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Journal of Experimental Marine Biology and Ecology 391 (2010) 2734

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Journal of Experimental Marine Biology and Ecology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e

Biochemical responses of red alga Gracilaria corticata (Gracilariales, Rhodophyta) to

salinity induced oxidative stress
Manoj Kumar, Puja Kumari, Vishal Gupta, C.R.K. Reddy , Bhavanath Jha
Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientic and Industrial Research (CSIR), Bhavnagar 364021, India

a r t i c l e

i n f o

Article history:
Received 26 February 2010
Received in revised form 31 May 2010
Accepted 1 June 2010
Keywords:
Antioxidant enzymes
Gracilaria corticata
Minerals
Oxidative stress
Phycobiliproteins
PUFAs
Salinity stress

a b s t r a c t
The biochemical responses of Gracilaria corticata (J. Agardh) J. Agardh to salinity induced oxidative stress were
studied following the exposure to different salinities ranging from 15, 25, 35 (control), 45 to 55 in laboratory
conditions. The growth was highest under 25 (3.14 0.69% DGR) and 35 (3.58 0.32% DGR) and decreased
signicantly in both extreme lower (15) and hyper (55) salinities. Both phycoerythrin (PE) and
allophycocyanin (APC) were signicantly higher in hyper-salinity (45) with an increase of almost 70% and
52% from their initial contents. Conversely, the level of increase of the same in hypo-salinities was considerably
lower as compared with that of hyper-salinity. Both hypo- and hyper-salinity treatments induced almost two
fold increase in the contents of polyphenols, proline and the activities of antioxidative enzymes such as
superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) especially for 6 d
exposure. The Na+ ions readily displaced the K+ and Ca2+ from their uptake sites at extreme hyper-salinity
(55) and accounted for substantial increase in the ratio of Na+/K+ and Na+/Ca2+ that impeded the
growth under long term exposure (N6 d). The survivability at salinity 45 even with considerably higher ratio of
Na+/K+ and Na+/Ca2+ suggests the compartmentalization of Na+ into the vacuoles. Further, the micro
nutrients such as Zn, Fe and Mn were decreased at both high and low end salinities with highest at extreme
hyper-salinity. The C18:1(n9) cis, C18:2(n6), C18:3(n3) and C20:4(n6) were found in signicant
amounts in hyper-salinities. The C18:1(n9) cis in particular increased by 60.25% and 70.51% for salinities 45
and 55, respectively from their initial amounts. The ratio of total unsaturated to saturated fatty acids (UFA/SFA)
also increased linearly with increasing salinity. These results collectively suggest the potential role of
antioxidative enzymes, phycobiliproteins, PUFAs and mineral nutrients to combat the salinity induced
oxidative stress in G. corticata.
2010 Elsevier B.V. All rights reserved.

1. Introduction
The red alga Gracilaria corticata (J. Agardh) J. Agardh is one of the
common algae of the Indian coast and occurs predominantly in the
lower littoral zone. It also inhabits occasionally in the intertidal rock
pools as submerged population. The intertidal algae often get exposed
to the atmosphere periodically during low tide regimes and experience an oxidative stress on regular basis with the turning tides. In
marine waters, salinity around 35 is the most common, but it could
also vary from 10 to 70 as a result of evaporation or precipitation/
freshwater inuxes (Graham and Wilcox, 2000). Osmotic stress most
often resulting from uctuating salinities exerts considerable oxidative stress on seaweeds in the intertidal zone. The previous studies
have investigated the responses of estuarine macroalgae for either
individual or combined abiotic factors (light, pH, temperature,

Corresponding author. Tel.: + 91 278 256 5801/256 3805x614; fax: + 91 278 256
6970 / 256 7562.
E-mail address: crk@csmcri.org (C.R.K. Reddy).
0022-0981/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2010.06.001

nutrient load and salinity) in the context of growth and photosynthetic

performance (Macler, 1988; Dawes et al., 1999; Israel et al., 1999; Choi
et al., 2006; Phooprong et al., 2007). Subsequent studies have also dealt
with the possible effects of environmental stresses on oristic
variations of intertidal benthic macro algal communities (Helmuth
et al., 2005).
It has been suggested that instant responses of marine plants to
adverse environmental conditions involve excess production of
reactive oxygen species (ROS) such as hydrogen peroxide (H2O2),
singlet oxygen (1O2), superoxide radical (O
2 ) and hydroxyl radical
(OH) (Dring, 2006). Increased physiological stress conditions lead to
the rapid formation of ROS that reacts with most cellular components
and thus they need to be neutralized instantly once formed.
Acclimation to altered osmotic conditions particularly to salinity
induced stress involves changes in physiological processes including
antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT),
ascorbate peroxidase (APX) and glutathione reductase (GR)] and nonenzymatic antioxidants (ascorbate, glutathione and carotenoids).
All these processes function in coordinated manner in order to
alleviate the cellular hypo/hyper osmolarity, ion disequilibrium and

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M. Kumar et al. / Journal of Experimental Marine Biology and Ecology 391 (2010) 2734

detoxication of ROS which otherwise cause oxidative destruction to

cell (Collen and Davidson, 1999; Liu and Phang, 2010; Choo et al.,
2004; Dring, 2006; Wu and Lee, 2008).
Hypo/hyper osmolarities primarily affect the external water
potential and disturb the turgor pressure, ions distribution and
organic solutes in the cell. Extended exposure to higher or lower
than the optimal salinities inhibits cell division and may result
in stunted growth (Graham and Wilcox, 2000). In halophytic plants
K+ are more important to maintain the turgor pressure in the cell
than production of organic solutes in terms of energy efciency. As
higher K+/Na+ ratio improves the salinity tolerance of the plants,
several genes and transporters contributing to cytosolic K+/Na+
homeostasis have been identied and characterized in higher plants
(Ligaba and Katsuhara, 2010). In addition, increased fatty acid
saturation in cell membranes is known to create a more condensed
lipid bilayer with lower permeability to small molecules. Changes in
the lipid environment can inuence the membrane protein
structure and uidity and consequently, stimulate or block the
ion channels activity and molecules transport. Recently, it has been
shown that membrane unsaturation is closely related to heavy
metal tolerance in Ulva lactuca (Manoj et al., 2010), salinity induced
stress in unicellular marine microalgae Dunaliella salina (Takagi
et al., 2006) and Porphyridium cruentum (Cohen et al., 1988). The
data on salinity induced variations in fatty acids and minerals
uptake is lacking more particularly in agarophytes such as Gracilaria
species.
The present study focused on determination of variations in
growth, lipid peroxidation, phycobiliproteins, antioxidant enzymes,
proline, polyphenols, PUFAs and minerals acquisition in G. corticata
grown in different salinity ranges in laboratory culture conditions. The
main objective of this study was to understand as how G. corticata
regulates its antioxidant, photosynthetic machinery, minerals uptake
and PUFAs composition to deal with salinity stress that commonly
prevails with the intertidal habitats.

determined by weighing the blot dry thalli. DGR was expressed in

percentage using the formula as given below:
h
i
1=t
DGR% = Wt =Wo 1 100

Where Wt represents fresh weight at time t, Wo represents initial

fresh weight, and t is time in days.
2.3. Determination of physiological parameter

2. Materials and methods

The level of lipid peroxidation in the thallus was determined by the

thiobarbituric acid reacting substances (TBARS) resulting from the
thiobarbituric acid reaction as described by Heath and Packer (1968).
The concentration of TBARS was expressed in terms of malondialdehyde (MDA) level by substracting the non specic absorbance
measured at A600 from A532 using an extinction coefcient of
155 mM 1 cm 1. Reactive oxygen species were determined by the
procedure as described by Contreras et al. (2005). The photosynthetic
pigments were estimated by following the method of Dawes et al.
(1999). Chlorophyll a (80 % acetone) and phycobiliproteins (100 mM
phosphate buffer, pH 6.5) were extracted by grinding the sample
(50 mg lyophilized tissue) in respective solutions (1:4 w/v) in the
dark and cold conditions followed by centrifugation at 3000 g at 4 C
for 10 min. Absorbance was recorded at 665 nm for chlorophyll a and
620, 650, and 565 nm for phycobiliproteins by using Shimadzu UV
spectrophotometer (UV-160, Shimadzu, Japan). Extinction coefcient
(11.9) was used for calculating chlorophyll a content. Phycocyanin,
allophycocyanin and phycoerythrin contents were estimated using
the equations described by Tandeau and Houmard (1988). Total
polyphenols were extracted by 80% ethanol and estimated according
to the procedures described by Parida et al. (2004) using phluroglucinol as standard. Free proline was extracted using 3% sulphosalicylic
acid and estimated following the method of Bates et al. (1973) using Lproline as standard.

2.1. Algal culture

2.4. Determination of ROS scavenging enzymes

The vegetative thalli of G. corticata were collected from intertidal

region during low tide periods from Veraval Coast (20 54 N, 70 22
E), Gujarat, India. Selected clean and young thalli were then brought
to the laboratory in a cool pack. In order to initiate unialgal culture, the
fronds were cleaned manually with brush in autoclaved seawater to
remove epiphytic foreign matters. The fronds thus cleaned were
acclimatized to laboratory conditions by culturing them in aerated
cultures in at bottom round asks with 3 L PES medium (Provasoli,
1968) supplemented with germanium dioxide (5 mg L 1) for 10 d.
During the acclimatization period, the medium was replenished twice
at 5 d interval and maintained under white cool uorescent tube
lights at 50 mol photons m 2 s 1 with a 12:12 h light: dark cycle at
22 1 C.

Extracts for measurement of superoxide dismutase (SOD),

glutathione reductase (GR), glutathione peroxidase (GPX), and
ascorbate peroxidase (APX) activities were prepared under icecold conditions in their respective extraction buffers at a proportion
of 1:2 (w/v) as described by Wu and Lee (2008). The SOD activity
was determined by photochemical inhibition of nitro blue tetrazolium (NBT) according to Beyer and Fridovich (1987). APX and GR
activities were measured by using the method of Rao et al. (1996).
One unit of enzyme activity is dened as 1 mol min 1 for APX and
GR. One unit (U) of SOD enzyme activity was taken as the quantity of
enzyme which reduced the absorbance reading of samples to 50% in
compare to control.
2.5. Determination of fatty acids and minerals

2.2. Salinity treatment and determination of growth

Following the acclimatization studies, the thalli of G. corticata were
grown in a series of salinities ranging from 15, 25, 35, 45 to 55 in
triplicate. The lower salinities were obtained by dilution of natural sea
water (35) with deionized water while higher ranges of salinities
were made by dissolving the sodium chloride in natural seawater. For
salinity treatment, the healthy thalli of 3 g fresh wt. was inoculated
and cultured in 1 L at bottom round asks with 900 mL PES medium
and cultured over a period of 12 d. The culture practice and conditions
were kept identical to the acclimatization period described earlier.
The measurement of wet weight for daily growth rate (DGR) was
done on every 4th d from the start of experiment. Fresh weight was

Prior to extraction of lipids, fresh thalli grown in different salinities

were washed with distilled water to remove any surface salt and then
blotted with paper towels. Lipids were extracted following the
method of Bligh and Dyer (1959). Fatty acids from lipids were
converted to respective methyl esters by trans-methylation using 1%
NaOH in methanol and heated for 15 min at 55 C, followed by the
addition of 5% methanolic HCl and again heated for 15 min at 55 C.
Fatty acid methyl esters (FAMEs) were extracted in hexane and
analyzed by GC-2010 coupled with GCMS-QP2010. Macro (Na, K, Ca
and Mg) and micro (Fe, Zn and Mn) minerals were measured in the
plants, dried to constant weight at 60 C. Dried tissues were acid
digested by HNO3/HClO4 (5:1, v/v) and then analyzed by inductively

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M. Kumar et al. / Journal of Experimental Marine Biology and Ecology 391 (2010) 2734

coupled plasma atomic emission spectroscopy (ICP-AES, PerkinElmer, Optima 2000).

2.6. Statistical analysis
Results are expressed as mean of three replicates with standard
deviation. Different letters on the gures indicate means values for a
parameter at different salinities for a particular day analyzed by one
way ANOVA were differed at p b 0.01.
3. Results
3.1. Effect of salinity on growth
The growth of G. corticata was found to get affected signicantly in
extreme salinity ranges (both hypo and hyper) and culture periods
investigated in the present study (Fig. 1A). Thalli grown at 15 salinity

29

showed loss of thallus rigidity, pigmentation after 9 d exposure while

at salinity 55 discoloration of thalli appeared early on 7 d in culture.
The higher daily growth rates (DGR) were consistently observed for
25 (3.14 0.69%) and 35 (3.58 0.32%) salinity (Fig.1A). At higher
salinity (45), the DGR was relatively lower (2.65 1.02%) as
compared with that of control salinity (35). The extreme low (15)
and higher (55) salinity showed signicant decrease in average DGRs
with 1.66 0.83% and 1.55 0.92% respectively.
In order to evaluate the oxidative stress generated by uctuating
salinities, lipid peroxidation in terms of MDA level and ROS generation
were determined after 4, 8 and 12 d of treatment. The MDA level and
ROS generation increased linearly with experimental duration in both
hypo- and hyper-salinities (Fig. 1B, C). Its level increased signicantly
(p b 0.01) by 1.70 fold for 15 and 1.91 fold for 55 salinity after 8 d
when compared with initial value of 0.023 mmol g1 FW (0 d). It
further increased by 2.13 and 2.30 fold for the same salinities after
12 d (Fig. 1B). Nonetheless, at 45 salinity, MDA level was marginally

Fig. 1. Effect of different salinities on the growth (A) lipid peroxidation (B) and accumulation of reactive oxygen species (C) of Gracilaria corticata. Different letters on the similar
shade columns indicate mean values for a particular day that were signicantly differed at p b 0.01. (Mean S.D, n3) analyzed by one way ANOVA. Shaded columns are indicated as
4d
8d
12 d.

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M. Kumar et al. / Journal of Experimental Marine Biology and Ecology 391 (2010) 2734

low compared with that of extreme hypo/hyper-salinities but

increased signicantly (p b 0.01) by 1.611.83 fold after 8 and 12 d
exposure when compared to initial value. At 25 salinity, MDA
concentration increased marginally and showed an average value of
0.030 mmol g1 FW over the studied period. The plants grown in

control salinity did not show signicant variation in the MDA level
over the studied period and accounted an average value of
0.023 mmol g 1 FW. The content of reactive oxygen species also
followed the similar trend as that of MDA and accumulated more in
both hypo and hyper-salinities with the culture duration when

Fig. 2. Effect of different salinities on chlorophyll (A) and phycobiliproteins like phycoerythrin (B) allophycocyanin (C) phycocyanin (D) of Gracilaria corticata. Different letters on the
similar shade columns indicate mean values for a particular day that were signicantly differed at p b 0.01. (Mean S.D, n3) analyzed by one way ANOVA. Shaded columns are
indicated as
0d
3d
6d
9 d.

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M. Kumar et al. / Journal of Experimental Marine Biology and Ecology 391 (2010) 2734

compared to the initial amount of 0.2 nmol DCF g 1 h 1 FW (Fig. 1 C).

Consequently, the culture period for subsequent experiments was
limited to 9 d only to minimize excess toxicity and cell death as a
result of extreme conditions and thus, data was collected at every 3 d
interval.
3.2. Changes in photosynthetic pigments
A decline in chlorophyll concentration was observed in almost all
tested salinities (15, 45 and 55) except for 25 and 35 salinity where it
increased with the cultured period and showed an average value of
0.60 0.05 and 0.68 0.06 mg g1 DW, respectively. In both extreme
low (15) and higher salinities (55), chlorophyll signicantly reduced
(p b 0.01) to almost half of their initial value of 0.63 0.08 mg g1 DW
(0 d) and attained a peak value after 3 d (Fig. 2A). Thalli exposed to 45
salinity showed an average decrease of 27% from the initial value and
reached the peak value 0.52 0.11 mg g1 DW after 3 d.
Phycobiliproteins such as phycoerythrin (PE), allophycocyanin
(APC) and phycocyanin (PC) showed varied responses at different
salinities (Fig. 2BD). The concentration of both PE and APC particularly
at 45 salinity were signicantly higher (pb 0.01) with a maximum
content of 3.96 0.26 mg g1 DW and 2.51 0.11 mg g1 DW, respectively (3 d). This correspond to an increase of 71.42 4.26% and
52.12 5.59% from their initial amount with 2.31 0.21 and 1.65
0.10 mg g1 DW, respectively. The degree of increase for the same at
hypo-salinities was considerably low when compared with hypersalinity.
The pigment phycocyanin showed a distinct pattern in response to
salinity stress. With increasing salinity it attained peak values in the
thalli when exposed to hyper-salinities (Fig. 2D). The salinities 45
and 55 registered the maximum values on 3 d with 2.63 0.17 and
2.15 0.15 mg g1 DW and accounted for an overall increase of
53.70 5.92% and 24.80 2.54% respectively from their initial
content 1.70 0.21 mg g1 DW. The thalli exposed to hypo-salinity
(15) experienced a 30% (pb 0.01) decrease in PC concentration from the
initial values and registered an average value of 1.27 0.23 mg g1 DW.
Nonetheless, at control salinity the concentration of PE, APC and PC did
not vary signicantly (pb 0.05) over the studied period from its initial
level (Fig. 2BD).
3.3. Changes in polyphenols and proline content
Polyphenols and free proline concentration at control salinity 35
did not show signicant variation throughout the studied period

31

(p b 0.01) and registered an average value of 0.57 0.07 and 0.48

0.08 mg g1 DW, respectively (Table 1). At hyper-salinities 45 and 55,
both polyphenols and proline were markedly higher (p b 0.01).
The peak values obtained on 6 d for polyphenols and proline at
salinity 45 were 1.34 0.08 and 0.89 0.10 mg g1 DW, respectively.
The corresponding peak values at salinity 55 were 0.92 0.06 and
0.67 0.05 mg g1 DW (3 d). This increase at both the hypersalinities have accounted to an increase of almost two fold from the
initial polyphenol (0.54 0.07 mg g1 DW) and proline (0.44
0.09 mg g1 DW) contents. On the other hand, thalli grown in
hypo-salinity (15) showed their maximum accumulation on 6 d
with 0.73 0.08 and 0.84 0.10 mg g1 DW respectively. A marginal
increment for the same has been observed at salinity 25 (Table 1).
3.4. Antioxidant enzyme activities
SOD, APX and GR were the enzymes selected to evaluate the
salinity induced oxidative stress. The specic activities of all these
enzymes increased in both hypo- and hyper-salinities and attained
peak values after 6 d and decreased thereafter gradually with the
culture period. The increase was noticeably higher (p b 0.01) at 45
salinity with the specic activity of 353 12 U mg 1 protein (SOD),
0.69 0.07 U mg 1 protein (APX) and 1.11 0.13 U mg 1 protein
(GR) after 6 d (Table 1) that corresponded to an increase of 2 fold
from the initial value of 139 12 (SOD), 0.33 0.09 (APX) and 0.42
0.06 (GR) U mg 1 protein. The increase of these enzyme activities at
salinities 15 and 55 were comparatively low but were signicant
(p b 0.01) when compared to the initial values (Table 1). In control
salinity (35) the enzyme activities did not differ signicantly
(p b 0.01) over the culture duration, therefore only average values
obtained for the enzyme activity over the studied period has been
shown in Table 1.
3.5. Minerals uptake
The ionic ratio of Na+/K+ and Na+/Ca2+ has greatly inuenced by
both hypo- and hyper-salinity treatments (Table 2). Their ratio
decreased constantly with culture duration in hypo-saline condition
(15). The decrease was more pronounced for Na+/Ca2+ which was
dropped by almost 47% (p b 0.01) compared with that of 32% (p b 0.01)
decrease for Na+/K+ ratio after 9 d of culture duration. The higher
concentration of Na+ in the external solution in case of hypersalinities resulted a decline in K+ and Ca2+ concentrations in the
tissue and consequently elevated the ratio of Na+/K+ (1.5 fold) and

Table 1
Effect of different salinities on antioxidative enzymes activities, proline and polyphenol contents in G. corticata. (Means S.D, n = 3).
Salinity

15

25

35
45

55

Time
(days)

SOD

0
3
6
9
0
3
6
9

139 12c
169 19b
263 11a
120 10c
139 12b
183 22a
170 16ab
177 14a
148 12
139 12c
255 10b
353 12a
149 14c
139 12b
259 18a
187 21ab
172 17ab

0
3
6
9
0
3
6
9

(U mg

APX
1

GR

protein)

Proline
(mg g

0.33 0.09b
0.42 0.06ab
0.55 0.10a
0.26 0.07c
0.33 0.09b
0.36 0.07b
0.44 0.10a
0.39 0.04ab
0.30 0.06
0.33 0.09c
0.58 0.11ab
0.69 0.07a
0.47 0.10b
0.33 0.09b
0.53 0.03a
0.36 0.06b
0.27 0.05c

0.42 0.06b
0.61 0.08a
0.70 0.11a
0.37 0.09b
0.42 0.06c
0.60 0.09ab
0.57 0.07b
0.66 0.07a
0.47 0.08
0.42 0.06c
0.96 0.07a
1.11 0.13a
0.50 0.08b
0.42 0.06b
0.81 0.06a
0.42 0.04b
0.36 0.06c

Mean values of a particular salinity in column with different superscripts are signicantly different at p b 0.01;
* indicate the average mean value of control for different parameters over the studied period.

Polyphenols
DW)

0.44 0.09b
0.61 0.10ab
0.73 0.08a
0.40 0.05b
0.44 0.09b
0.52 0.07a
0.51 0.07a
0.50 0.06a
0.48 0.08
0.44 0.09c
0.77 0.10ab
0.89 0.10a
0.62 0.06b
0.44 0.09b
0.67 0.05a
0.48 0.06b
0.36 0.04c

0.54 0.07bc
0.72 0.05b
0.84 0.10a
0.47 0.07c
0.54 0.07b
0.75 0.06a
0.67 0.10ab
0.72 0.10a
0.57 0.07
0.54 0.07c
1.09 0.08ab
1.34 0.08a
0.84 0.04b
0.54 0.07bc
0.92 0.06a
0.68 0.07b
0.41 0.05c

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M. Kumar et al. / Journal of Experimental Marine Biology and Ecology 391 (2010) 2734

Table 2
Effect of different salinities on minerals acquisition in G. corticata (Means S.D, n = 3).
Salinity

Time (Days)

Na+/K+

Na+/Ca2+

Mg

Fe

Zn

Mn

15

0
3
6
9
0
3
6
9

0.77a
0.70ab
0.65b
0.52c
0.77a
0.74a
0.78a
0.74a
0.78
0.77b
0.90a
0.94a
0.96a
0.77c
1.03b
1.14a
1.19a

12.87a
9.03b
7.64bc
6.76c
12.87a
11.64a
10.08ab
9.61b
12.72
12.87c
13.32b
12.70c
16.63a
12.87c
16.14bc
20.46b
29.18a

0.53 0.06bc
0.72 0.08a
0.63 0.06b
0.46 0.06c
0.53 0.06b
0.56 0.06b
0.62 0.10a
0.57 0.09b
0.56 0.04
0.53 0.06a
0.49 0.11ab
0.48 0.05ab
0.41 0.05b
0.53 0.06a
0.46 0.05b
0.38 0.08c
0.34 0.06c

5.88 0.30a
4.58 0.26b
5.08 0.51a
4.28 0.45b
5.88 0.30a
4.63 0.66c
5.56 0.47a
5.09 0.69b
6.04 0.33
5.88 0.30a
3.92 0.57c
4.73 0.42b
4.33 0.37bc
5.88 0.30a
3.72 0.29c
4.13 0.13b
3.92 0.11bc

1.93 0.15bc
3.03 0.18a
2.58 0.31b
1.64 0.31c
1.93 0.15c
2.15 0.33b
2.55 0.41a
2.23 0.41a
1.95 0.26
1.93 0.15b
2.41 0.37a
2.45 0.20a
1.77 0.17c
1.93 0.15ab
2.07 0.18a
1.57 0.22b
1.36 0.07c

2.39 0.21a
1.39 0.12b
1.45 0.22b
1.20 0.09c
2.39 0.21a
1.71 0.29c
1.94 0.42b
1.96 0.22b
2.38 0.25
2.39 0.21a
2.37 0.15a
2.32 0.16a
1.82 0.42b
2.39 0.21a
1.86 0.33b
2.05 0.34a
1.41 0.28c

25

35*
45

55

0
3
6
9
0
3
6
9

Mean values of a particular salinity in column with different superscripts are signicantly different at p b 0.01; * indicate the average mean value of control for different parameters
over the studied period.

Na+/Ca2+ (N2 fold) (p b 0.01) after 9 d exposure. The Mg content after

attaining the initial rise of 0.72 mg/100 g DW (3 d) compared to initial
value (0.53 mg/100 g DW, 0 d), decreased gradually with the exposure time at 15 salinity. This decrease was more prominent at hypersalinity (55) where it decreased by almost 32% at the end of culture
period. Among the micronutrients investigated, Zn accumulated
signicantly in the thalli exposed to both hypo (15) and hypersalinities (45) (Table 2), though at extreme hyper-salinity (55) it
consistently decreased with the culture duration. The level of Mn
decreased sharply during the culture duration (1.52 fold, p b 0.01) in
both extreme end salinities from their initial value, though its level in
salinity 45 was almost similar to the control salinity (35) till the
bleaching appeared on peripheral margins after 9 d exposure. The Fe
content decreased marginally in hypo-salinities but decreased sharply
(p b 0.01) in hyper-salinities (Table 2).
3.6. Changes in fatty acids composition
The fatty acid composition of G. corticata grown in different
salinities is shown in Table 3. Palmitic (16:0), stearic (18:0), palmitoleic
(C16:1, n7), oleic (C18:1, n9cis), linoleic (C18:2, n6), linolenic
(C18-3, n3) and arachidonic acids (C20:4, n6) were the foremost
fatty acids that showed major uctuations in response to salinity stress.
At hypo-salinity (15), among the monounsaturated fatty acids,
palmitoleic acid C16:1(n7) particularly increased by 44.61% while
unsaturated fatty acids such as C18:1(n9) cis; C18:2(n6) and C18:3
(n3) decreased by 16.67%, 28.41% and 32.23% respectively from their
initial content (Table 3). In contrast, at hyper-salinities (N35) the
contents of C18:1(n9) cis; C18:2(n6), C18:3(n3) and C20:4(n6)
fatty acids were increased by 60.25%, 31.81%, 32.75% and 12.17% for
45 salinity and 70.51%, 54.92%, 22.70%, 11.05% for 55 salinity from their
initial content. Of the total fatty acids (TFA), the amount of total
saturated fatty acids (SFAs) declined considerably with increasing
salinity and thus was in contrast to total unsaturated fatty acids
(UFAs) content. This in turn increased the ratio of total UFA/SFA and
registered an average value of 1.30, 1.43, 1.68, 2.61 and 2.56 for salinities
15, 25, 35, 45 and 55 respectively, compared to their respective initial
value 1.66.
4. Discussion
It is evident from the growth experiments that G. corticata has a
narrow salinity tolerance range and can sustain its growth well in
the salinity ranges between 25 and 35 (Fig. 1A). The growth rate of
G. corticata obtained in the present study for normal salinity (35) is

somewhat the same as described for other Gracilaria species from

open sea coasts (Nelson et al., 1980; Phooprong et al., 2007). The
higher DGR values observed for 25 and 35 salinities could be
Table 3
Fatty acids composition (% of the total FAMEs) of G. corticata exposed to different
salinities.
Fatty acid

Salinity Days (Mean, n3)

0

C16:0

15
25
35
45
55
C18:0
15
25
35
45
55
C16:1(n7) 15
25
35
45
55
C18:1(n9) 15
cis
25
35
45
55
C18:2(n6) 15
25
35
45
55
C18:3(n3) 15
25
35
45
55
C20:4(n6) 15
25
35
45
55
UFA/SFA 15
25
35
45
55

33.96 39.20
37.14
33.79
25.06
23.25
2.21
2.86
2.21
2.19
1.80
1.50
0.65
0.85
0.87
0.61
0.55
0.53
0.78
0.67
0.65
0.77
1.26
1.33
2.64
1.98
2.15
2.55
3.34
4.24
5.77
3.91
4.28
5.86
7.49
7.23
52.18 48.39
50.37
52.40
59.20
60.72
1.66
1.29
1.43
1.68
2.61
2.93

39.07
36.94
33.54
25.21
24.25
2.84
2.32
2.26
1.85
1.46
0.88
0.84
0.66
0.55
0.54
0.63
0.70
0.75
1.16
1.29
1.91
2.16
2.43
3.45
4.40
3.96
4.15
5.88
7.68
7.39
48.56
50.63
52.53
58.67
59.49
1.30
1.44
1.68
2.58
2.79

38.78
37.29
33.58
24.80
30.81
2.87
2.25
2.17
1.88
1.80
1.09
0.85
0.70
0.58
0.57
0.65
0.68
0.78
1.35
1.38
1.79
2.27
2.54
3.65
3.62
3.87
4.32
5.88
7.82
6.62
48.70
49.98
52.41
58.53
53.64
1.31
1.42
1.69
2.63
1.95

Overall
Mean SD

% change
over 0DAT

39.01 0.21a
37.13 0.17a
33.72 0.13a
25.02 0.21b
26.10 4.11b
2.86 0.01a
2.26 0.06a
2.21 0.05a
1.84 0.04b
1.59 0.19b
0.94 0.13a
0.86 0.02ab
0.66 0.04bc
0.56 0.02c
0.55 0.02c
0.65 0.02c
0.68 0.02c
0.77 0.02bc
1.25 0.09a
1.33 0.04a
1.89 0.10b
2.20 0.07b
2.54 0.06b
3.48 0.16a
4.09 0.01a
3.91 0.05c
4.25 0.09c
5.85 0.01b
7.66 0.17a
7.08 0.41a
48.55 0.16b
50.33 0.33b
52.38 0.07ab
58.80 0.35a
57.95 3.78a
1.30 0.01c
1.43 0.01c
1.68 0.01bc
2.61 0.02a
2.56 0.53ab

+ 14.87
+ 9.33
0.70
26.32
23.14
+ 29.41
+ 2.26
0
16.74
28.05
+ 44.61
+ 24.41
+ 1.54
13.84
15.38
16.67
12.82
1.28
+ 60.25
+ 70.51
28.41
16.67
3.78
+ 31.81
+ 54.92
32.23
26.34
+ 1.39
+ 32.75
+ 22.70
6.96
3.55
+ 0.38
+ 12.17
+ 11.05
21.69
13.86
+ 1.20
+ 57.22
+ 54.21

Overall mean values for each fatty acid in the respective column with different
superscript are signicantly different at p b 0.01.

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M. Kumar et al. / Journal of Experimental Marine Biology and Ecology 391 (2010) 2734

attributable to its adaptation for oceanic conditions exclusively. We

also observed its distribution in nature exclusively conning to open
sea coasts. Gracilaria chorda and G. salicornia have also been reported
to grow well at salinities between 25 and 35 (Choi et al., 2006;
Phooprong et al., 2007). In contrast, G. verrucosa and G. tenuistipitata
have shown a broad salinity tolerance ranging from 5 to 47 extending
their distribution boundaries from brackish waters to marine waters
(Israel et al., 1999). The lower DGR values in extreme hypo- and
hyper-salinities (15 and 55) observed here can likely be accredited to
the increased lipid peroxidation indicated by higher MDA level and
ROS accumulation.
Chl a and the phycobiliproteins are the key photosynthetic
pigments in the red algae. Their cellular level could be an important
physiological parameter for assessment of stress environment. The
present study showed decreased chlorophyll content in both extreme
hypo- and hyper-salinity while marginal uctuations at 25 and 35
salinities. Among the phycobiliproteins PE and APC were found
accumulated in both hypo- and hyper-salinities, however PC attained
peak value in hyper-salinities only. The accumulation of phycobiliproteins could be a possible acclamatory mechanism for G. corticata in
response to salinity induced stress as these proteins function as
storage proteins for biosynthesis during the adaptation period under
adverse conditions. Recently, Kim et al. (2007) reported the storage
function of PE in Porphyra tissue cultured in a media supplemented with
higher doses of ammonia (250mol L 1). Moreover, Cano-Europa et al.
(2010) suggested the phycobiliproteins as good antioxidants prevent
oxidative stress because of their nucleophilic ability to neutralize
reactive oxidants. The increase in phycobiliproteins especially in hypersalinities has also been reported for G. tenuistipitata cultures when
shifted from 20 to 40 salinities (Israel et al., 1999). In contrast,
Macler (1988) observed a decrease in the level of both chlorophyll
and phycobiliproteins in both hypo- and hyper-salinities (550).
Interestingly, in the present study both phycoerythrin and phycocyanin
contents did not increase signicantly in hypo-saline conditions as
compared to hyper-saline conditions (Fig. 2B & D) despite the fact
that these pigments function as a nitrogen reserves in hypo-saline
conditions. Therefore, hypo- and hyper-saline conditions are not
specic to induce or inhibit a particular site of photosynthetic machinery
rather affect many sites involved in this process in one or other manner
and bring about signicant changes in the photosynthetic performance
of the algae.
A consistent increase in the content of both polyphenols and
proline particularly at salinity 45 could be an adaptive feature to
improve the turgor required for maintaining the ionic balance during
salinity induced osmotic stress. The higher level of their contents in
both the extreme end salinities (15 and 55) were not enough to
sustain the growth of plants under these salinity conditions as
evidenced by decreased daily growth rate, higher MDA and ROS
generation. Interestingly, polyphenol responded more distinctly to
both hypo- and hyper-salinity conditions than did free proline as
evident by their increased level of polyphenol (Table 1). From these
results, it can be speculated that polyphenols may play a major role to
scavenge the ROS generated by salinity induced oxidative stress.
Parida et al. (2004) reported an enhanced level of polyphenol as an
acclimatization mechanism that ameliorating the ionic effect resulting
from hypo- and hyper-salinity conditions. A considerable accumulation of proline in both hypo- and hyper-salinity conditions has
also been reported for Ulva pertusa (Kakinuma et al., 2006) and
G. tenuistipitata (Lee et al., 1999).
Recently, Dring (2006) reported that oxidative stresses tend to
result in a gradual buildup of ROS in macroalgae. SOD, APX and GR are
the important antioxidative enzymes in the plant cell to protect cell
against the peroxidation system and to maintain the redox state of the
cell. Enhanced activities of all three antioxidative enzymes in the
present study especially at 15 and 45 salinities compared to control
during initial 6 days suggest the generation of all kind of reactive

33

oxygen species viz. O

2 , H2O2 and OH in the plant which would have
detoxied by these enzymes. Thus, G. corticata could resist the stress
of adversity and maintain the normal construction and function of cell
membrane as evident by a reduction of only 2530% in chlorophyll
pigment during the initial days of culture period at salinities 15 and
45. Thereafter, a sharp decline observed in activities of antioxidative
enzymes could be due to formation of ROS beyond the critical limit
which might pose serious threat to plant cell, causing higher lipid
peroxidation in hypo- and hyper-salinities. Supporting to our results,
Collen and Davidson (1999); Choo et al. (2004) found a positive
relation between low peroxidative damage and a more efcient
antioxidative system in intertidal red and green seaweeds, respectively. A signicant higher activity of antioxidative enzymes in
U. lactuca was shown for better acclimatization to the changing
environments than the conspecics in the subtidal habitats (Ross and
Van Alstyne, 2007).
Further, a sizeable increase in GR activity with 1.11 0.17 U mg 1
protein in 45 salinity compared to the activity of 0.70 0.06 U mg 1
protein at 15 salinity after 6 d exposure suggests greater demand for
the supply of reduced glutathione. Reduced glutathione helps in
ascorbate regeneration, regulate the protein thiol-disulde exchange
reactions, maintain the redox state of the cell and thus increases the
stress tolerance. On the other hand, inadequate antioxidative
enzymes activities at extreme high salinity (55) might also be
correlated with early onset of bleaching and tissue necrosis
symptoms. Lu et al. (2006) also showed a decreased antioxidant
activity in Ulva fasciata during long term (4 d) exposure to higher
salinity (90) due to increased H2O2, and MDA level. Enhanced activity
of either individual or combined SOD, APX and GR enzymes have also
been observed in U. fasciata and U. lactuca (Sung et al., 2009; Manoj et al.,
2010), Grateloupia turuturu and Palmaria palmata (Liu and Phang, 2010)
when subjected to oxidative stress such as hyper-salinity, heavy metal
and/or other adverse environment conditions.
Among the most common effects of altered salinity is the growth
inhibition which mainly occurs due to change in external water
potential. Elevated salinity (55) greatly affect the homeostasis of the
cell as indicated by bleaching symptoms and enhanced Na+/K+ and
Na+/Ca2+ ratio. This could be explained due to the antagonistic
behavior of Na+ and K+ at the uptake sites which in turn impaired the
ion selectivity and integrity of the cell membrane and permit the
passive and excess accumulation of Na+ in the thalli. Moreover, high
Na+/K+ ratio has been suggested to disturb various metabolic
processes such as protein synthesis in the cytoplasm. A substantial
tolerance capacity of plants grown at salinity 45 could be attributed to
the compartmentalization of Na+ into the vacuole before its
concentration increases beyond the threshold concentration in the
cytoplasm. In addition a considerable accumulation of intracellular
Ca2+ at salinity 45 below the toxic level as compared to 55 improved
the tolerance ability of the plants. Lee and Liu (1999) found that Ca2+
involves in signaling mechanism of salinity induced proline accumulation for osmotic adjustment. Therefore, Na+/K+ and Na+/Ca2+ ratio
might be promising indicators for salt resistance. A two fold increase
for polyphenols and proline in the present study may be another
reason to cope with this hyper osmolarity condition. On the other
hand, at salinity 15 a relatively lower ratio of Na+/K+ and Na+/Ca2+
together with accumulated Mg indicate the gradual accumulation of
K+ and Ca2+ that eventually accumulated beyond their critical
concentration and thus plants collapsed when exposed for longer
duration under this salinity.
The most signicant difference in fatty acid composition is the
enhanced relative proportion of C18:1(n9) cis; C18:2(n6) and
C18:3(n3) by approximately 1.31.6 fold over their initial content
with a parallel decrease of palmitoleic acid C16:1(n7) at hypersalinities. A possible explanation for this change could be the salinity
induced partial inhibition of 9 desaturase enzyme that inserts the rst
double bond in C16:0 and the induction of 9 desaturase enzyme

Author's personal copy

34

M. Kumar et al. / Journal of Experimental Marine Biology and Ecology 391 (2010) 2734

responsible for inducing double bonds in stearic acid to convert most of

the C18:0 to C18:1(n9) which later become a substrate for the
succeeding enzyme reactions and resulted in more PUFA synthesis.
Higher content of these PUFAs could be an adaptive strategy for
maintaining the requisite greater membrane uidity, to stabilize the
protein complexes of photosystem II and to control membrane physicochemical properties such as the increased activity of Na+/H+ antiporter
system of the plasma membrane to cope with hyper-salinity stress
(Khotimchenko and Yakovleva, 2005). Interestingly, Leu et al. (2006)
reported a similar pattern of decreased C16 MUFA (16:1; n7) in the
marine diatoms Thalassiosira antarctica and Bacterosira bathyomphala
when exposure to UV radiation. In conditions of drought, a decrease of
C18 PUFAs reect membrane damage, while an increase in the level of
these fatty acid, as observed here, may represent a defense component
(Zhang et al., 2005). On the other hand, at hypo-salinity (15) relatively
more rigid membranes during initial 6 d culture period would have
prevented the ion loss to certain extent and thus increased membrane
uidity brought by higher PUFAs level was not needed. The prolonged
exposure to these adverse hypo-saline conditions impaired the cell
function due to disrupted growth and synthesis of photosynthetic
membranes leading to lower levels of PUFAs (Floreto et al., 1994).
5. Conclusion
The overall ndings presented in this study suggest that G.
corticata regulates its antioxidant machinery to eliminate ROS under
long term stress conditions. Maintenance of Na+/K+ and Na+/Ca2+
ratio to the optimum level and compartmentalization of Na+ into the
vacuoles together with the higher level of polyphenols and proline
could be the tolerant strategies to combat the salt stress particularly at
salinity 45. In addition, higher concentration of PUFAs such as oleic,
linoleic, linolenic and phycobiliproteins particularly phycoerythrin
full the demand of higher energy required for biosynthesis,
reorganization of cell and to maintained the requisite membrane
uidity under salinity stress condition.
Acknowledgements
The nancial support received from the Council of Scientic and
Industrial Research (NWP 018), New Delhi is gratefully acknowledged. The authors are grateful to Dr. Dilip Ghosh, Director, Nutri
Connect, Australia for scientic editing of the manuscript. The rst
author (MK) and second author (PK) gratefully acknowledges the
CSIR, New Delhi for awarding the Senior and Junior Research
Fellowships respectively. The third author (VG) also expresses his
gratitude to Department of Science and Technology, New Delhi for
nancial support. We would also like to thank Mr. Harshad R.
Brahmbhatt for technical assistance in analysis of fatty acids using GCMS.
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