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Biochemical responses of red alga Gracilaria corticata (Gracilariales, Rhodophyta) tosalinity induced oxidative stress

Manoj Kumar, Puja Kumari, Vishal Gupta, C.R.K. Reddy ⁎, Bhavanath JhaDiscipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), Bhavnagar 364021, India

a b s t r a c ta r t i c l e i n f o

Article history:Received 26 February 2010Received in revised form 31 May 2010Accepted 1 June 2010

Keywords:Antioxidant enzymesGracilaria corticataMineralsOxidative stressPhycobiliproteinsPUFAsSalinity stress

The biochemical responses of Gracilaria corticata (J. Agardh) J. Agardh to salinity induced oxidative stress werestudied following the exposure to different salinities ranging from 15, 25, 35 (control), 45 to 55 in laboratoryconditions. The growth was highest under 25 (3.14±0.69% DGR) and 35 (3.58±0.32% DGR) and decreasedsignificantly in both extreme lower (15) and hyper (55) salinities. Both phycoerythrin (PE) andallophycocyanin (APC) were significantly higher in hyper-salinity (45) with an increase of almost 70% and52% from their initial contents. Conversely, the level of increase of the same in hypo-salinitieswas considerablylower as compared with that of hyper-salinity. Both hypo- and hyper-salinity treatments induced almost twofold increase in the contents of polyphenols, proline and the activities of antioxidative enzymes such assuperoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) especially for 6 dexposure. 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 thegrowth under long term exposure (N6 d). The survivability at salinity 45 evenwith considerably higher ratio ofNa+/K+ and Na+/Ca2+ suggests the compartmentalization of Na+ into the vacuoles. Further, the micronutrients such as Zn, Fe and Mn were decreased at both high and low end salinities with highest at extremehyper-salinity. The C18:1(n−9) cis, C18:2(n−6), C18:3(n−3) and C20:4(n−6) were found in significantamounts in hyper-salinities. The C18:1(n−9) cis in particular increased by 60.25% and 70.51% for salinities 45and 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 ofantioxidative enzymes, phycobiliproteins, PUFAs and mineral nutrients to combat the salinity inducedoxidative 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 thecommon algae of the Indian coast and occurs predominantly in thelower littoral zone. It also inhabits occasionally in the intertidal rockpools as submerged population. The intertidal algae often get exposedto the atmosphere periodically during low tide regimes and experi-ence an oxidative stress on regular basis with the turning tides. Inmarine waters, salinity around 35 is the most common, but it couldalso vary from 10 to 70 as a result of evaporation or precipitation/freshwater influxes (Graham and Wilcox, 2000). Osmotic stress mostoften resulting from fluctuating salinities exerts considerable oxida-tive stress on seaweeds in the intertidal zone. The previous studieshave investigated the responses of estuarine macroalgae for eitherindividual or combined abiotic factors (light, pH, temperature,

nutrient load and salinity) in the context of growth andphotosyntheticperformance (Macler, 1988; Dawes et al., 1999; Israel et al., 1999; Choiet al., 2006; Phoopronget al., 2007). Subsequent studies have also dealtwith the possible effects of environmental stresses on floristicvariations of intertidal benthic macro algal communities (Helmuthet al., 2005).

It has been suggested that instant responses of marine plants toadverse environmental conditions involve excess production ofreactive oxygen species (ROS) such as hydrogen peroxide (H2O2),singlet oxygen (1O2), superoxide radical (O2

•−) and hydroxyl radical(OH−) (Dring, 2006). Increased physiological stress conditions lead tothe rapid formation of ROS that reacts with most cellular componentsand thus they need to be neutralized instantly once formed.Acclimation to altered osmotic conditions particularly to salinityinduced stress involves changes in physiological processes includingantioxidant enzymes [superoxide dismutase (SOD), catalase (CAT),ascorbate peroxidase (APX) and glutathione reductase (GR)] and non-enzymatic antioxidants (ascorbate, glutathione and carotenoids).All these processes function in coordinated manner in order toalleviate the cellular hypo/hyper osmolarity, ion disequilibrium and

Journal of Experimental Marine Biology and Ecology 391 (2010) 27–34

⁎ Corresponding author. Tel.: +91 278 256 5801/256 3805x614; fax: +91 278 2566970 / 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

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detoxification of ROS which otherwise cause oxidative destruction tocell (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 waterpotential and disturb the turgor pressure, ions distribution andorganic solutes in the cell. Extended exposure to higher or lowerthan the optimal salinities inhibits cell division and may resultin stunted growth (Graham and Wilcox, 2000). In halophytic plantsK+ are more important to maintain the turgor pressure in the cellthan production of organic solutes in terms of energy efficiency. Ashigher K+/Na+ ratio improves the salinity tolerance of the plants,several genes and transporters contributing to cytosolic K+/Na+

homeostasis have been identified and characterized in higher plants(Ligaba and Katsuhara, 2010). In addition, increased fatty acidsaturation in cell membranes is known to create a more condensedlipid bilayer with lower permeability to small molecules. Changes inthe lipid environment can influence the membrane proteinstructure and fluidity and consequently, stimulate or block theion channels activity and molecules transport. Recently, it has beenshown that membrane unsaturation is closely related to heavymetal tolerance in Ulva lactuca (Manoj et al., 2010), salinity inducedstress in unicellular marine microalgae Dunaliella salina (Takagiet al., 2006) and Porphyridium cruentum (Cohen et al., 1988). Thedata on salinity induced variations in fatty acids and mineralsuptake is lacking more particularly in agarophytes such as Gracilariaspecies.

The present study focused on determination of variations ingrowth, lipid peroxidation, phycobiliproteins, antioxidant enzymes,proline, polyphenols, PUFAs and minerals acquisition in G. corticatagrown in different salinity ranges in laboratory culture conditions. Themain objective of this study was to understand as how G. corticataregulates its antioxidant, photosynthetic machinery, minerals uptakeand PUFAs composition to deal with salinity stress that commonlyprevails with the intertidal habitats.

2. Materials and methods

2.1. Algal culture

The vegetative thalli of G. corticata were collected from intertidalregion during low tide periods from Veraval Coast (20° 54′ N, 70° 22′E), Gujarat, India. Selected clean and young thalli were then broughtto the laboratory in a cool pack. In order to initiate unialgal culture, thefronds were cleaned manually with brush in autoclaved seawater toremove epiphytic foreign matters. The fronds thus cleaned wereacclimatized to laboratory conditions by culturing them in aeratedcultures in flat bottom round flasks with 3 L PES medium (Provasoli,1968) supplemented with germanium dioxide (5 mg L−1) for 10 d.During the acclimatization period, the mediumwas replenished twiceat 5 d interval and maintained under white cool fluorescent tubelights at 50 μmolphotons m−2 s−1 with a 12:12 h light: dark cycle at22±1 °C.

2.2. Salinity treatment and determination of growth

Following the acclimatization studies, the thalli of G. corticataweregrown in a series of salinities ranging from 15, 25, 35, 45 to 55 intriplicate. The lower salinities were obtained by dilution of natural seawater (35) with deionized water while higher ranges of salinitieswere made by dissolving the sodium chloride in natural seawater. Forsalinity treatment, the healthy thalli of 3 g fresh wt. was inoculatedand cultured in 1 L flat bottom round flasks with 900 mL PES mediumand cultured over a period of 12 d. The culture practice and conditionswere kept identical to the acclimatization period described earlier.The measurement of wet weight for daily growth rate (DGR) wasdone on every 4th d from the start of experiment. Fresh weight was

determined by weighing the blot dry thalli. DGR was expressed inpercentage using the formula as given below:

DGR% = Wt=Woð Þ1= t−1h i

× 100

Where Wt represents fresh weight at time t, Wo represents initialfresh weight, and t is time in days.

2.3. Determination of physiological parameter

The level of lipid peroxidation in the thalluswas determined by thethiobarbituric acid reacting substances (TBARS) resulting from thethiobarbituric acid reaction as described by Heath and Packer (1968).The concentration of TBARS was expressed in terms of malondialde-hyde (MDA) level by substracting the non specific absorbancemeasured at A600 from A532 using an extinction coefficient of155 mM−1 cm−1. Reactive oxygen species were determined by theprocedure as described by Contreras et al. (2005). The photosyntheticpigments were estimated by following the method of Dawes et al.(1999). Chlorophyll a (80 % acetone) and phycobiliproteins (100 mMphosphate buffer, pH 6.5) were extracted by grinding the sample(50 mg lyophilized tissue) in respective solutions (1:4 w/v) in thedark and cold conditions followed by centrifugation at 3000 g at 4 °Cfor 10 min. Absorbance was recorded at 665 nm for chlorophyll a and620, 650, and 565 nm for phycobiliproteins by using Shimadzu UVspectrophotometer (UV-160, Shimadzu, Japan). Extinction coefficient(11.9) was used for calculating chlorophyll a content. Phycocyanin,allophycocyanin and phycoerythrin contents were estimated usingthe equations described by Tandeau and Houmard (1988). Totalpolyphenols were extracted by 80% ethanol and estimated accordingto the procedures described by Parida et al. (2004) using phluroglu-cinol as standard. Free proline was extracted using 3% sulphosalicylicacid and estimated following themethod of Bates et al. (1973) using L-proline as standard.

2.4. Determination of ROS scavenging enzymes

Extracts for measurement of superoxide dismutase (SOD),glutathione reductase (GR), glutathione peroxidase (GPX), andascorbate peroxidase (APX) activities were prepared under ice-cold conditions in their respective extraction buffers at a proportionof 1:2 (w/v) as described by Wu and Lee (2008). The SOD activitywas determined by photochemical inhibition of nitro blue tetrazo-lium (NBT) according to Beyer and Fridovich (1987). APX and GRactivities were measured by using the method of Rao et al. (1996).One unit of enzyme activity is defined as 1 μmol min−1 for APX andGR. One unit (U) of SOD enzyme activity was taken as the quantity ofenzyme which reduced the absorbance reading of samples to 50% incompare to control.

2.5. Determination of fatty acids and minerals

Prior to extraction of lipids, fresh thalli grown in different salinitieswere washed with distilled water to remove any surface salt and thenblotted with paper towels. Lipids were extracted following themethod of Bligh and Dyer (1959). Fatty acids from lipids wereconverted to respective methyl esters by trans-methylation using 1%NaOH in methanol and heated for 15 min at 55 °C, followed by theaddition of 5% methanolic HCl and again heated for 15 min at 55 °C.Fatty acid methyl esters (FAMEs) were extracted in hexane andanalyzed by GC-2010 coupled with GCMS-QP2010. Macro (Na, K, Caand Mg) and micro (Fe, Zn and Mn) minerals were measured in theplants, dried to constant weight at 60 °C. Dried tissues were aciddigested by HNO3/HClO4 (5:1, v/v) and then analyzed by inductively

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

2.6. Statistical analysis

Results are expressed as mean of three replicates with standarddeviation. Different letters on the figures indicate means values for aparameter at different salinities for a particular day analyzed by oneway ANOVA were differed at pb0.01.

3. Results

3.1. Effect of salinity on growth

The growth of G. corticatawas found to get affected significantly inextreme salinity ranges (both hypo and hyper) and culture periodsinvestigated in the present study (Fig. 1A). Thalli grown at 15 salinity

showed loss of thallus rigidity, pigmentation after 9 d exposure whileat salinity 55 discoloration of thalli appeared early on 7 d in culture.The higher daily growth rates (DGR) were consistently observed for25 (3.14±0.69%) and 35 (3.58±0.32%) salinity (Fig.1A). At highersalinity (45), the DGR was relatively lower (2.65±1.02%) ascompared with that of control salinity (35). The extreme low (15)and higher (55) salinity showed significant decrease in average DGRswith 1.66±0.83% and 1.55±0.92% respectively.

In order to evaluate the oxidative stress generated by fluctuatingsalinities, lipid peroxidation in terms ofMDA level and ROS generationwere determined after 4, 8 and 12 d of treatment. The MDA level andROS generation increased linearly with experimental duration in bothhypo- and hyper-salinities (Fig. 1B, C). Its level increased significantly(pb0.01) by 1.70 fold for 15 and 1.91 fold for 55 salinity after 8 dwhen compared with initial value of 0.023 mmol g−1 FW (0 d). Itfurther increased by 2.13 and 2.30 fold for the same salinities after12 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 similarshade columns indicate mean values for a particular day that were significantly differed at pb0.01. (Mean±S.D, n−3) analyzed by oneway ANOVA. Shaded columns are indicated as

4 d 8 d 12 d.

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low compared with that of extreme hypo/hyper-salinities butincreased significantly (pb0.01) by 1.61–1.83 fold after 8 and 12 dexposure when compared to initial value. At 25 salinity, MDAconcentration increased marginally and showed an average value of0.030 mmol g−1 FW over the studied period. The plants grown in

control salinity did not show significant variation in the MDA levelover the studied period and accounted an average value of0.023 mmol g−1 FW. The content of reactive oxygen species alsofollowed the similar trend as that of MDA and accumulated more inboth 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 thesimilar shade columns indicate mean values for a particular day that were significantly differed at pb0.01. (Mean±S.D, n−3) analyzed by one way ANOVA. Shaded columns areindicated as 0 d 3 d 6 d 9 d.

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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 waslimited to 9 d only to minimize excess toxicity and cell death as aresult of extreme conditions and thus, data was collected at every 3 dinterval.

3.2. Changes in photosynthetic pigments

A decline in chlorophyll concentration was observed in almost alltested salinities (15, 45 and 55) except for 25 and 35 salinity where itincreased with the cultured period and showed an average value of0.60±0.05 and 0.68±0.06 mg g−1 DW, respectively. In both extremelow (15) and higher salinities (55), chlorophyll significantly reduced(pb0.01) to almost half of their initial value of 0.63±0.08 mg g−1 DW(0 d) and attained a peak value after 3 d (Fig. 2A). Thalli exposed to 45salinity showed an average decrease of 27% from the initial value andreached the peak value 0.52±0.11 mg g−1 DW after 3 d.

Phycobiliproteins such as phycoerythrin (PE), allophycocyanin(APC) and phycocyanin (PC) showed varied responses at differentsalinities (Fig. 2B–D). The concentration of both PE and APC particularlyat 45 salinity were significantly higher (pb0.01) with a maximumcontent of 3.96±0.26 mg g−1 DW and 2.51±0.11 mg g−1 DW, res-pectively (3 d). This correspond to an increase of 71.42±4.26% and52.12±5.59% from their initial amount with 2.31±0.21 and 1.65±0.10 mg g−1 DW, respectively. The degree of increase for the same athypo-salinities was considerably low when compared with hyper-salinity.

The pigment phycocyanin showed a distinct pattern in response tosalinity stress. With increasing salinity it attained peak values in thethalli when exposed to hyper-salinities (Fig. 2D). The salinities 45and 55 registered the maximum values on 3 d with 2.63±0.17 and2.15±0.15 mg g−1 DW and accounted for an overall increase of53.70±5.92% and 24.80±2.54% respectively from their initialcontent 1.70±0.21 mg g−1 DW. The thalli exposed to hypo-salinity(15) experienced a 30% (pb0.01) decrease in PC concentration from theinitial values and registered anaverage valueof 1.27±0.23 mg g−1 DW.Nonetheless, at control salinity the concentration of PE, APC and PC didnot vary significantly (pb0.05) over the studied period from its initiallevel (Fig. 2B–D).

3.3. Changes in polyphenols and proline content

Polyphenols and free proline concentration at control salinity 35did not show significant variation throughout the studied period

(pb0.01) and registered an average value of 0.57±0.07 and 0.48±0.08 mg g−1 DW, respectively (Table 1). At hyper-salinities 45 and 55,both polyphenols and proline were markedly higher (pb0.01).The peak values obtained on 6 d for polyphenols and proline atsalinity 45 were 1.34±0.08 and 0.89±0.10 mg g−1 DW, respectively.The corresponding peak values at salinity 55 were 0.92±0.06 and0.67±0.05 mg g−1 DW (3 d). This increase at both the hyper-salinities have accounted to an increase of almost two fold from theinitial polyphenol (0.54±0.07 mg g−1 DW) and proline (0.44±0.09 mg g−1 DW) contents. On the other hand, thalli grown inhypo-salinity (15) showed their maximum accumulation on 6 dwith 0.73±0.08 and 0.84±0.10 mg g−1 DW respectively. A marginalincrement 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 thesalinity induced oxidative stress. The specific activities of all theseenzymes increased in both hypo- and hyper-salinities and attainedpeak values after 6 d and decreased thereafter gradually with theculture period. The increase was noticeably higher (pb0.01) at 45salinity with the specific activity of 353±12 Umg−1 protein (SOD),0.69±0.07 Umg−1 protein (APX) and 1.11±0.13 Umg−1 protein(GR) after 6 d (Table 1) that corresponded to an increase of ≥2 foldfrom 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 atsalinities 15 and 55 were comparatively low but were significant(pb0.01) when compared to the initial values (Table 1). In controlsalinity (35) the enzyme activities did not differ significantly(pb0.01) over the culture duration, therefore only average valuesobtained for the enzyme activity over the studied period has beenshown in Table 1.

3.5. Minerals uptake

The ionic ratio of Na+/K+ and Na+/Ca2+ has greatly influenced byboth hypo- and hyper-salinity treatments (Table 2). Their ratiodecreased constantly with culture duration in hypo-saline condition(15). The decrease was more pronounced for Na+/Ca2+ which wasdropped by almost 47% (pb0.01) comparedwith that of 32% (pb0.01)decrease for Na+/K+ ratio after 9 d of culture duration. The higherconcentration of Na+ in the external solution in case of hyper-salinities resulted a decline in K+ and Ca2+ concentrations in thetissue and consequently elevated the ratio of Na+/K+ (˜1.5 fold) and

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

Salinity Time(days)

SOD APX GR Proline Polyphenols

(U mg−1 protein) (mg g−1 DW)

15 0 139±12c 0.33±0.09b 0.42±0.06b 0.44±0.09b 0.54±0.07bc

3 169±19b 0.42±0.06ab 0.61±0.08a 0.61±0.10ab 0.72±0.05b

6 263±11a 0.55±0.10a 0.70±0.11a 0.73±0.08a 0.84±0.10a

9 120±10c 0.26±0.07c 0.37±0.09b 0.40±0.05b 0.47±0.07c

25 0 139±12b 0.33±0.09b 0.42±0.06c 0.44±0.09b 0.54±0.07b

3 183±22a 0.36±0.07b 0.60±0.09ab 0.52±0.07a 0.75±0.06a

6 170±16ab 0.44±0.10a 0.57±0.07b 0.51±0.07a 0.67±0.10ab

9 177±14a 0.39±0.04ab 0.66±0.07a 0.50±0.06a 0.72±0.10a

35⁎ 148±12 0.30±0.06 0.47±0.08 0.48±0.08 0.57±0.0745 0 139±12c 0.33±0.09c 0.42±0.06c 0.44±0.09c 0.54±0.07c

3 255±10b 0.58±0.11ab 0.96±0.07a 0.77±0.10ab 1.09±0.08ab

6 353±12a 0.69±0.07a 1.11±0.13a 0.89±0.10a 1.34±0.08a

9 149±14c 0.47±0.10b 0.50±0.08b 0.62±0.06b 0.84±0.04b

55 0 139±12b 0.33±0.09b 0.42±0.06b 0.44±0.09b 0.54±0.07bc

3 259±18a 0.53±0.03a 0.81±0.06a 0.67±0.05a 0.92±0.06a

6 187±21ab 0.36±0.06b 0.42±0.04b 0.48±0.06b 0.68±0.07b

9 172±17ab 0.27±0.05c 0.36±0.06c 0.36±0.04c 0.41±0.05c

Mean values of a particular salinity in column with different superscripts are significantly different at pb0.01;* indicate the average mean value of control for different parameters over the studied period.

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Na+/Ca2+ (N2 fold) (pb0.01) after 9 d exposure. TheMg content afterattaining the initial rise of 0.72 mg/100 gDW (3 d) compared to initialvalue (0.53 mg/100 gDW, 0 d), decreased gradually with the expo-sure time at 15 salinity. This decrease was more prominent at hyper-salinity (55) where it decreased by almost 32% at the end of cultureperiod. Among the micronutrients investigated, Zn accumulatedsignificantly in the thalli exposed to both hypo (15) and hyper-salinities (45) (Table 2), though at extreme hyper-salinity (55) itconsistently decreased with the culture duration. The level of Mndecreased sharply during the culture duration (1.5–2 fold, pb0.01) inboth extreme end salinities from their initial value, though its level insalinity 45 was almost similar to the control salinity (35) till thebleaching appeared on peripheral margins after 9 d exposure. The Fecontent decreasedmarginally in hypo-salinities but decreased sharply(pb0.01) in hyper-salinities (Table 2).

3.6. Changes in fatty acids composition

The fatty acid composition of G. corticata grown in differentsalinities is shown in Table 3. Palmitic (16:0), stearic (18:0), palmitoleic(C16:1, n−7), oleic (C18:1, n−9cis), linoleic (C18:2, n−6), α linolenic(C18-3, n−3) and arachidonic acids (C20:4, n−6) were the foremostfatty acids that showedmajor fluctuations in response to salinity stress.At hypo-salinity (15), among the monounsaturated fatty acids,palmitoleic acid C16:1(n−7) particularly increased by 44.61% whileunsaturated fatty acids such as C18:1(n−9) cis; C18:2(n−6) and C18:3(n−3) decreased by 16.67%, 28.41% and 32.23% respectively from theirinitial content (Table 3). In contrast, at hyper-salinities (N35) thecontents of C18:1(n−9) cis; C18:2(n−6), C18:3(n−3)andC20:4(n−6)fatty acids were increased by 60.25%, 31.81%, 32.75% and 12.17% for45 salinity and 70.51%, 54.92%, 22.70%, 11.05% for 55 salinity from theirinitial content. Of the total fatty acids (TFA), the amount of totalsaturated fatty acids (SFAs) declined considerably with increasingsalinity and thus was in contrast to total unsaturated fatty acids(UFAs) content. This in turn increased the ratio of total UFA/SFA andregistered anaverage valueof 1.30, 1.43, 1.68, 2.61 and 2.56 for salinities15, 25, 35, 45 and 55 respectively, compared to their respective initialvalue 1.66.

4. Discussion

It is evident from the growth experiments that G. corticata has anarrow salinity tolerance range and can sustain its growth well inthe salinity ranges between 25 and 35 (Fig. 1A). The growth rate ofG. corticata obtained in the present study for normal salinity (35) is

somewhat the same as described for other Gracilaria species fromopen sea coasts (Nelson et al., 1980; Phooprong et al., 2007). Thehigher DGR values observed for 25 and 35 salinities could be

Table 2Effect 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 0.77a 12.87a 0.53±0.06bc 5.88±0.30a 1.93±0.15bc 2.39±0.21a

3 0.70ab 9.03b 0.72±0.08a 4.58±0.26b 3.03±0.18a 1.39±0.12b

6 0.65b 7.64bc 0.63±0.06b 5.08±0.51a 2.58±0.31b 1.45±0.22b

9 0.52c 6.76c 0.46±0.06c 4.28±0.45b 1.64±0.31c 1.20±0.09c

25 0 0.77a 12.87a 0.53±0.06b 5.88±0.30a 1.93±0.15c 2.39±0.21a

3 0.74a 11.64a 0.56±0.06b 4.63±0.66c 2.15±0.33b 1.71±0.29c

6 0.78a 10.08ab 0.62±0.10a 5.56±0.47a 2.55±0.41a 1.94±0.42b

9 0.74a 9.61b 0.57±0.09b 5.09±0.69b 2.23±0.41a 1.96±0.22b

35* 0.78 12.72 0.56±0.04 6.04±0.33 1.95±0.26 2.38±0.2545 0 0.77b 12.87c 0.53±0.06a 5.88±0.30a 1.93±0.15b 2.39±0.21a

3 0.90a 13.32b 0.49±0.11ab 3.92±0.57c 2.41±0.37a 2.37±0.15a

6 0.94a 12.70c 0.48±0.05ab 4.73±0.42b 2.45±0.20a 2.32±0.16a

9 0.96a 16.63a 0.41±0.05b 4.33±0.37bc 1.77±0.17c 1.82±0.42b

55 0 0.77c 12.87c 0.53±0.06a 5.88±0.30a 1.93±0.15ab 2.39±0.21a

3 1.03b 16.14bc 0.46±0.05b 3.72±0.29c 2.07±0.18a 1.86±0.33b

6 1.14a 20.46b 0.38±0.08c 4.13±0.13b 1.57±0.22b 2.05±0.34a

9 1.19a 29.18a 0.34±0.06c 3.92±0.11bc 1.36±0.07c 1.41±0.28c

Mean values of a particular salinity in column with different superscripts are significantly different at pb0.01; * indicate the average mean value of control for different parametersover the studied period.

Table 3Fatty acids composition (% of the total FAMEs) of G. corticata exposed to differentsalinities.

Fatty acid Salinity Days (Mean, n−3) OverallMean±SD

% changeover 0DAT

0 3 6 9

C16:0 15 33.96 39.20 39.07 38.78 39.01±0.21a +14.8725 37.14 36.94 37.29 37.13±0.17a +9.3335 33.79 33.54 33.58 33.72±0.13a −0.7045 25.06 25.21 24.80 25.02±0.21b −26.3255 23.25 24.25 30.81 26.10±4.11b −23.14

C18:0 15 2.21 2.86 2.84 2.87 2.86±0.01a +29.4125 2.21 2.32 2.25 2.26±0.06a +2.2635 2.19 2.26 2.17 2.21±0.05a 045 1.80 1.85 1.88 1.84±0.04b −16.7455 1.50 1.46 1.80 1.59±0.19b −28.05

C16:1(n−7) 15 0.65 0.85 0.88 1.09 0.94±0.13a +44.6125 0.87 0.84 0.85 0.86±0.02ab +24.4135 0.61 0.66 0.70 0.66±0.04bc +1.5445 0.55 0.55 0.58 0.56±0.02c −13.8455 0.53 0.54 0.57 0.55±0.02c −15.38

C18:1(n−9)cis

15 0.78 0.67 0.63 0.65 0.65±0.02c −16.6725 0.65 0.70 0.68 0.68±0.02c −12.8235 0.77 0.75 0.78 0.77±0.02bc −1.2845 1.26 1.16 1.35 1.25±0.09a +60.2555 1.33 1.29 1.38 1.33±0.04a +70.51

C18:2(n−6) 15 2.64 1.98 1.91 1.79 1.89±0.10b −28.4125 2.15 2.16 2.27 2.20±0.07b −16.6735 2.55 2.43 2.54 2.54±0.06b −3.7845 3.34 3.45 3.65 3.48±0.16a +31.8155 4.24 4.40 3.62 4.09±0.01a +54.92

C18:3(n−3) 15 5.77 3.91 3.96 3.87 3.91±0.05c −32.2325 4.28 4.15 4.32 4.25±0.09c −26.3435 5.86 5.88 5.88 5.85±0.01b +1.3945 7.49 7.68 7.82 7.66±0.17a +32.7555 7.23 7.39 6.62 7.08±0.41a +22.70

C20:4(n−6) 15 52.18 48.39 48.56 48.70 48.55±0.16b −6.9625 50.37 50.63 49.98 50.33±0.33b −3.5535 52.40 52.53 52.41 52.38±0.07ab +0.3845 59.20 58.67 58.53 58.80±0.35a +12.1755 60.72 59.49 53.64 57.95±3.78a +11.05

∑UFA/ΣSFA 15 1.66 1.29 1.30 1.31 1.30±0.01c −21.6925 1.43 1.44 1.42 1.43±0.01c −13.8635 1.68 1.68 1.69 1.68±0.01bc +1.2045 2.61 2.58 2.63 2.61±0.02a +57.2255 2.93 2.79 1.95 2.56±0.53ab +54.21

Overall mean values for each fatty acid in the respective column with differentsuperscript are significantly different at pb0.01.

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attributable to its adaptation for oceanic conditions exclusively. Wealso observed its distribution in nature exclusively confining to opensea coasts. Gracilaria chorda and G. salicornia have also been reportedto grow well at salinities between 25 and 35 (Choi et al., 2006;Phooprong et al., 2007). In contrast, G. verrucosa and G. tenuistipitatahave shown a broad salinity tolerance ranging from 5 to 47 extendingtheir distribution boundaries from brackish waters to marine waters(Israel et al., 1999). The lower DGR values in extreme hypo- andhyper-salinities (15 and 55) observed here can likely be accredited tothe increased lipid peroxidation indicated by higher MDA level andROS accumulation.

Chl a and the phycobiliproteins are the key photosyntheticpigments in the red algae. Their cellular level could be an importantphysiological parameter for assessment of stress environment. Thepresent study showed decreased chlorophyll content in both extremehypo- and hyper-salinity while marginal fluctuations at 25 and 35salinities. Among the phycobiliproteins PE and APC were foundaccumulated in both hypo- and hyper-salinities, however PC attainedpeak value in hyper-salinities only. The accumulation of phycobili-proteins could be a possible acclamatory mechanism for G. corticata inresponse to salinity induced stress as these proteins function asstorage proteins for biosynthesis during the adaptation period underadverse conditions. Recently, Kim et al. (2007) reported the storagefunction of PE in Porphyra tissue cultured in amedia supplementedwithhigher doses of ammonia (250μmol L−1). Moreover, Cano-Europa et al.(2010) suggested the phycobiliproteins as good antioxidants preventoxidative stress because of their nucleophilic ability to neutralizereactive oxidants. The increase in phycobiliproteins especially in hyper-salinities has also been reported for G. tenuistipitata cultures whenshifted from 20 to 40 salinities (Israel et al., 1999). In contrast,Macler (1988) observed a decrease in the level of both chlorophylland phycobiliproteins in both hypo- and hyper-salinities (5–50).Interestingly, in the present study both phycoerythrin and phycocyanincontents did not increase significantly in hypo-saline conditions ascompared to hyper-saline conditions (Fig. 2B & D) despite the factthat these pigments function as a nitrogen reserves in hypo-salineconditions. Therefore, hypo- and hyper-saline conditions are notspecific to induceor inhibit a particular site of photosyntheticmachineryrather affect many sites involved in this process in one or other mannerand bring about significant changes in the photosynthetic performanceof the algae.

A consistent increase in the content of both polyphenols andproline particularly at salinity 45 could be an adaptive feature toimprove the turgor required for maintaining the ionic balance duringsalinity induced osmotic stress. The higher level of their contents inboth the extreme end salinities (15 and 55) were not enough tosustain the growth of plants under these salinity conditions asevidenced by decreased daily growth rate, higher MDA and ROSgeneration. Interestingly, polyphenol responded more distinctly toboth hypo- and hyper-salinity conditions than did free proline asevident by their increased level of polyphenol (Table 1). From theseresults, it can be speculated that polyphenols may play a major role toscavenge the ROS generated by salinity induced oxidative stress.Parida et al. (2004) reported an enhanced level of polyphenol as anacclimatizationmechanism that ameliorating the ionic effect resultingfrom hypo- and hyper-salinity conditions. A considerable accumula-tion of proline in both hypo- and hyper-salinity conditions hasalso been reported for Ulva pertusa (Kakinuma et al., 2006) andG. tenuistipitata (Lee et al., 1999).

Recently, Dring (2006) reported that oxidative stresses tend toresult in a gradual buildup of ROS in macroalgae. SOD, APX and GR arethe important antioxidative enzymes in the plant cell to protect cellagainst the peroxidation system and tomaintain the redox state of thecell. Enhanced activities of all three antioxidative enzymes in thepresent study especially at 15 and 45 salinities compared to controlduring initial 6 days suggest the generation of all kind of reactive

oxygen species viz. O2•−, H2O2 and •OH in the plant which would have

detoxified by these enzymes. Thus, G. corticata could resist the stressof adversity andmaintain the normal construction and function of cellmembrane as evident by a reduction of only 25–30% in chlorophyllpigment during the initial days of culture period at salinities 15 and45. Thereafter, a sharp decline observed in activities of antioxidativeenzymes could be due to formation of ROS beyond the critical limitwhich might pose serious threat to plant cell, causing higher lipidperoxidation in hypo- and hyper-salinities. Supporting to our results,Collen and Davidson (1999); Choo et al. (2004) found a positiverelation between low peroxidative damage and a more efficientantioxidative system in intertidal red and green seaweeds, respec-tively. A significant higher activity of antioxidative enzymes inU. lactuca was shown for better acclimatization to the changingenvironments than the conspecifics in the subtidal habitats (Ross andVan Alstyne, 2007).

Further, a sizeable increase in GR activity with 1.11±0.17 Umg−1

protein in 45 salinity compared to the activity of 0.70±0.06 Umg−1

protein at 15 salinity after 6 d exposure suggests greater demand forthe supply of reduced glutathione. Reduced glutathione helps inascorbate regeneration, regulate the protein thiol-disulfide exchangereactions, maintain the redox state of the cell and thus increases thestress tolerance. On the other hand, inadequate antioxidativeenzymes activities at extreme high salinity (55) might also becorrelated with early onset of bleaching and tissue necrosissymptoms. Lu et al. (2006) also showed a decreased antioxidantactivity in Ulva fasciata during long term (4 d) exposure to highersalinity (90) due to increased H2O2, and MDA level. Enhanced activityof either individual or combined SOD, APX and GR enzymes have alsobeenobserved inU. fasciata andU. 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 metaland/or other adverse environment conditions.

Among the most common effects of altered salinity is the growthinhibition which mainly occurs due to change in external waterpotential. Elevated salinity (55) greatly affect the homeostasis of thecell as indicated by bleaching symptoms and enhanced Na+/K+ andNa+/Ca2+ ratio. This could be explained due to the antagonisticbehavior of Na+ and K+ at the uptake sites which in turn impaired theion selectivity and integrity of the cell membrane and permit thepassive and excess accumulation of Na+ in the thalli. Moreover, highNa+/K+ ratio has been suggested to disturb various metabolicprocesses such as protein synthesis in the cytoplasm. A substantialtolerance capacity of plants grown at salinity 45 could be attributed tothe compartmentalization of Na+ into the vacuole before itsconcentration increases beyond the threshold concentration in thecytoplasm. In addition a considerable accumulation of intracellularCa2+ at salinity 45 below the toxic level as compared to 55 improvedthe tolerance ability of the plants. Lee and Liu (1999) found that Ca2+

involves in signaling mechanism of salinity induced proline accumu-lation for osmotic adjustment. Therefore, Na+/K+ and Na+/Ca2+ ratiomight be promising indicators for salt resistance. A two fold increasefor polyphenols and proline in the present study may be anotherreason to cope with this hyper osmolarity condition. On the otherhand, at salinity 15 a relatively lower ratio of Na+/K+ and Na+/Ca2+

together with accumulated Mg indicate the gradual accumulation ofK+ and Ca2+ that eventually accumulated beyond their criticalconcentration and thus plants collapsed when exposed for longerduration under this salinity.

The most significant difference in fatty acid composition is theenhanced relative proportion of C18:1(n−9) cis; C18:2(n−6) andC18:3(n−3) by approximately 1.3–1.6 fold over their initial contentwith a parallel decrease of palmitoleic acid C16:1(n−7) at hyper-salinities. A possible explanation for this change could be the salinityinduced partial inhibition of Δ9 desaturase enzyme that inserts the firstdouble bond in C16:0 and the induction of Δ9 desaturase enzyme

33M. Kumar et al. / Journal of Experimental Marine Biology and Ecology 391 (2010) 27–34

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responsible for inducing double bonds in stearic acid to convert most ofthe C18:0 to C18:1(n−9) which later become a substrate for thesucceeding enzyme reactions and resulted in more PUFA synthesis.Higher content of these PUFAs could be an adaptive strategy formaintaining the requisite greater membrane fluidity, to stabilize theprotein complexes of photosystem II and to control membrane physico-chemical properties such as the increased activity of Na+/H+ antiportersystem 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; n−7) in themarine diatoms Thalassiosira antarctica and Bacterosira bathyomphalawhen exposure to UV radiation. In conditions of drought, a decrease ofC18 PUFAs reflect membrane damage, while an increase in the level ofthese fatty acid, as observed here, may represent a defense component(Zhang et al., 2005). On the other hand, at hypo-salinity (15) relativelymore rigid membranes during initial 6 d culture period would haveprevented the ion loss to certain extent and thus increased membranefluidity brought by higher PUFAs level was not needed. The prolongedexposure to these adverse hypo-saline conditions impaired the cellfunction due to disrupted growth and synthesis of photosyntheticmembranes leading to lower levels of PUFAs (Floreto et al., 1994).

5. Conclusion

The overall findings presented in this study suggest that G.corticata regulates its antioxidant machinery to eliminate ROS underlong term stress conditions. Maintenance of Na+/K+ and Na+/Ca2+

ratio to the optimum level and compartmentalization of Na+ into thevacuoles together with the higher level of polyphenols and prolinecould be the tolerant strategies to combat the salt stress particularly atsalinity 45. In addition, higher concentration of PUFAs such as oleic,linoleic, α linolenic and phycobiliproteins particularly phycoerythrinfulfil the demand of higher energy required for biosynthesis,reorganization of cell and to maintained the requisite membranefluidity under salinity stress condition.

Acknowledgements

The financial support received from the Council of Scientific andIndustrial Research (NWP 018), New Delhi is gratefully acknowl-edged. The authors are grateful to Dr. Dilip Ghosh, Director, NutriConnect, Australia for scientific editing of the manuscript. The firstauthor (MK) and second author (PK) gratefully acknowledges theCSIR, New Delhi for awarding the Senior and Junior ResearchFellowships respectively. The third author (VG) also expresses hisgratitude to Department of Science and Technology, New Delhi forfinancial support. We would also like to thank Mr. Harshad R.Brahmbhatt for technical assistance in analysis of fatty acids using GC-MS.

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