the primary lineages of photosynthetic eukaryotes
(Delwiche 1999), with features resembling metazoan apoptosis.
Although it has been assumed that PCD was developed by
multicellular organisms to regulate growth and development,
recent reports also recognized a role for PCD mechanisms in
unicellular algae (Zuppini et al. 2007, Affenzeller et al. 2009a,
Affenzeller et al. 2009b, Zuppini et al. 2009). Evidence for a cell
suicide pathway in unicellular organisms has been shown
during stress response to light deprivation, CO2, ROS, UV-C
radiation, heat shock and salt stress (Vardi et al. 1999, Segovia
et al. 2003, Moharikar et al. 2006, Zuppini et al. 2007,
Co
pre-treatment longer than 24 h did not abolish HS-induced

PCD: a 48 h incubation of C. saccharophila cells with 0.2 or 0.3 M
enhanced the effect of HS (data not shown).
S
S
+H
+H
3M
2M
.
.
0
0
Co
24
24
HS (2h/44C)
90
0.2 M NaCl (24 h) + HS (2h/44C)
80
0.3 M NaCl (24 h) + HS (2h/44C)
rbcL
70
60
Dead cells (relative %)
100
50
b
40
30
20
PR
a a
a
a a
Proc3
10
0
0
Co
24
48
Time from the HS (h)
PR
1.0
3.0
0.2 M NaCl (24 h) + HS (2h/44C)
2.0
0.6
1.5
0.4
Chl (mg g.f.w.-1)
Carotenoids (mg g.f.w.-1)
2.5
0.3 M NaCl (24 h) + HS (2h/44C)
0.8
1.0
0.2
0.5
Co
0
24
48
Co
24
48
Fig. 6 Effect of combined stresses on C. saccharophila cells. (A) Cell viability of the untreated cell population (Co); heat shock (HS)-treated (44C
for 2 h) cells (black bars) analyzed immediately after the HS (0) and at 4, 24 and 48 h after the treatment; NaCl-treated cells (0.2 and 0.3 M for 24 h)
subjected to a subsequent HS (0.2 M, white bars; 0.3 M, gray bars) and analyzed immediately after the HS (0) and at 4, 24 and 48 h after the
treatment; the 100% value corresponds to cells treated for 10 min at 100C. Bars labeled with a different letter differ signicantly (P < 0.05) by
Students t-test. (B) Immunoblot analysis of total protein extracts (1030 g) from control cells (Co) and cells treated for 24 h with 0.2 or 0.3 M
NaCl followed by HS (0.2 M + HS, 0.3 M + HS) and analyzed at 4 and 24 h after the HS using an antibody raised against the Rubisco large subunit
(rbcL) and procaspase 3-like (Proc3) proteins. Co, untreated cells; PR, Ponceau red staining. (C) Time course of the pigment contents in untreated
cells (Co) and cells treated with 0.2 M (white bars) and 0.3 M (gray bars) NaCl followed by an HS (44C for 2 h) and analyzed immediately after the
HS (0) and after 4, 24 and 48 h; chl, total chlorophyll amount; data are means SD of ve independent replicates.
891
A. Zuppini et al.
892
Overall, we can conclude that NaCl treatment induces activation of the PCD process and, at least in part of the cell population, it prompts acclimation competence.
Psychrophilic communities include organisms that experience the simultaneous occurrence of several abiotic stresses
in their environment rather than a single stress condition.
Chlorella saccharophila has a widespread distribution mainly
in extreme environments (Huss et al. 2002), and the snow strain
used in this work turned out to be particularly resistant to light,
temperature (Zuppini et al. 2007) and salinity variations.
Salinity is considered to be one of the most important constraints on species diversity and productivity of natural populations of algae (Booth and Beardall 1991). Despite the existence
of studies on the effect of single abiotic stresses on microorganisms, there are few data on the effects of co-occurrence of
different stresses (Skandamis et al. 2008, Gustavs et al. 2009).
In the present work we tried to analyse the effect of a combination of environmental conditions on C. saccharophila. We used
two abiotic stresses, salt stress and HS, to analyse the response
of a unicellular chlorophyte to environmental changes. The
typical HS regime that works well with this snow strain of
C. saccharophila is 44C for 2 h (Zuppini et al. 2007). Our data
reveal the occurrence of a heat-induced PCD-like process
which is completely inhibited by a preliminary administration
of a salt stress, highlighting a probable cross-talk between
the two signaling pathways. The application of these subsequent conditions abolished alterations in chloroplast pigment
content, Rubisco degradation and caspase 3-like activation,
features occurring during the application of the single HS.
Thus, the NaCl-treated cell population seems to tolerate HS.
Tolerance to a combination of different stresses in plants is
likely to be a complex mechanism involving cross-talk between
different sensors and signal transduction pathways (Mittler
2006). The receptors for abiotic stresses have not been identied yet, neither have the intermediates of the signal transduction pathways (Chinnusamy et al. 2004). However, it is likely
that some signaling and transduction pathways are unique for
a specic stress, whereas for abiotic stresses a stress-mediated
cross-talk between the different signaling pathways could exist.
Our data show that the tolerance is induced by NaCl treatment
for 24 h but not by longer NaCl incubation (data not shown),
suggesting that the possibility of inducing the cross-tolerance
between the two signaling pathway is time limited. These
ndings prompt the idea that different stresses can result in the
activation of overlapping stress response pathways that culminate either in PCD, acclimation or the so-called cross-tolerance.
Due to the importance of salt-induced PCD in unicellular
organisms for maintenance of population integrity and in plants
for tolerance induction (Huh et al. 2002), unraveling the components of this model will enable future molecular dissection of
the heat tolerance mechanism. Moreover, given the signicance
of microalgae communities in maintaining glacier ecosystems,
future studies will provide a comprehensive paradigm of
how algae will integrate the responses to changes in global
environment.
Darehshouri et al. 2008, Affenzeller et al. 2009a, Affenzeller

et al. 2009b) and suggests an evolutionary meaning for PCD in
microorganisms (Gordeeva et al. 2004). A rather large number
of PCD-related sequences was probably present early in the
evolution of eukaryotes (Nedelcu 2009). PCD has been demonstrated in cyanobacteria, chlorophytes and dinoagellates
(Gordeeva et al. 2004), involving H2O2 production, caspase-like
proteases (Moharikar et al. 2006, Zuppini et al. 2007), chromatin condensation, nuclear alteration and DNA fragmentation
(Segovia et al. 2003, Gordeeva et al. 2004, Zuppini et al. 2007). In
particular, caspase 3 has been shown to be instrumental in
mammalian apoptosis. Although no caspase orthologs have
been identied in plant genomes, the presence of caspase-like
activity has been reported in several plant and algae PCD processes (Moharikar et al. 2006, Zuppini et al. 2007, Bonneau et al.
2008), but the cellular roles and activities of these enzymes
remain an open question in unicellular organisms. Recently,
salt-induced PCD has been observed in Micrasterias cells occurring without the involvement of caspase enzymes. Instead, our
ndings highlight a signicant increase in caspase 3-like activity
following NaCl treatment in Chlorella, suggesting the possible
existence of multiple transduction pathways involved in PCD.
A cysteine protease activity is a recognized key event in PCD
mechanisms induced by various environmental stresses in
phytoplankton species (Vardi et al. 1999, Segovia et al. 2003,
Moharikar et al. 2006, Zuppini et al. 2007). Moreover, immunoblot analysis clearly shows the presence of a cross-reacting protein band corresponding to active caspase 3 in C. saccharophila
NaCl-treated protein extracts. Caspase 3-like activity correlated
well with other PCD markers such as chromatin condensation,
DNA fragmentation and morphological alterations, as well as
the detachment of the plasma membrane from the cell wall
and nuclear shrinkage. ROS are produced during the cell
response to abiotic stresses and are recognized important signals in the activation of plant PCD. Moreover, a key role for ROS
has been documented in PCD induced by salt stress (Lin et al.
2006, Affenzeller et al. 2009a, Affenzeller et al. 2009b, Chen et al.
2009), although the ROS signaling network in salinity-induced
PCD has not be claried so far. In the Chlorella salt-induced
PCD pathway we found a production of ROS species during the
rst 10 min after the stress, supporting an involvement of
these molecules in the salt-induced response in unicellular
algae. Taken together our data suggest an induction of PCD
by NaCl treatment in part of the Chlorella cell population,
with a reduction of viability within the rst 36 h: after longer
times, the population seems to acclimate to the new growth
conditions. The C. saccharophila cell population consists of cells
in different developmental stages, thus a speculative hypothesis
could be that the different developmental stages (such as vegetative cells, autospore mother cells and autospores) respond
to salt treatment with a different sensitivity. Nevertheless,
due to the difculty in discriminating between the different
kinds of cells (especially vegetative and autospore mother cells)
and in creating synchronous cell populations, it is not clear if
there is a real dissimilarity in NaCl sensing of the cell population.
Pigment concentration
Materials and Methods

Growth conditions and treatments
Western blot analyses

Equal amounts of protein extracts (1030 g) were loaded on
a 12% SDSPAGE gel according to the method of Laemmli
(1970). The proteins were transferred to polyvinyl diuoride
(PVDF) membrane with the Semidry-system (BioRad, Oakland,
CA, USA) according to the manufacturers instructions and
then visualized by staining with Ponceau red to verify protein
loading. Membranes were blocked with 5% (w/v) skim milk
in phosphate-buffered saline (PBS) buffer for 1 h at room temperature and immunoblotted with antibodies raised against
wheat rbcL (diluted 1 : 7,000; from N. La Rocca, Padova, Italy)
and human caspase 3 (diluted 1 : 500; Calbiochem, San Diego,
CA, USA) for 2 h at room temperature. After washing in 0.02%
Tween/PBS and PBS, membranes were incubated with the secondary antibodies. Labeling was detected by chemiluminescence (CDP-star, Biolabs, Hitchin, UK) and autoradiography
lms (Sigma-Aldrich, St Louis, MO, USA).
Evans blue analysis

Equal amount of C. saccharophila cells in exponential growth
phase were incubated in 0.05% Evans blue solution for 15 min
and then extensively washed with deionized water (Levine
et al. 1996). The dye bound to dead cells was solubilized in
50% methanol/1% SDS for 30 min at 55C and quantied by
absorbance at 600 nm. The percentage of dead cells in the
population was calculated considering as the 100% reference
value the absorbance of an equal amount of cells treated for
10 min at 100C.
HO staining
Control and treated cell suspensions were stained (10 min
at room temperature) with 8 g ml1 HO (Sigma-Aldrich,
St Louis, MO, USA). Chromatin uorescence was observed
under a UVlight microscope. About 600 cells were analyzed
for each experiment and cells which had undergone PCD
were morphologically dened by cytoplasmic and nuclear
shrinkage and chromatin condensation. HO experiments
were conducted in triplicate. The cells from ve random
microscopic elds were counted to determine the number of
stained nuclei.
Caspase 3 activity assay

Caspase 3 activity was assayed using a caspase-3 colorimetric
activity assay kit (Chemicon International, Inc., Temecula, CA,
USA), following the manufacturers protocol. Briey, cytosolic
extracts (30 g) from untreated and treated cells were incubated with Ac-DEVD-pNA for 2 h at 37C in dark. Developed
color in the reaction was measured at 405 nm.
The green alga C. saccharophila was grown in Bristol GR+

liquid medium (www.bio.utexas.edu/research/utex/) and kept
under a 16 h photoperiod at 4C. Cultures were subcultured
every 4 weeks, during the exponential growth phase, by making
a 1 : 2 dilution in fresh medium. For salt and HS treatments,
0.050.3 M NaCl was added to the culture medium of cells
in the exponential growth phase and/or cells were incubated
in a 44C pre-warmed circulating water bath in the light for
2 h followed by incubation under normal growth conditions
for 048 h.
Chl and carotenoid contents were determined using the

extinction coefcients proposed by Porra et al. (1989). Pigments
were extracted from cells (300 mg) with 1 ml of N,Ndimethylformamide. Concentrations of Chl and carotenoids
were calculated on a fresh weight basis.
Transmission electron microscopy

Samples were xed overnight at 4C in 0.1 M cacodylate buffer
containing 3% glutaraldehyde (v/v) and post-xed in 1% OsO4
in the same buffer for 2 h. The samples were then dehydrated
through a graded ethanol series [25, 50, 75 and 100% (v/v in
ddH2O); 15 min at each concentration] at room temperature
and embedded in araldite resin (Sigma-Aldrich, St Louis, MO,
USA). Sections (0.05 m), obtained with an ultramicrotome,
were stained in uranyl acetate in ethanol for 30 min at room
temperature and viewed at 75 kV in a Hitachi 300 transmission
electron microscope.
In situ detection of DNA fragmentation

(TUNEL analysis)
Cells were analysed using the In situ cell death detection kit,
uorescein (Roche Diagnostic Spa, Milan, Italy) according to
the manufacturers instructions. NaCl-treated C. saccharophila
cells were xed for 24 h in 4% formaldehyde at room temperature, permeabilized with 0.1% Triton X-100/0.1% sodium
citrate and incubated at 37C for 60 min with TdT and uorescein-conjugated nucleotides. Negative controls were produced
as described above but without adding TdT to the reaction mix.
Positive controls were obtained by treating cells with DNase I,
grade I (5,000 U ml1 in 50 mM TrisHCl, pH 7.5) for 10 min
prior to incubation with TdT. Slides were observed with an
epiuorescence microscope (Leica DMR), and a total of
600 cells for each sample were analyzed for TUNEL positivity.
DNA fragmentation experiments were conducted in triplicate
and the percentage of TUNEL-positive cells (apoptotic index,
AI% = No. of apoptotic nuclei/number of nuclei scored 100),
using 40 magnication, and the mean SD was calculated.
Measurements of intracellular ROS

Extracellular H2O2 production was determined according to
Wolff (1994). Briey, untreated and treated cells were incubated with an assay solution (0.5 mM ammonium ferrous
sulfate, 50 mM H2SO4, 0.2 mM xylenol orange, 200 mM sorbitol)
for 45 min at room temperature in the dark. The assay is based
on a colorimetric reaction due to the peroxide-mediated
893
A. Zuppini et al.
Statistical analysis
Values were expressed as the mean SD of 35 independent
experiments, using the Students t-test to analyze the differences between the control and treated group. Statistical
signicance was set at P < 0.05.
Funding
This work was supported by the University of Padova, Progetto
di Ateneo CDPA075925/07 (to B.B.).
Acknowledgments
We thank C. Andreoli (Padova, Italy) for kindly providing
C. saccharophila cells, and N. La Rocca (Padova, Italy) for the
kind gift of Rubisco large subunit antibody.
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Programmed Cell Death and Adaptation: Two Different Types

of Abiotic Stress Response in a Unicellular Chlorophyte

nutrient uptake (Huh et al. 2002, Mahajan and Tuteja 2005),

C. saccharophila abiotic stress responses

(Donnan et al. 1985). Chlorella vulgaris is believed to replicate

environmental changes because of their exposure to seasonal

Salt stress-induced PCD markers

Cell number 106

Cell number 106

Time of the treatment (h)

Time of the treatment (h)

Time of the treatment (h)

(Fig. 2A). It was not possible to detect any differences between

after 8 h treatment (4.3 0.2-fold compared with the control)

Dead cells (relative %)

C. saccharophila abiotic stress responses

Fluorescence (arbitrary units)

Time of the treatment (min)

caspase 3-like activity (relative %)

Time of the treatment (h)

8 h treatment; for time treatments of 4, 24 and 36 h we found

was conducted, showing normal subcellular morphology in

Cell nuclei (%)

C. saccharophila abiotic stress responses

Salt treatment abolishes HS-induced PCD

Time of the treatment (h)

Time of the treatment (h)

Fig. 5 Time course of the pigment contents in untreated cells (Co)

in the population was comparable with that in the untreated

pre-treatment longer than 24 h did not abolish HS-induced

C. saccharophila abiotic stress responses

0.2 M NaCl (24 h) + HS (2h/44C)

0.3 M NaCl (24 h) + HS (2h/44C)

Dead cells (relative %)

Time from the HS (h)

Chl (mg g.f.w.-1)

Carotenoids (mg g.f.w.-1)

0.3 M NaCl (24 h) + HS (2h/44C)

Time from the HS (h)

Time from the HS (h)

Darehshouri et al. 2008, Affenzeller et al. 2009a, Affenzeller

C. saccharophila abiotic stress responses

Materials and Methods

Western blot analyses

Evans blue analysis

Caspase 3 activity assay

The green alga C. saccharophila was grown in Bristol GR+

Chl and carotenoid contents were determined using the

Transmission electron microscopy

In situ detection of DNA fragmentation

Measurements of intracellular ROS

Chinnusamy, V., Schumaker, K. and Zhu, J.K. (2004) Molecular genetic

oxidation of Fe2+ followed by the reaction of Fe3+ with xylenol

C. saccharophila abiotic stress responses

salt-tolerant Plantago maritime and salt-sensitive Plantago media.

Mahajan, S. and Tuteja, N. (2005) Cold, salinity and drought stresses:

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