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INTRODUCTION
Marine diatoms are some of the most successful
groups of unicellular eukaryotic photosynthetic organ-
isms, forming massive blooms in coastal and open waters
and having a key role in global marine primary production
(Armbrust, 2009). Elucidating the mechanisms that regu-
late diatom bloom dynamics and mainly their decay de-
terminants is therefore fundamental to understand nutrient
and carbon cycling so as energy flow at the global as well
as the local scale.
Programmed cell death (PCD) is an active, self-con-
trolled death mechanism well known in higher organisms
which has been also detected in unicellulars (Bidle et al.,
2007; Segovia, 2011). However, its occurrence in unicel-
lular organisms is still considered a matter of ongoing de-
bate (Deponte, 2008; Jimenez et al., 2009), and the specific
mode of action in which PCD is initiated remains to be elu-
cidated. PCD has been suggested to act in phytoplankton
populations to eliminate aged cells and to reduce population
size in order to cope with nutrient limitation, for instance,
in the final stages of blooms (Bidle and Falkowski, 2004;
Vardi et al., 1999, 2006, 2007). After a certain threshold of
limitation of environmental factors (mainly nutrients), PCD
induces the massive decay of the bloom, sometimes
through massive production of resting or resistance stages
(Ellegaard et al., 2013; McQuoid et al., 2002). These fac-
tors include nitrogen and light limitation (Berges and
Falkowski, 1998), iron starvation (Bidle and Bender, 2008;
Thamatrakoln et al., 2012), virus infection (Bidle et al.,
2007; Vardi et al., 2009), CO2 limitation (Vardi et al., 1999)
and several secondary metabolites (Cervia et al., 2009;
Costas et al., 1993). Reactive oxygen species (ROS) are
often involved in inducing PCD in reaction to a stress factor
in a wide range of organisms and cell lines, from bacteria
to mammalians and plants (Andrianasolo et al., 2007;
Jones, 2008; Kuwabara et al., 2008; Lam, 2008; Rocken-
feller and Madeo, 2008; Scherz-Shouval and Elazar, 2007),
mainly at intermediate concentrations. In fact, several cell
reactions to ROS strongly depend upon the ROS concen-
tration and may range from production of antioxidant com-
pounds to necrotic cell death (Chandra et al., 2000), or PCD
(Costas et al., 1993; Vardi et al., 2009). Apart from ROS
concentrations, several variables come into play by modu-
lating and regulating the cell response to ROS. These in-
clude the cell type, the stress type, its magnitude and its
duration in time (Martindale and Holbrook, 2002).
Different case studies report the co-occurrence of
ROS and PCD in marine phytoplankton and suggest a
correlation between them (Butow et al., 1997; Segovia
and Berges, 2009; Vardi et al., 1999). In the dinoflagel-
late Peridinium gatunense, PCD leading to the collapse
Advances in Oceanography and Limnology, 2015; 6(1/2): 13-20 ORIGINAL ARTICLE
DOI: 10.4081/aiol.2015.5466
Expression of death-related genes and reactive oxygen species production in
Skeletonema tropicum upon exposure to the polyunsaturated aldehyde octadienal
Alessandra A. Gallina,1# Chih-Ching Chung,2,3 Raffaella Casotti1*
1Stazione Zoologica Anton Dohrn di Napoli, Villa Comunale, 80121 Naples, Italy; 2Institute of Marine Environmental Chemistry and
Ecology, National Taiwan Ocean University, Keelung 20224, Taiwan, China; 3Center of Excellence for the Oceans, National Taiwan
Ocean University, Keelung 20224,Taiwan, Republic of China
#Present address: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
*Corresponding author: raffaella.casotti@szn.it
ABSTRACT
The effects of 4E/Z-octadienal (OCTA) on ScDSP-1 and ScDSP-2 gene expression and reactive oxygen species (ROS) production
were investigated in the marine diatom Skeletonema tropicum (formerly costatum) using qRTPCR and flow cytometry. ScDSP-1 and
ScDSP-2 genes have been previously shown to be involved in cell death in ageing cells and in response to photosynthetic stress. OCTA in-
duced a differential, concentration-dependent DSP gene expression associated to ROS production, 821.6 and 97.7 folds higher for ScDSP-
1 and ScDSP-2, respectively. Among the concentrations tested, only 8 μM OCTA, which caused a reduction of 50% in cell concentrations
at 24 h, was able to elicit an expression pattern consistent with a signalling role. Interestingly, only intermediate levels of reactive oxygen
species (ROS) (i.e., 1.5±0.1 increase) were observed to be elicited by such concentration. These results suggest that ROS are key components
of the molecular cascade triggered by polyunsaturated aldehydes (PUA) and leading to cell death. This could have implications for bloom
final stages at sea, where PUA may act as effectors of diatom population dynamics through ROS acting as modulators.
Key words: Diatom; chemical ecology; bloom evolution; Programmed cell death; ROS.
Received: July 2015. Accepted: October 2015.
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14DSP expression in response to PUA
of the algal population is mediated by oxidative stress in-
duced by carbon dioxide (CO2) limitation (Vardi et al.,
1999). Similarly, ROS-mediated cell death was observed
at the end of a bloom of the same species, despite an in-
crease in antioxidant activity (Butow et al., 1997). PCD-
like cell death concomitant with accumulation of ROS is
also reported to be induced by the toxic cyanobacterium
Microcystis sp. (Vardi et al., 2002), or H2O2 (Vardi et al.,
2007). In the chlorophyte Dunaliella tertiolecta, dark-in-
duced PCD, involving the activity of caspase-like en-
zymes has been linked to an increase in ROS production
(Segovia and Berges, 2009). In the prymnesiophyte Emil-
iana huxley viral infection has been associated to an en-
hanced production of ROS, possibly leading to PCD
(Evans et al., 2006).
Interestingly, the secondary metabolite Euplotin C de-
rived from the marine ciliated protist Euplotes crassus, is
found to induce PCD in the congeneric Euplotes vannus,
which does not produce this metabolite (Cervia et al.,
2009). This suggests that specific secondary metabolites
can play an ecological role in broadening phytoplankton
niche size through different mechanisms, including PCD
(Cervia et al., 2009).
In the marine diatom Phaeodactylum tricornutum, the
exposure to diatom-derived secondary metabolites,
polyunsaturated aldehydes (PUA), results in an altered ex-
pression of metacaspases through the overexpression of a
gene associated to NO generation (Vardi et al., 2008). In
the marine diatom Thalassiosira weissflogii, PUA were
shown to induce PCD-like cell death, suggesting for a role
in activating the cell death cascade, therefore regulating
population dynamics and species succession (Casotti et
al., 2005). In Phaeodactylum tricornutum it is speculated
that a sophisticated stress surveillance system exists, in-
volving NO and Calcium, in response to PUA, which
could lead to bloom termination and population-level cell
death at specific PUA concentrations (Vardi et al., 2006).
Even PUA precursors, the polyunsaturated fatty acids
(PUFAs), have been reported to inhibit growth of Candida
albicans and Candida dubliniensi by inducing ROS ac-
cumulation and consequent PCD (Thibane et al., 2012).
In the marine diatom Skeletonema tropicum (formerly
costatum) two genes have been identified, coding for two
identical Ca-regulated proteins, named S. costatum Death-
Specific Proteins (ScDSP-1 and ScDSP-2) (Chung et al.,
2005), which are both composed of a transmembrane do-
main and a pair of EF-hand motifs (Luan et al., 2002;
Means and Dedman, 1980). Both ScDSP gene expressions
were shown to be strongly upregulated in aging cells and
also during light stress. Their expression is correlated to
DNA fragmentation, suggesting for a possible role of the
encoded protein in the signal transduction of stress to the
cell death machinery (Chung et al., 2005, 2008). In partic-
ular, ScDSP expression has been related to the blockage of
the electron flow between PSII and cytochrome b6f and it
was found to be NO dependent (Chung et al., 2008). More-
over, two DSP-like proteins (TpDSP1 and TpDSP2), have
been recently identified in the marine diatom Thalassiosira
pseudonana and have been shown to be upregulated both
under iron limitation and starvation, suggesting for a role
in Fe-induced stress and consequent death (Thamatrakoln
et al., 2012). However, they have also recently been shown
to enhance growth under Fe limitation and to be controlled
by light intensity (Thamatrakoln et al., 2013).
In this study we report the gene expression of the two
death-related genes ScDSP-1 and ScDSP-2 in ST exposed
to the polyunsaturated aldehyde octadienal, and relate it
to ROS production. The overall aim is to relate cell death
to chemical stress induced by PUA and to advance hy-
potheses on bloom decay mechanisms at sea.
METHODS
Experimental setup and cultures
A unialgal culture of Skeletonema tropicum Cleve (for-
merly costatum) was grown at 23°C on a 12h-12h light-
dark cycle under a photon flux density of 110 µmol quanta
m–2 s–1 (Chung et al., 2005). Natural seawater, amended
with f/2 nutrients (Guillard, 1975) was used as medium.
For gene expression measurements, S. tropicum was
grown in 10 L polycarbonate carboys (Nalgene). When the
culture reached the late exponential phase of growth (LE,
Fig. 1), cells were transferred into 2 L polycarbonate bot-
tles (Nalgene) and treated with different concentrations of
4E/Z-octadienal (OCTA) or H2O2. Samples were collected
at 1,2,3,4,5 and 1,2,3,4,12 days since the beginning of the
Fig. 1. Effect of OCTA and H2O2 on cell concentrations of S.
tropicum. LE is the time point indicated as Late Exponential
growth phase. Data are means of triplicates ±SD. When the bar
is not visible, it is smaller than the symbol.
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15 A.A. Gallina et al.
experiment for cell counts and total RNA extraction, re-
spectively. Cell concentrations were determined by using
a Sedgwick-Rafter counting chamber (Hausser Scientific
Partnership). At least 200 cells from each sample were
counted using a light microscope (BX60; Olympus, Center
Valley, USA) at 200× magnification. Growth rates were
calculated using the following equation:
µ=ln [(N1/N0)/t] (eq. 1)
where N0 and N1 are cell density at the start and the end
of the growth period, and t is the time between measure-
ments (in days). Since no dead vs alive cells assay has
been used, it is possible that the cells counts reported in
the study might include damaged or dying cells.
Gene expression experiments were performed using
three OCTA concentrations, namely the concentration
which caused a reduction of 50% in cell concentration at
24 h, half this concentration and twice this concentration
(4, 8 and 16 µM). For ROS detection, three independent
cultures were grown in 1 L glass flasks until the late ex-
ponential growth phase. Cells were harvested and directly
exposed to the same concentrations of OCTA used for
gene expression measurements and analysed through flow
cytometry as described below. The controls were dye-
loaded samples processed exactly as the other samples ex-
cept for PUA inoculation.
Each experiment was performed at least twice, but
generally three times, i.e. the data presented are the results
of biological replicates.
Chemicals
The PUA used in this study was 2E,4E/Z-octadienal
(OCTA, from Sigma Aldrich Inc. Milan, Italy). This was
chosen because it is the main and most commonly pro-
duced PUA by diatoms, and, in particular, by Skeletonema
spp. (Wichard et al., 2005). The concentrations used were
chosen accounting for multiples of the concentration caus-
ing a reduction of 50% in cell concentrations at 24h, as
empirically determined for S. tropicum (data not shown)
resulting to be 8 μM. The reference value of 8 μM has
been chosen also based on Ribalet et al., (2007), where 8
μM represented the EC50 for growth in the congeneric
species S. marinoi. OCTA working solutions were dis-
solved in HPLC-grade pure Methanol (JT Baker, Phillips-
burg, NJ, USA) at room temperature. Since Methanol can
be toxic, it was tested on growing cultures and cultures
showed no reaction up to 7 microliters per mL. Subse-
quently, and conservatively, no more than 4 microliters of
Methanol plus OCTA were ever inoculated into the cul-
tures. Before incubation, the exact OCTA concentration
in the working solution was assessed spectrophotometri-
cally (Hewlett-Packard 8453- Hewlett-Packard Co., Palo
Alto, CA, USA) by measuring absorption at 274 nm and
a specific molar absorption coefficient of 31,000 (Pippen
and Nonaka, 1958). Hydrogen peroxide (H2O2, 30 wt. %
from Sigma Aldrich, Milan, Italy) was used as positive
control, after dilution 1/200 in filtered seawater and added
to samples as a 50 μL:1 mL ratio.
The ROS scavenger, 4-Hydroxy-2,2,6,6-tetram-
ethylpiperidine 1-oxyl (TEMPOL, Sigma-Aldrich, Milan,
Italy) was used as negative control, so as sodium di-
ethyldithiocarbamate trihydrate (DETC, Sigma-Aldrich),
an inhibitor of superoxide dismutase (SOD) at a final con-
centration of 5 mM and 1 mM, respectively, in filtered sea
water (FSW). Samples with DETC were incubated for
120 min prior to PUA addition. The ROS-sensitive dye
dihydrorodamine 123 (DHR, Molecular Probes, Leiden,
NL) (5 mM stock in DMSO) was used to monitor ROS
production at the single-cell level. DHR can be oxidized
by different ROS, including hydrogen peroxide (H2O2)
and peroxynitrite (ONOO–), to form the fluorescent de-
rivative rhodamine 123 (excitation WL 492-495 nm,
emission WL=517-527 nm), and is commonly used to in-
vestigate oxidative stress in vivo (Ischiropoulos et al.,
1999). DHR has been previously used in different mi-
croalgal species to measure oxidative burst (Jamers et al.,
2009; Vardi et al., 1999). Samples were incubated with
both 10 μM DHR and PUA in the dark at room tempera-
ture and analysed using flow cytometry (see below).
Flow cytometry
To quantify ROS production in vivo, a flow cytometer
(FACScalibur, Becton Dickinson, Palo Alto, CA, USA)
equipped with a 488-nm laser as excitation source and fil-
tered sea water as sheath was used. A 530/30-nm beam-
pass emission filter was used for detection of DHR-derived
green fluorescence. Both the sheath fluid and the sample
velocity (65 μLmin–1) were kept constant during all the ex-
periments. As a trigger signal, red fluorescence was used
with a threshold at channel 52. Red fluorescence was col-
lected through a 650-nm long-pass filter and was also used
as a proxy for chlorophyll cell fluorescence. Fluorescent
beads (Coulter FlowSet Fluorospheres, Beckman Coulter,
Fullerton, CA, USA) were used as internal standard. Green
fluorescence values were normalized to the green fluores-
cence of the beads, and all data were expressed as fold
changes of green fluorescence relative to the control val-
ues. Data acquisition (104 cells on average for each sample)
was performed using CellQuest software (Becton-Dikin-
son). Data analysis was performed using FCS4 Express
(De Novo Softwares, Los Angeles, CA, USA).
Total RNA extraction
Two-hundred mL of culture were collected through a fil-
tration system using a 2-µm pore size polycarbonate filter
(Nucleopore; Whatman, Maidstone, UK), placed over a
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16DSP expression in response to PUA
GF/F glass microfiber filter (Whatman). Filtration never
lasted longer than 30 min. Cells were then resuspended in
0.7 mL guanidine isothiocyanate buffer (RLT buffer, Qia-
gen, Valencia, CA, USA) containing 1% β-mercaptoethanol
(Sigma Aldrich, St. Louis, MO, USA), immediately frozen
at -80°C, and stored for a few days. After disrupting the cells
by sonication on ice (Sonicator ultrasonic processor XL,
Heat System Ultrasonics, Farmingdale, NY, USA), total
RNA was extracted using the silica-membrane spin column
included in the RNeasy Plant Mini Kit (Qiagen). The resid-
ual genomic DNA was removed by an additional treatment
with the RNase-Free DNase Set (Qiagen) according to the
manufacturer’s instruction. Total RNA concentration and
purity were determined with a spectrophotometer (ND-100;
NanoDrop Technologies, Wilmington, DE, USA) by meas-
uring absorption at 260 and 280 nm.
Reverse transcription and Real time quantitative
polymerase chain reaction
One μg of total RNA was treated with DNase I and re-
verse transcribed into first-strand complementary DNA
(cDNA) fragments by using random hexamer oligonu-
cleotides and the High Capacity cDNA Reverse Transcrip-
tion Kit (Applied Biosystems, Foster City, CA, USA). The
conditions for the retro-transcription were set as in Chung
et al. (2005): 25°C for 10 min, 37°C for 120 min, and 85°C
for 5 min. Real time quantitative polymerase chain reac-
tions (Q-PCR) were performed using 2× SYBR Green
PCR master mix (Applied Biosystems) with 250 nM of
each of the forward and reverse primers. The nucleotide
sequences of all primer pairs used in the Q-PCRs are indi-
cated in Tab. 1. The GeneAmp 7000 sequence detection
system (Applied Biosystems) was used to carry out all the
reactions. The threshold cycle at which the fluorescence
intensity exceeded a preset threshold was used to calculate
the target gene mRNA and 18S rRNA expression levels.
The RNA molar ratio of ScDSP mRNA and 18S rRNA was
calculated using the formula described in Chung et al.,
(2005), and the data normalized to the control values. The
levels of ScDSP mRNA expression were expressed as fold
changes of mRNA abundance relative to the control. All
data reported are means of three technical replicates but
the experiments were repeated at least twice.
Statistical analyses
Pairwise comparisons among treatments and control
were assessed by Student’s t-test using the Excel spread-
sheet (Microsoft, Redmond, Washington, MA, USA).
RESULTS AND DISCUSSION
Cultures were affected by OCTA exposure. 16 μM
OCTA reduced cell concentrations to 51.17% of initial val-
ues after 24 h (Fig. 1; Tab.2). H2O2 also strongly affected
cell concentrations which decreased to 50% as early as 2
h after inoculation and to zero 24 h later (Fig. 1).
ScDSP-1 and ScDSP-2 gene expression and ROS lev-
els increased in S. tropicum cultures exposed to different
concentrations of OCTA. A peak in ScDSP-1 and ScDSP-
2 expression was visible as early as 1 h after exposure and
its intensity for cells in LE exposed to the concentration
of 8 μM OCTA was ca. 822 and 98 folds that in the control
for ScDSP-1 and ScDSP-2, respectively (Fig. 2 a,b). Gene
expression decreased afterward and showed no further
peak until the end of the experiment. In the same strain,
an increase in ScDSP expression has been previously re-
ported when cultures entered senescence (Chung et al.,
2005) and also in response to a light stress involving the
blockage of electron flow at the level of the photosystem
II (Chung et al., 2008). The latter also involved NO in-
crease in the gene regulation and involvement of a PCD
machinery (Chung et al., 2008). Overall, these results pro-
vide evidence that the DSP genes have a role in regulating
the molecular mechanism of mortality in S. tropicum
under stress.
In our experiments, gene expression was highest at the
concentrations where ROS production was intermediate,
suggesting that there exists a threshold for the activation
of the two death-related genes investigated. A peak in
ROS was observed 20 min after inoculation with OCTA
(Fig. 3), followed by a decrease. The intensity of the peak
Tab. 1. Primer pairs used for the qRT PCR reactions, as in
Chung et al. (2008).
Target gene Primer name Nucleotide sequence (5’-end to 3’-end)
ScDSP-1 ScDSP-SG-F GAACA AGCAA ACTGC ACTCG TC
ScDSP-SG-R GTCAA GAATG TTGGT CGTCG CG
ScDSP-2 ScDSP-SG-F GAACA AGCAA ACTGC ACTCG TC
ScDSP2-SG-R GTAGG CATCT GCTAT TCTTT CTG
18S rRNA Ske-18S-F GAATT CCTAG ATATC GCAGT TCATC
Ske-18S-R GCTAA TCCAC AATCT CGACT CCTC
Tab. 2. Effect of OCTA on growth rate of S. tropicum cultures after
24 h. Initial concentration (t0, i.e., LE) was 2.53 105 cell mL–1.
Growth rate (day–1)
Concentration (µM) LE
OCTA° 0 0.30±0.10
4 0.03±0.08
8* -0.63±0.39
16 -0.80±0.22-
16H2O2 2.70±0.35
LE, Late exponential growth phase (as in Fig. 1); °2E,4E/Z-octadienal;
*concentration causing a reduction of 50% in cell concentrations at 24
h (see text). Data are means ±SD, n=3.
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17 A.A. Gallina et al.
was PUA concentration-dependent, and was highest for
16 μM OCTA (1.9±0.2 increase; P<0.001, n=3). Both the
ROS scavenger Tempol and the SOD inhibitor DETC re-
duced the intensity of the peak, therefore supporting the
conclusion that the peak was really due to an increase of
ROS. No increase in DHR-derived green fluorescence
was observed when methanol only was added to the cul-
tures, excluding an artifact due to the solvent used (data
not shown). Hence, it is suggested that cell death is trig-
gered in S. tropicum only by specific PUA concentrations,
which are those causing a reduction of 50% in cell con-
centrations at 24 h (8 μM OCTA). This is supported by
the observation that 8 μM OCTA induced intermediate
levels of ROS production (1.5±0.1 increase in LE cul-
tures), while a higher concentration (16 μM OCTA)
elicited a considerably lower ScDSP expression, even
though it induced a higher ROS production (1.9±0.2 in-
crease). This could be related to different molecular sig-
nals triggered in the two cases.
In our experiments, while the addition of H2O2 to the
cultures induced an increased DSP gene expression, the
levels were not as high as those elicited by OCTA, even
though the DHR-ROS derived green fluorescence was the
highest observed (7.5±0.9 times the control at 40 min after
exposure, not shown). This suggests that ROS could be
important modulators in the pathway leading to cell death.
Even though evidence for the occurrence of apoptotic-like
features in S. tropicum in response to PUA was not pro-
vided in this study, it has been previously reported that
PUA are able to induce apoptotic-like cell death in the ma-
rine diatom Thalassiosira weissflogii (Casotti et al.,
2005). In addition, in the marine diatom Phaeodactylum
tricornutum PUA exposure induced overexpression of a
gene associated to NO production (designated as PtNOA),
that resulted in an altered expression of superoxide dis-
mutase and an increased metacaspase activity, which are
known factors in the stress responses and PCD pathway
(Vardi et al., 2008). Indeed, in our OCTA-exposed S. trop-
icum cultures, PUA had a toxic effect drastically affecting
growth and inducing cell death.
ROS are generally indicated in the literature as PCD-
inducing factors (Aldsworth et al., 1999; Simon et al.,
2000). However, we suggest that a difference exists be-
tween the intracellular effectors of a PCD-like process
(e.g., NO and ROS), and other factors and stimuli that,
causing an alteration of NO and ROS, are able to modulate
the response leading to cell death. Based on the results re-
ported here, it is proposed that PUA act as such factors,
which induce modifications of the intracellular ROS lev-
els, which then result in a differential activation of ScDSP
genes. The successive reaction depends then on the amount
of ROS produced and possibly on the cell physiological
and growth status. This scenario is supported by the ob-
servation that the increase in DSP gene expression in our
experiments was not linearly correlated to PUA concen-
trations. While it is possible that the experimental design
was not appropriate to detect sudden peaks occurring at
intermediate time points, it is unlikely that these lasted for
shorter than 1 h. Indeed, when a further time point at 30
min after inoculation was included, the expression levels
were not even comparable to the highest expression meas-
ured during the experiments (data not shown). Therefore,
Fig. 2. ScDSP1 (a) and ScDSP2 (b) mRNA expression in S. tropicum cultures exposed to different OCTA concentrations and H2O2 in
LE cultures. Data are expressed as mean of fold changes of mRNA abundance (relative to control) from technical triplicates. The ex-
periments were repeated at least twice.
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18DSP expression in response to PUA
considering that a burst in ROS production always oc-
curred 20 min after exposure to all the different PUA
tested, it is possible to conclude that the concentration of
8 μM OCTA represents a threshold value below which the
cell activates defences mechanisms to cope with the re-
lated oxidative stress. (Chung et al., 2005, 2008).
Based on the results reported here, it is suggested that in
the case of PUA-induced chemical stress, DSP genes ex-
pression is regulated by ROS. Although we cannot exclude
a role for NO in the process, data from the congeneric S.
marinoi show that ROS and not NO have a main role in the
response to PUA in this diatom and probably in the con-
generic S. tropicum (Gallina et al., 2014). In addition, con-
sidered that DSP is reported to be a Ca2+ regulated protein
(Chung et al., 2005), it is then possible that in S. tropicum
ROS are acting as Ca2+ mobilizing agents, as also happens
in other biological systems (Gonzalez et al., 2002;
Mallilankaraman et al., 2011; Wendehenne et al., 2004).
CONCLUSIONS
The reported findings are only preliminary and need
to be substantiated by further experiments aimed at prov-
ing a clear connection between ROS and DSP expression.
In addition, direct data on PCD activation have to be ac-
quired, possibly using the genomic approach used by Lau-
ritano et al., (2015). However, based on these preliminary
results and combining them on the vast literature on PUA
in diatoms, we propose that in Skeletonema spp. the ROS
downstream response to PUA activates either a protection
pathway (i.e., antioxidant defence) or a cell death cascade
either through activation of specific genes or through non-
genetically controlled cell death, depending on the PUA
used, its concentration, the time of exposure, the concen-
tration of ROS produced and the physiological state of the
cell. This has important implications for bloom regulation
and population dynamics and induction of cell lysis, as
instance, immediately after the peak stage of a diatom
bloom, when cell concentrations are highest but environ-
mental factors become limiting and do not support further
growth (Vanboekel et al., 1992).
It has been proposed that the evolution of PCD in uni-
cellular organisms as a byproduct of selection might be
explained by the possibility that PCD could be a maladap-
tive stress-induced process that can be selected under cer-
tain conditions to benefit only some organisms or part of
a population (Nedelcu et al., 2010). As related to this con-
cept, if an intrapopulation signal (e.g., PUA) triggers the
production of PCD-inducing signal (e.g., ROS), and if this
intrapopulation signal-dependent death is beneficial for a
part of the population (for instance, by fitting cell density
to the environmental conditions), then PCD could be
adaptively selected for and consequently evolve into al-
truistic adaptation (Nedelcu et al., 2010). This hypothesis
needs to be tested in situ, where PCD has been shown to
exist, for instance in reaction to a viral attack (Bidle et al.
2007), supporting its role as population regulatory mech-
anism, or, triggering the production of survival forms such
as cysts or resting stages, using high ROS levels as signals
(Vardi et al., 1999), so to assure perpetuation of the
species later on, when the environment conditions become
favourable again (McQuoid et al., 2002).
ACKNOWLEDGMENTS
AAG deeply acknowledges SH Hung and SF. Wang
for their assistance during experiments and technical sup-
port. Travel fund for AAG has been supported by the Na-
tional Science Council of Taiwan (NSC) through the
Summer Program for Italian Graduate Students fellow-
ship. AAG has been supported by a SZN-OU PhD fellow-
ship. Materials for this study were partly supported by the
Taiwan National Science Council (NSC 99-2611-M-019
-014 -MY2).
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