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Journal of Plant Physiology 165 (2008) 306—316

0176-1617/$ - sdoi:10.1016/j.

AbbreviationDCPIP, 2, 6-dicGS, glutaminePS, photosyste�CorrespondE-mail addr

www.elsevier.de/jplph

Impact of different abiotic stresses on growth,photosynthetic electron transport chain, nutrientuptake and enzyme activities of Cu-acclimatedAnabaena doliolum

Riti Thapar, Ashish Kumar Srivastava, Poonam Bhargava,Yogesh Mishra, Lal Chand Rai�

Molecular Biology Section, Laboratory of Algal Biology, Center of Advanced Study in Botany,Banaras Hindu University, Varanasi 221005, India

Received 12 January 2007; received in revised form 6 May 2007; accepted 8 May 2007

KEYWORDSAnabaena doliolum;Copper acclimated;Glutaminesynthetase;Photosyntheticelectron transportactivity;Photosystem II

ee front matter & 2007jplph.2007.05.002

s: ATP, adenosine triphhlorophenol indophenolsynthetase; MV, methyl vm; UV-B, ultraviolet-B ring author. Tel.: +91 542esses: lcrai@bhu.ac.in,

SummaryThis study provides a comparative account of the effects of cadmium, temperature,ultraviolet-B and sodium chloride on the growth, photosynthesis, nutrient uptakeand enzyme activities of untreated control and copper-acclimated Anabaenadoliolum. Reduction in all the studied parameters, except carotenoids, wasmaximum for sodium chloride followed by ultraviolet-B, temperature and cadmiumtreatments, the reduction being greater in control than acclimated A. doliolum.Among the various parameters, photosystem II was most sensitive for all the stressesin both control and acclimated A. doliolum. Likewise, O2 evolution was moresusceptible to various stressors than 14C uptake. Ammonium uptake and glutaminesynthetase (GS, EC 6.3.1.2) were the least affected parameters. As compared tocontrol, acclimated Anabaena exhibited higher ATP content under normal condi-tions. These results attest our hypotheses that acclimated Anabaena wasphysiologically more robust than control and that salinity was more injurious tothe test organism than other abiotic stresses investigated.& 2007 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights reserved.

osphate; Car, carotenoid; Chl, chlorophyll; DCMU, 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea;; ETC, electron transport chain; GOGAT, glutamine 2-oxo-glutarate amino transferase;iologen; NR, nitrate reductase; PAR, photosynthetically active radiation; PBQ, p-benzoquinone;adiation (280–320 nm)2307146; fax: +91 542 2368174.lcrai_bhu@yahoo.co.in (L.C. Rai).

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Response of copper-acclimated Anabaena to stress 307

Introduction

Accelerated anthropogenic activities and indus-trialization are continuously dumping huge amountsof waste and contaminating our aquatic resources,e.g. rivers, ponds, lakes, etc. These aquaticresources, besides borewells and canals, are themajor sources of irrigation in India, and responsiblefor adding a host of xenobiotics including heavymetals to crop fields. Nevertheless, the use ofcopper-containing pesticides further adds to theexisting burden of the soil and may negativelyaffect the survival of micro-organisms in such soils.In order to continue to fix atmospheric nitrogen,cyanobacteria must adapt to increasing levels ofcopper. In addition to this, the accelerated globalwarming by heat-trapping gases (CH4, CO2, NO2 andCFC (chlorofluorocarbons)) not only increases theaverage temperature but also leads to a continuousdepletion of the stratospheric ozone layer, result-ing in increased solar ultraviolet-B radiation (UV-B)(280–320 nm) at the earth’s surface (Crutzen, 1992;Smith et al., 1992). A multitude of these stressesmay affect the very survival of different livingorganisms, including cyanobacteria (Yruela, 2005),which are not only key players in aquatic produc-tivity but are also responsible for fixing atmo-spheric nitrogen and thus increasing the soilfertility of rice fields. Among various heterocystouscyanobacteria, Anabaena doliolum is known to be apromising candidate for dinitrogen fixation in thewaterlogged paddy fields of India (Singh, 1961).

A critical perusal of the literature revealedchanges in growth, and physiological and biochem-ical processes of cyanobacteria exposed to heavymetals (Prasad et al., 1991; Rai et al., 1995;Bhargava et al., 2005), temperature (Mishra et al.,2005; Srivastava et al., 2006) and salinity (Srivas-tava et al., 2005, 2006). Copper-containing pesti-cides used for controlling pests as well as forincreasing crop productivity have been used since1761 (http://www.copper.org/applications/com-pounds/agricultural.html); thus cyanobacteriaought to acclimate to high levels of copper fortheir survival. It is relevant to state that reports areavailable in the literature on copper-acclimatedpea (Palma et al., 1987), mustard (Wang et al.,2004), yeast (Miura et al., 2002) and algae such asA. doliolum (Rai et al., 1991) and Enteromorphacompressa (Ratkevicius et al., 2003).

However, no effort seems to have been made tocompare the toxic effects of different abioticstresses, including Cd2+, temperature, UV-B andNaCl, on untreated control and Cu2+-acclimated A.doliolum as well as the cross protection offered byCu2+-acclimated cyanobacterium to different abio-

tic stresses. It is also not known as to which of theemployed abiotic stresses should be adjudged asthe most potential culprit of cyanobacterium inarresting its growth and metabolic processes.

In view of the report of Rai et al. (1991) thatmetabolic variables of Cu2+-acclimated A. doliolumwere less affected by Cu2+ than control, wehypothesized that Cu2+-acclimated A. doliolumshould be more tolerant than control cells todifferent abiotic stresses. Keeping in mind the vitalrole of NaCl in the maintenance of cellular home-ostasis, it was further hypothesized that salt stressshould be more injurious than other stresses. Takingrecourse to the fact that Cu2+ and Cd2+ are divalentcations and may compete for common binding sites,it was also hypothesized that cadmium toxicityshould be minimum in Cu2+-acclimated A. doliolumthan the other stresses.

Thus, this study was designed to test the abovehypotheses and to provide first-hand data on thecomparative assessment of the effect of variousabiotic stresses on control and Cu2+-acclimated A.doliolum.

Materials and methods

Test organism and culture conditions

The nitrogen-fixing filamentous cyanobacterium A.doliolum Bhardawaja was isolated from a rice field ofBanaras Hindu University, Varanasi, and grown axenicallyin a modified Allen and Arnon (1955) medium bufferedwith Tris/HCl at 2472 1C under 72 mmol photonm�2 s�1

photosynthetically active radiation (PAR) with a photo-period of 14:10 h (light:dark) at pH 7.5. The cultureswere shaken by hand two to three times daily. Cu2+-acclimated A. doliolum was obtained by successivecultivation (15 times) at increasing doses of Cu2+ up to250 mM (in the form of CuCl2) as described by Rai et al.(1991). Hereafter, this strain is referred to as theacclimated strain and the other as control strain.

Experimental design, stress application andbiochemicals

The experiments were done in four sets, wherein cellsof control and acclimated A. doliolum were subjected to(i) Cd2+ (1mM), (ii) temperature (48 1C) for 1 h, (iii) UV-Birradiation (12.9mWm�2 nm�1) for 22min and then keptin the dark for 24 h and (iv) NaCl (150mM). Thephysiological parameters were evaluated at regularintervals of 48 h up to 11 days. All the experiments wereconducted in triplicate using exponential phase culturesand repeated at least twice to confirm the reproducibilityof the results. A stock solution of CdCl2 � 2H2O (1mM) wasprepared in glass distilled water and sterilized by passingthrough Millipore membrane filters (0.22 mm). Tempera-

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R. Thapar et al.308

ture treatment to culture suspension was given in atemperature-controlled incubator under continuous irra-diance of 72 mmol photonm�2 s�1 PAR provided by fluor-escent lamps throughout the treatments. For UV-B stress,culture suspensions were transferred to 75mm glass Petridishes agitated with a magnetic stirrer to ensure uniformexposure to artificial UV-B radiation (from a UV-B lamp,Cat. No. 34408, Fotodyne, Inc., USA, giving maximumoutput at 310 nm). The UV light was passed through a UVfilter for removing all incident UV-C radiation (o280 nm).The desired dose (12.9mWm�2 nm�1) was obtained byadjusting the distance between the UV-B light source andthe cyanobacterial suspension kept in an open Petri dish.The above dose was selected keeping in view the latitudeof our workstation (251N) and the mean percentagedepletion of ozone layer as calculated by Crutzen (1992)and Smith et al. (1992). NaCl autoclaved separately wasadded directly into the sterilized medium to achieve theappropriate concentration. All biochemicals were pro-cured from Sigma Chemical Co., USA.

Measurement of survival and growth

To measure the survival, cyanobacterial cells weretreated with different concentrations of Cd2+

(0.0–50.0 mM), temperature (40–60 1C), UV-B (0–50min)and NaCl (0.0–250mM). The LC50 and lethal doses forCd2+, temperature, UV-B and NaCl against control A.doliolum were determined by the plate colony countmethod of Rai and Raizada (1985). In this method, thecyanobacterial suspension was homogenized to generatefilaments of almost equal length (10–15 cells perfilament). Thirty such filaments were counted and theaverage number of cells per filament was calculated(Lawton et al., 1999). Aliquots of this culture suspension(0.03mL) containing 4.39� 104 cellsmL�1 were platedonto agar plates supplemented with various doses of theabove-mentioned stresses. Colonies were counted after 3weeks and the survival percentage was scored (data notshown).

Approximately 50% survival of the test cyanobacteriumwas observed at 1mM (Cd2+) cadmium, 48 1C (T)temperature, 22min (UV) UV-B exposure and 150 mM(S) NaCl. Growth was estimated by measuring opticaldensity of the cyanobacterial cultures at 663 nm in a UV/vis spectrophotometer (Systronics, India) on every secondday up to the 11th day by using a reference blank of basalculture medium. Specific growth rate was calculated byusing the following equation: m ¼ [ln (n2/n1)]/[t2–t1],where m stands for specific growth rate and n1 and n2are absorbances of culture suspension at the beginning(t1) and the end (t2) of the selected time interval.

Protein estimation

Total cell protein was estimated using the method ofBradford (1976).One hundred microliters of cell extractwas mixed with 1mL of Bradford reagent, which wasprepared by adding 2mL of 0.2% Coomassie Brilliant blue

G-250 and 4mL of orthophosphoric acid in 14mL ofMillipore water. Absorbance was recorded at 595 nm.

Photosynthetic pigment determination

For estimation of photosynthetic pigment, 5mL ofcyanobacterial culture was centrifuged and the pelletwas suspended in a desired volume of acetone (80%).After overnight incubation at 4 1C, the suspension wascentrifuged and the supernatant was used for themeasurement of chlorophyll a and carotenoid contentsby determining absorbance at 663 and 480 nm, respec-tively. Chlorophyll and carotenoid were calculatedaccording to Myers and Kratz (1955).

Estimation of O2 evolution and carbon fixation

Photosynthetic O2 evolution in control and treatedcyanobacterial cells was measured in a polarographicoxygen electrode enclosed in an airtight reaction vesseland connected to an oxygen analyzer (Digital OxygenSystem, Model-10, Rank Brothers, UK) at 2572 1C underan illumination of 300 mmolm�2 s�1 PAR. The tempera-ture of the vessel was maintained by a thermocirculator.O2 evolution was recorded for 5min with the help of anO2 analyzer and expressed in mmol O2 evolved mg�1

protein h�1. Carbon fixation was measured according tothe method of Rai and Raizada (1985). A 0.2mL NaH14CO3

(specific activity 18.5� 105 Bq) solution was added to1mL algal culture and kept in light for 2 h. The reactionwas stopped by adding 0.2mL 50% acetic acid andbubbling with air for 5min, followed by the addition of5mL scintillation cocktail. Counting was done in aBeckman model-LS6500 liquid scintillation counter.

Measurement of photosynthetic electron transportchain

Electron transport activities were measured accordingto the methods described by Rai and Raizada (1985) andLien (1978). The activities were determined by three basicassays. A cell-free thylakoid membrane was prepared bysonication following the method of Lien (1978). Activity ofphotosystem I (PS I) was determined in the presence ofDCMU (10�5mM) by recording O2 consumption after theaddition of ascorbate (5mM), 2,6-dichlorophenol indophe-nol (DCPIP, 50mM) and methyl viologen (MV, 1.4mM).Likewise, photosystem II (PS II) activity was measured interms of O2 evolution in the presence of p-benzoquinone(PBQ, 2mM). The whole-chain electron transport (PS I andPS II dependent) was measured in the presence ofpotassium ferricyanide (K3 Fe (CN)6; 80mM) as electronacceptor and H2O as electron donor.

The overall reaction can be expressed as

ascorbate! DCPIP! MV ðPS IÞ, (1)

H2O! PBQ ðPS IIÞ, (2)

H2O! ferricyanide ðwhole chainÞ. (3)

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1 3 5 7 9 11

Days

0.0

0.1

0.2

0.3

0.4 ControlCadmiumTemperatureUV-BSalt

A6

63

A6

63

0.0

0.1

0.2

0.3

0.4

A

B

Figure 1. Effects of cadmium, temperature, UV-B andsalt on the growth behavior of (A) control and (B) copper-acclimated Anabaena doliolum. Error bars indicate SD ofthree replicates.

Response of copper-acclimated Anabaena to stress 309

Nutrient uptake

The uptake of nitrate (NO3�), ammonium (NH4

+) andphosphate (PO4

3�) from the medium by control andacclimated A. doliolum under different treatments wasestimated using the brucine sulfuric acid method(Nicholas and Nason, 1957), Nessler’s reagent (Herbertet al., 1971) and the stannous chloride method (APHA,1985), respectively.

Enzyme assays

The activity of nitrate reductase (NR, EC 1.6.6.1) wasestimated by measuring the formation of nitrite fromnitrate (Camm and Stein, 1974). The reagents used weresulfanilamide and (N-1)-naphthylethylene diamine dihy-drochloride. Estimation of whole-cell glutamine synthe-tase (GS, EC 6.3.1.2) activity was done by measuring theformation of g-glutamylhydroxamate at pH 7.0 with animidazole–HCl buffer (Stacey et al., 1977). Alkalinephosphatase (APase, EC 3.1.3.1) activity was analyzedby the method of Ihlenfeldt and Gibson (1975).

Estimation of ATP content

The size of the adenosine triphosphate (ATP) pool ofthe test cyanobacterium, immediately after treatment,was measured according to the method of Larson andOlsson (1979). ATP was extracted in 4% TCA supplemen-ted with 2mM EDTA and total content was measured by aluciferin–luciferase assay using an LKB 1250 luminometer.

Statistical analysis

Results were statistically analyzed using a one-wayANOVA and correlation coefficients (r). The number ofindependent variables for each experiment was three.

Results

Growth behavior

A continuous decline in the growth of control andacclimated A. doliolum under different stresses,viz. Cd2+, temperature, UV-B and NaCl, is shown inFig. 1. The control cells registered appreciable(56.25%) reduction in specific growth rate at150mM NaCl, while growth inhibition by UV-B(37.5%), temperature (21.88%) and Cd2+ (9.38%)was mild. Cu2+-acclimated A. doliolum exhibited alower level of inhibition in the specific growth rateas compared to the control (Table 1).

Photosynthetic pigments

Chlorophyll a and carotenoid contents ofA. doliolum markedly decreased at LC50 doses of

Cd2+ and temperature. UV-B and NaCl were foundto increase carotenoid content by 103.61% and101.2% in control and 84.85% and 83.84% in Cu2+-acclimated A. doliolum, respectively. However, theinhibitory impact of different stresses on chloro-phyll and carotenoid contents was comparativelyless in Cu2+-acclimated A. doliolum in comparisonto control. It is interesting to note that comparedto control, Cu2+-acclimated A. doliolum exhibited ahigher carotenoid/chlorophyll a ratio. This ratiowas highest (1.52) for NaCl treated Cu2+-acclimatedA. doliolum, followed by UV-B (1.45). The inhibitionin pigment content in different treatments in boththe strains suggested that chlorophyll a was moresensitive than carotenoid content.

O2 evolution and carbon fixation

Table 2 compiles data on the O2 evolution andcarbon fixation of control and Cu2+-acclimated A.doliolum exposed to different stresses. After 24 h

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Table 1. Effects of cadmium, temperature, UV-B and salt on specific growth rate and pigment content of control andcopper-acclimated Anabaena doliolum

Treatment Specific growth rate (m) Chlorophyll a (mgmL�1) Carotenoid (mgmL�1) Car/Chl a

C 0.3270.02 1.6070.53 0.8370.04 0.52CA 0.2570.04 1.4570.12 0.9970.10 0.60C+Cd 0.2970.03 (9.38) 1.3270.17* (17.5) 0.7470.02* (10.84) 0.56CA+Cd 0.2370.01 (8) 1.4070.11 (3.45) 0.8970.17* (10.10) 0.64C+T 0.2570.05* (21.88) 1.2270.37* (23.75) 0.7170.03* (14.46) 0.58CA+T 0.2070.04* (20) 1.2970.06* (11.03) 0.8570.09* (14.14) 0.65C+UV-B 0.2070.03* (37.5) 1.1870.09* (26.25) 1.6970.07* (103.61) 1.43*CA+UV-B 0.1870.02* (28) 1.2670.01* (13.10) 1.8370.02* (84.85) 1.45*C+salt 0.1470.09* (56.25) 1.1570.15* (28.13) 1.6770.08* (101.20) 1.45*CA+salt 0.1570.05* (40) 1.2070.02* (17.24) 1.8270.14* (83.84) 1.52*

C ¼ control, CA ¼ copper-acclimated.Data in parentheses indicate percent change in content. All the values are means7SD of three replicates. *Indicates significant change(Po 0.05) as compared to their respective controls.

R. Thapar et al.310

exposure to stresses used, O2 evolution wasdrastically reduced in both the strains. Significantreduction (43.64%, 45.89%, 48.35% and 56.71%) inO2 evolution was observed in control A. doliolumfollowing treatment of Cd2+, temperature, UV-Band NaCl, respectively, after 11 days of exposure.This reduction, however, was relatively less in Cu2+-acclimated A. doliolum (2.58%, 17.21%, 10.13% and33.44%) following exposure to Cd2+, temperature,UV-B and NaCl, respectively, as compared to theuntreated acclimated A. doliolum. ANOVA indi-cated that the inhibition was significant (Po0.05).In tune with O2 evolution, 14CO2 fixation wasreduced significantly in control (71.15%, 71.73%,83.14% and 88.69%) and Cu2+-acclimated A. dolio-lum (27.65%, 41.89%, 66.02% and 77.26%) whensubjected to LC50 doses of Cd

2+, temperature, UV-Band NaCl, respectively, after 11 days of exposure.

Electron transport system

Table 3 shows the inhibition of photosyntheticelectron transport activity in both the strains ofA. doliolum subjected to Cd2+, temperature, UV-Band salt stress. The percent inhibition for PSI, PSIIand whole chain increased with the duration ofexposure to temperature, UV-B and salinity. How-ever, in the case of Cd treatment, the percentinhibition of PSI was 13.51 after 24 h and 9.19 after11 days. All the three components of the electrontransport chain (ETC) of the Cu2+-acclimatedAnabaena were less susceptible to selected stressesthan the control cells. Interestingly, NaCl producedthe highest reduction of 70.4%, 74.2% and 66.3% ofPSI, PSII and whole chain, respectively, after 11days of treatment of the control A. doliolum.However, the hierarchy (NaCl4UV-B4temperature

4Cd2+) of inhibition of the ETC by differentstresses was the same in both the strains. Further,PSII appeared to be slightly more sensitive than PSIto the stresses used in this study.

Nutrient uptake

Fig. 2 compiles data on the effects of LC50 dosesof Cd2+, temperature, UV-B and NaCl on nutrientuptake (NO3

�, NH4+ and PO4

3�) of control and Cu2+-acclimated A. doliolum. Reduction in nutrientuptake was dependent on the duration of exposureand the stresses used. Both the strains exhibitedconsiderable variability in uptake of NO3

�, NH4+ and

PO43� from the external medium. NO3

� uptake wasquite sensitive, showing more inhibition by differ-ent stresses than NH4

+ and PO43� uptake. Like

photosynthesis, the nutrient uptake of the controlwas more sensitive to different treatments incomparison to Cu2+-acclimated A. doliolum. Con-trol A. doliolum treated with NaCl exhibitedmaximum inhibition (74.49%, 20.35% and 66.67%)in nitrate, ammonium and phosphate uptake ascompared to 67.44%, 19.58% and 55.56%. respec-tively, in Cu2+-acclimated A. doliolum after 11 daysof exposure. Further, salinity produced maximumreduction in NO3

� and PO43� uptake than NH4

+

uptake.

Enzyme activities

Significant changes were registered in activitiesof nitrate reductase, glutamine synthetase andalkaline phosphatase when both control and Cu-acclimated A. doliolum were subjected to differentstresses. Data relating to the effect of differentstresses on the enzyme activities are compiled in

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Table 2. Effects of cadmium, temperature, UV-B and salt on photosynthetic O2 evolution,14C incorporation and ATP

content of control and copper-acclimated Anabaena doliolum after 24 h and 11 days of exposure

Treatment O2 evolution (mmol O2

evolved mg�1 protein h�1)

14C uptake (CPM� 103) ATP content(mg ATP mg protein�1)

After 24 hC 1.5270.02 29.1570.04 0.7570.01CA 1.0470.05 22.2370.10 0.8670.03C+Cd 0.0070.00* (100) 17.8070.07* (38.94) 0.6970.07 (8)CA+Cd 0.0070.00* (100) 21.4270.02 (3.64) 0.8070.04 (6.98)C+T 0.0070.00* (100) 15.2470.13* (47.72) 0.3670.08* (52)CA+T �1.4970.01* (243.27) 18.6670.29* (16.06) 0.4570.02* (47.67)C+UV-B �0.7470.02* (148.68) 13.8170.15* (52.62) 0.1470.01* (81.33)CA+UV-B �2.6270.04* (351.92) 16.3370.06* (26.54) 0.2270.02* (74.42)C+salt �1.5070.06* (198.68) 16.2970.05* (44.12) 0.0770.01* (90.67)CA+salt �3.1770.03* (404.81) 19.6270.11 (11.74) 0.1070.04* (88.37)

After 11 daysC 31.6070.01 87.2370.05 0.9270.08CA 27.5470.02 74.8070.01 1.0270.48C+Cd 17.8170.05* (43.64) 25.1770.04* (71.15) 0.7570.05* (18.48)CA+Cd 26.8370.18 (2.58) 54.1270.49* (27.65) 0.8670.02* (15.69)C+T 17.1070.10* (45.89) 24.6670.21* (71.73) 0.3770.04* (59.78)CA+T 22.8070.49* (17.21) 43.4670.06* (41.89) 0.6870.03*(33.33)C+UV-B 16.3270.20* (48.35) 14.7170.50* (83.14) 0.1870.01* (80.43)CA+UV-B 24.7570.01 (10.13) 25.4270.01* (66.02) 0.2570.02* (75.49)C+salt 13.6870.05* (56.71) 9.8770.03* (88.69) 0.0970.01* (90.22)CA+salt 18.3370.03* (33.44) 17.0170.05* (77.26) 0.1170.05* (89.22)

C ¼ control, CA ¼ copper-acclimated.Data in parentheses indicate percent change in content. All the values are mean7SD of three replicates. *Indicates significant change(Po0.05) as compared to their respective controls.

Response of copper-acclimated Anabaena to stress 311

Fig. 3. Relatively higher activities of nitratereductase, glutamine synthetase and APase wereobserved in Cu2+-acclimated A. doliolum than incontrol. However, maximum inhibition (24.24%,19.80% and 27.42%, respectively) was found incontrol A. doliolum when treated with NaCl. Aninteresting observation was the meager decrease inNR activity and significant inhibition of NO3

� uptakefollowing treatment with different stresses.Further, glutamine synthetase activity declinedwith different stresses as well as time duration.Cd2+, temperature, UV-B and NaCl-induced inhibi-tion of alkaline phosphatase activity remainedunchanged up to 72 h in both control and accli-mated strains as compared to significant reductionin their respective controls. The NaCl stressdepicted maximum toxicity on all the enzymes(NR, GS and APase), followed by UV-B, temperatureand Cd2+.

ATP content

Data pertaining to ATP content of both the strainsunder different treatments are summarized in

Table 2. Exposure of control and Cu2+-acclimatedA. doliolum to different stresses produced asignificant decrease in cellular ATP content. How-ever, the inhibition in ATP content in Cu2+-accli-mated A. doliolum was meager in comparison tocontrol A. doliolum. Inhibition in the ATP contentwas also time dependent. Like photosynthesis, ATPcontent registered significant reduction (90.22%) incontrol A. doliolum treated with NaCl (150 mM),over its respective control, followed by UV-B(80.43), temperature (59.78) and Cd2+(18.48) after11 days of exposure.

Discussion

The results revealed that the abiotic stresses(Cd2+, temperature, UV-B and NaCl) generatedsignificant reduction in growth and metabolicvariables in both control and acclimatedA. doliolum, but reduction was higher in thecontrol than in the acclimated A. doliolum. Thismay be because Cu2+-induced changes enabled thecells to regulate alterations in physiological vari-ables. Stress-induced inhibition in growth of both

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Table 3. Effects of cadmium, temperature, UV-B and salt on photosynthetic electron transport activity of control andcopper-acclimated Anabaena doliolum after 24 h and 11 days of exposure

Treatment PS-I activity (ascorbate-DCPIP-MV) (mmol O2

consumed mg�1 protein h�1)

PS-II activity (H2O-PBQ)(mmol O2

evolvedmg�1 protein h�1)

Whole chain (H2O-ferricyanide) (mmol O2

evolved mg�1 protein h�1)

After 24 hC 12.2170.02 10.1270.01 20.1570.01CA 15.6870.03 11.9970.06 22.0870.02C+Cd 10.5670.04* (13.51) 8.8770.08* (12.35) 15.2470.04* (24.37)CA+Cd 14.1870.07 (9.57) 10.9270.01 (8.92) 18.7370.09* (15.17)C +T 9.8570.05* (19.33) 7.4070.03* (26.88) 14.5170.04* (27.99)CA+T 12.8170.07* (18.30) 8.8370.05* (26.36) 16.2470.01* (26.45)C+UV-B 8.7970.09* (28.01) 6.6370.01* (34.49) 9.3170.03* (53.79)CA+UV-B 11.5370.02* (26.47) 7.9670.06* (33.61) 11.6070.06* (47.46)C+salt 7.5370.03* (38.33) 5.0970.07* (49.70) 7.2670.08* (63.97)CA+salt 10.0670.01* (35.84) 6.4170.05* (46.54) 8.9970.03* (59.28)

After 11 daysC 34.9270.02 37.2970.05 43.8170.01CA 36.8370.01 39.6070.12 45.6270.03C+Cd 31.7170.05 (9.19) 28.3270.01* (24.05) 29.4070.02* (32.89)CA+Cd 34.7070.12 (5.78) 30.1170.04* (23.96) 32.3270.16* (29.15)C+T 27.6470.01* (20.85) 20.1270.03* (46.04) 24.5970.13* (43.87)CA+T 30.2270.15* (17.95) 22.9670.02* (42.02) 27.2270.01* (40.33)C+UV-B 14.9770.03* (57.13) 13.8270.01* (62.94) 15.8270.04* (63.89)CA+UV-B 23.7270.11* (35.59) 16.2270.03* (59.04) 26.9170.18* (41.01)C+salt 10.3370.06* (70.42) 9.6170.05* (74.23) 14.7470.02* (66.35)CA+salt 15.8970.07* (56.86) 12.4570.02* (68.56) 19.3670.10* (57.56)

C ¼ control, CA ¼ copper-acclimated.Data in parentheses indicate percent change in content. All the values are mean7SD of three replicates. *Indicates significant change(Po0.05) as compared to their respective controls.

R. Thapar et al.312

the strains might be due to damage of the cellularconstituents or inactivation of vital processes suchas nutrient uptake, enzyme activities and photo-synthesis as observed by Altamirano et al. (2000) inUlva rigida. A profound toxicity of salinity may bedue to Na+-induced disruption of cellular home-ostasis and osmoticum (Srivastava et al., 2005).However, a meager toxicity due to temperature canbe ascribed to the induction of heat-shock proteins(molecular chaperonin), which help in maintainingthe active conformation of house-keeping proteins(Suzuki and Mittler, 2006). In contrast to the above,failure of Cd2+ to induce appreciable changes in thegrowth of acclimated A. doliolum may be due tothe chelation of Cd2+ by Cu2+-induced phytochela-tin (De Vos et al., 1992).

The reduction in chlorophyll content (Table 1)due to different stresses may result from inhibitionof chlorophyll biosynthesis brought about by inhibi-tion of d-aminolevulinic acid dehydrogenase andprotochlorophyllide reductase (Ouzounidou, 1995).Specifically, NaCl-induced decrease in contentappears to be due to damage of the thylakoid

membrane, a major target of salt stress (Srivastavaet al., 2005), and photo-oxidation and bleaching ofdifferent photosynthetic pigments by UV-B (Lao andGlazer, 1996) and Cd2+-induced replacement ofMg2+ from chlorophyll molecules (Kupper et al.,1996).

Cadmium and temperature caused a decline incarotenoid content, whereas it increased after UV-B and NaCl stress. Temperature is known to inhibitthe violaxanthin–zeaxanthin cycle of carotenoidsynthesis (Tran and Raymundo, 1999). An increasein carotenoid by UV-B and NaCl may be due to itsrole as protectant against reactive oxygen species(Bhargava et al., 2005; Srivastava et al., 2005).

Pronounced inhibition of nutrient uptake by NaCl(Fig. 2) may be due to alterations in the membranepermeability as well as cellular homeostasis.Reduction in NO3

� uptake is linked with thereduction in NH4

+ uptake as both the processes arelinked through the GS–GOGAT pathway and theenzymes of this pathway are highly sensitive todifferent stresses. It may also be due to depletionin ATP content and carbon assimilation, which are

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A

B

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Figure 2. Effects of cadmium, temperature, UV-B and salt on the uptake of (A) NO3� (mgNO3

� mg protein�1), (B) NH4+

(mgNH4+ mg protein�1) and (C) PO4

3� (mg PO43� mg protein�1) of control and copper-acclimated Anabaena doliolum. Error

bars indicate SD of three replicates. An asterisk on the bars represents significant change (Po0.05) in nutrient contentas compared to their respective controls.

Response of copper-acclimated Anabaena to stress 313

essential for nitrogen metabolism (Oelmuelleret al., 1988).

It is very interesting to mention that while NO3�

uptake was reduced significantly, its assimilatoryenzyme NR was less affected. This result findssupport from Shukla and Rai (2007), who opinedthat conditions favoring high NH4

+ uptake lead toincreased NR activity in Microcystis. So a meagerreduction in NH4

+ and profound reduction in NO3�

uptake make the cellular environment NH4+ dom-

inating. Since nitrate reductase is bound to the

chlorophyll-containing membrane fractions, inhibi-tion of chlorophyll as observed in the present studymay directly affect the nitrate reductase activity.Ammonium assimilation in cyanobacteria ismediated by ATP-dependent glutamine synthetasewhose inhibition may be attributed to either directaction of stressors on the enzyme complexesor depletion of the ATP pool. Inhibition of NH4

+

uptake as observed in this study could simply bedue to either inhibition of growth or GS activity.Instances of increased APase activity, as noticed in

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µM

–γg

luta

myl

hydro

xam

ate

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rote

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C

B

A

Figure 3. Effects of cadmium, temperature, UV-B and salt on the activities of (A) NR (mgNO2� mg protein�1), (B) GS (mM-

g-glutamyl hydroxamate mg protein�1) and (C) APase (nmol PNPmg protein�1 h�1) of control and copper-acclimatedAnabaena doliolum. Error bars indicate SD of three replicates. An asterisk on the bars represents significant change(Po0.05) in nutrient content as compared to their respective controls.

R. Thapar et al.314

Cu2+-acclimated A. doliolum, are also available inCd- and Pb-tolerant strains of Pseudomonas marina(Chan and Dean, 1988). Considerable reduction inAPase activity may be due to a stress-induceddecrease in PO4

3� uptake.The trend (NaCl4UV-B4temperature4Cd2+) of

inhibition of O2 evolution indicated damage of thePS II reaction center. This supports the contentionthat PS II is a very sensitive site, as also reported inSpirulina platensis (Murthy et al., 1989). Inhibition

of PS II activity may be due to loss of D1 protein(Matoo et al., 1989). An enhanced sensitivity of PSII toward salinity and UV-B could be due tointeraction of these stresses with the oxygenevolving complex, carriers of oxidizing as well asreducing sides of PS II and the reaction center itself(Renger et al., 1989). The inhibitory effect of NaClon O2 evolution, function of PS II electron transportand water-splitting cycle has also been observed bySudhir et al. (2005). UV-B inhibits expression and

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Response of copper-acclimated Anabaena to stress 315

activity of ribulose-1,5-bisphosphate carboxylase/oxygenase, the key enzyme of CO2 fixation (Lao andGlazer, 1996). Temperature-induced inhibition ofO2 evolution may be ascribed to either damage ofthe O2-evolving complex or alteration in theorganization of the thylakoid membrane. Relativelyless inhibition of PS I than PS II may be due to thestability of PS I of the thylakoid membrane as wellas less abundance than PS II (Almog et al., 1991).Cadmium affects the lipid structure around PS II,especially the light-harvesting chlorophyll a/bprotein complex II, thereby crippling the photo-synthetic process (Peters and Chin, 2003). Further,Cd2+ interacts with Ca2+, Mn2+ and Cl�, which areessential for the function of the O2-evolvingcomplex (Sersen and Kralova, 2001). A relativelypronounced inhibition of PS II than PS I and whole-chain activity in both the strains suggests thatcyanobacterium may take electrons from H2 orsulfur-containing compounds.

Inhibition of carbon fixation by different abioticstressors (Table 2) may be due to disruption ofthylakoid integrity. Further, reduced phosphate up-take, APase activity and inhibition of ETS arecollectively responsible for reduction in ATP contentin both the strains under stress. Decrease in ATP poolin Nostoc muscorum, Anabaena doliolum and Micro-cystis after UV-B and Cd2+ treatments is known (Atriand Rai, 2003). ATP synthesis was found to be blockedby NaCl, and UV-B exhibited a deleterious effect onthe photosynthetic apparatus, leading to a reducedsupply of ATP and NADPH. A significant positivecorrelation (P40.05) between 14C uptake and theATP pool supports this postulate. Therefore, inhibi-tion of all the metabolic and enzymatic processes dueto ATP depletion seems quite likely.

Conclusion

Based on the results of the present study and thesynthesis of available information, we can concludethat the metabolic processes of copper-acclimatedA. doliolum were affected by different stresses butthe magnitude was quite low as compared tocontrol cells. The acclimated A. doliolum showeda cross tolerance to abiotic stresses as compared tothe control, salinity emerged as highly injuriousand Cd2+ showed minimum toxicity.

Acknowledgments

L.C. Rai is thankful to DST for financial supportand Poonam Bhargava, Ashish Kumar Srivastava and

Yogesh Mishra are thankful to UGC and CSIR for theaward of SRF.

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