Transcript
Page 1: Tolerance of soil algae and cyanobacteria to drought stress

TOLERANCE OF SOIL ALGAE AND CYANOBACTERIA TO DROUGHT STRESS1

Ching-Su Lin

Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei 10660, Taiwan

and Jiunn-Tzong Wu2

Biodiversity Research Center, Academia Sinica, Taipei 11529, Taiwan

Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei 10664, Taiwan

Tolerance to drought stress in soil crustmicroorganisms is essential for exploiting suitableorganisms for restoring soil. In this study, theresponses to drought stress of two drought-tolerantspecies, a green alga and a cyanobacterium, werecompared with those of two non-tolerant green algae.In response to drought stress, induced by treatmentwith polyethylene glycol, the intracellular prolinelevels increased and were associated with increases inmalondialdehye, pigment contents, and enzymeactivities such as superoxide dismutase (SOD) andperoxidase (POD). Our results suggest that toleranceto drought stress could be indicated by theintracellular levels of proline, SOD, and carotenoids.This study provides insights into the droughtphysiology of the photosynthetic microorganisms andsuggests that Leptolyngbya boryana and Chlorellavulgaris are suitable pioneer organisms for soilrestoration.

Key index words: biological soil crusts; drought stress;soil algae; soil cyanobacteria; tolerance physiology

Abbreviations: APC, allophycocyanin; BSA, bovineserum albumin; BSC, biological soil crusts; chl-a,chlorophyll a; EPS, extracellular polysaccharides;EtBr, ethidium bromide; MDA, malondialdehyde;NCBI, National Center for Biotechnology Informa-tion; PC, phycocyanin; PEG, polyethylene glycol;POD, peroxidase; ROS, reactive oxygen species;RWC, relative water content; SOD, superoxidedismutase; TBA, 2-thiobarbituric acid; VIF, varianceinflation factor; WL, water loss; l, specific growthrate

Soil algae and cyanobacteria are usually the pio-neer colonizers in bare soils. They form BSCs andexert crucial influences on the development ofpedo-ecosystems (Moore 1998, Belnap 2003). Adap-tive mechanisms that enhance tolerance to stress arerequired for BSCs to survive stressful conditions suchas desiccation, extreme temperature, high incident

solar radiation, and low nutrients. Desiccation is oneof the most important stresses for BSCs (Lange2003). Osmotic stress often results in cellular andphoto-oxidative damage caused by accumulation ofROS (Peltzer et al. 2002). ROS attack a variety ofbiomolecules, resulting in enzyme inhibition, chloro-phyll degradation, DNA damage, and lipid peroxida-tion, which may lead to irreparable metabolicdysfunction and cell death (Apel and Hirt 2004).Cellular defense systems against ROS include

enzymatic scavenging through increased activity ofSOD, POD, catalase, ascorbate peroxidase, glutathi-one peroxidase, glutathione reductase, and peroxire-doxin (Mittler et al. 2004). Non-enzymatic defensesystems include elevation of cellular proline, carote-noids, tocopherols, ascorbic acid, and glutathione.The defense systems triggered in cells vary fromorganism to organism (Takeda et al. 1995, AbdEl-Baky et al. 2004). Numerous studies on droughtstress have been done with plants (Morgan 1984,Apel and Hirt 2004). Plants tend to accumulatespecific substances for osmoregulation under osmoticstress (Morgan 1984). This allows cells to keep watereven at low soil water potentials, so that the turgorpressure, metabolic activity, and growth of plants aremaintained during prolonged water deficits (Hansonand Hits 1982).Recently, the establishment and use of BSCs to

ameliorate desertification, to restore acrid or pol-luted lands, and to improve soil texture havereceived great interest (Yang et al. 2001, Jusu et al.2004, Guo et al. 2008), due to the fact that BSCsmay increase soil aggregation and stability, therebyreducing wind and water erosion (Mazor et al. 1996,Eldridge and Kinnell 1997). BSCs would increasesoil fertility by N- and C-fixations (Starks et al. 1981,Eldridge and Greene 1994, Lange et al. 1994).Numerous strains in BSCs are capable of toleranceor resistance to drought by maintaining a constantimbalance between the internal water content andexternal water availability. Nevertheless, in compari-son with plants, less is known about drought-tolerancemechanisms in soil algae and cyanobacteria. Tobetter understand the mechanism underlyingdrought stress injury in these organisms, we com-pared the physiological response of drought-tolerant

1Received 8 October 2012. Accepted 12 September 2013.2Author for correspondence: e-mail [email protected] Responsibility: T. Mock (Associate Editor)

J. Phycol. 50, 131–139 (2014)© 2013 Phycological Society of AmericaDOI: 10.1111/jpy.12141

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soil species with non-tolerant ones. We also attemptedto determine the key compounds responsible forthe tolerance to drought stress and assessed organ-isms suitable for stabilizing bare soils.

MATERIALS AND METHODS

Test organisms and culture conditions. BSCs were collectedfrom Hoyen Mountain (24°35′ N, 121°24′ E) that is situatedin the middle of Taiwan. They were crushed and passedthrough 1.0 mm pore size sieve. Five grams of samples weresuspended in 100 mL of sterile distilled water. Then, the mix-tures were incubated under shaking in darkness for 2 h toget soil suspensions. Subsequently, 0.1 mL of the soil suspen-sion was inoculated in 100 mL BG11 medium (Stanier et al.1971) or Chlorella (NC) medium (Kuhl 1962) and illuminatedunder 75 mol photons � m�2 � s�1. After about 1 week, thesamples were prepared for isolation and for further purifica-tion on agar plates.

After isolations, the cultures were first starved for 48 h indarkness, followed by dilution (1:10) with TYG broth andincubated for another 2 h in darkness. Then, 1 mg � mL�1

cycloserine (Sigma-Aldrich, St. Louis, MO, USA) was addedand incubated in darkness at 28°C for 24 h. Subsequently, cul-tures were plated on BG11 or NC agar plates. After ~2 weeks,the colonies on plates were isolated for subculture and furtherpurification to axenic status (Vaara et al. 1979). The axenicisolates were cultured in liquid medium, cyanobacteria inBG11 and chlorophytes in NC medium, by shaking at28 � 1°C under illumination of 75 mol photons � m�2 � s�1

with light:dark photoperiod of 14:10 h. Four axenic cultures,a cyanobacterium, Leptolyngbya boryana (Gomont) Anagnostidisand Kom�arek (IR-01), and three chlorophytes, Chlamydomonasreinhardtii P.A. Dangeard (MI-01), Chlorella vulgaris Beijerinck(SLE-01), and Klebsormidium flaccidum (K€utzing) P.C. Silva,K.R. Mattox and W.H. Blackwell (SLE-02) were used for thisstudy (Fig. S1 in the Supporting Information). These strainsare known to be widespread terrestrial strains (Casamatta et al.2005, Li and Brand 2007, Rindi et al. 2008). L. boryana andK. flaccidum have a filamentous morphology, while C. rein-hardtii and C. vulgaris are coccoid, single cells. The identifica-tion of these organisms was based on their morphology andDNA sequences.

DNA extraction, PCR amplification and sequencing. The DNAof the studied strains were extracted using the phenol–chloroform protocol (Saunders 1993). Amplification wascarried out by means of PCR as described by Sherwood andPresting (2007), using primers pair p23SrV_f1 and p23SrV_r1,flanking Domain V of the 23S plastid rDNA gene fragment ineukaryotic algae and cyanobacteria. The PCR products werevisualized on 1% agarose gel stained with EtBr and furtherpurified, using the Qiagen PCR purification kit (Stratagene,Santa Clara, CA, USA). DNAs were sequenced commercially inboth directions, and ambiguous bases were checked andaltered using the BioEdit program. Sequences were comparedto known cultured and environmental sample sequences usingthe BLAST search tool on the NCBI website (http://www.ncbi.nlm.nih.gov). The consensus sequences were then deposited atNCBI under the accession numbers: JX877619, JX877620,JX877621, and JX877624. All the strains were archived andavailable in the Phycological Laboratory, the BiodiversityResearch Center, Academia Sinica, Taiwan.

Estimation of RWC. RWC is used to measure the water-retention capacity of cells. For measurement, 250 mL of cul-tures were filtered through a cellulose acetate filter (pore sizeof 0.45 lm; Sartorius, G€ottingen, Germany) under reducedpressure. The filters were placed in an oven (60°C) to dry.The WL of cells was estimated using the following equation:

WL (%) = 100 9 [(W0 � Wt)/(W0 � Wd)], where W0 is theinitial wet weight after removing water drops by lightlyblotting with tissue paper; Wt is the instantaneous weightmeasured at every 30 min; Wd is the dry weight measuredafter the strains had been dried at 60°C for 48 h and cooledin a desiccators. RWC was then calculated as following: RWC(%) = 100 � WL (Gao and Ye 2007).

Growth under drought stress. Drought stress was achieved bythe addition of PEG 6000 (PEG-6000, MW 6000; Merck,Darmstadt, Germany) to culture medium. Different concen-trations of PEG were added to 100 mL cultures of testedstrains to give water potentials of 0, �0.15, �0.30, �1.03, and�1.76 MPa (equivalent to 0, 15, 20, 30, and 40% (w/v) ofPEG-6000, respectively) according to the methods of Moham-madkhani and Heidari (2008). Algal cells grown under expo-nential growth phase were used for this study. The growthrate was calculated on the basis of incubation for 3 d. Chl-awas used as a measure of growth. Ratios of chl-a to dry weightwere checked to confirm the suitability of using chl-a as ameasure for the studied organisms.

Determination of chl-a, carotenoids, phycobilins, specific growthrate, and lipid peroxidation. Algal cells were harvested by cen-trifugation (4,000g 9 10 min). Dry weight was measured byfiltering a 100 mL sample through pre-weighted 0.45 lmWhatman GF/C filters and drying the cell mass at 70°C for24 h (Fan et al. 1994). Chl-a and carotenoids from sampleswere extracted in 90% acetone and the abundance of the pig-ments was determined from absorbance at 450, 645, and663 nm, using the methods of Inskeep and Bloom (1985).Specific growth rate (l, d�1) was determined using the equa-tion suggested by Myers and Kratz (1955) as follows: l = ln(N1/N0)/(T1 � T0), where N1 and N0 are the final and ini-tial concentrations of chl-a at time T1 and T0, respectively.The growth rate per day was used as a basis of comparison inthis study.

The methods of Tandeau de Marsac & Houmard (1988)were used to estimate the concentration of PC and APC incyanobacterial cells. The cells were soaked in 20 mM sodiumacetate buffer (pH 5.5) and subjected to disruption with abead beater (Biospec, Bartlesville, OK, USA). After centrifu-gation (10,000g 9 10 min), 1% (w/v) streptomycin sulfatewas mixed with the supernatant for 30 min at 4°C. Sampleswere centrifuged (10,000g 9 10 min) at 4°C and the absor-bance at 620 and 650 nm of the supernatant was measuredby a Beckman Coulter DU800 spectrophotometer (Brea, CA,USA).

Lipid peroxidation was estimated by measuring the forma-tion of MDA with TBA, according to the methods of Heathand Packer (1968) modified by De Vos et al. (1989). Thesamples were suspended in 1 mL H2O (bi-distillated) andmixed with an equal volume of sea sand. Subsequently, thecells were disrupted with a bead beater and centrifuged toseparate the aqueous extracts from debris. Then, 1 mL of0.25% TBA dissolved in 10% trichloroacetic acid was addedand incubated at 95°C–100°C for 30 min, then chilled on ice.Samples were centrifuged, and MDA was determined by sub-tracting absorbance of the supernatant at 600 nm from thatat 532 nm. To calculate the concentration, an absorbancecoefficient of 155 mM�1 � cm�1 was used (Kwon et al. 1965).

Assay of SOD and POD activities. The methods of Chanceand Maehly (1955) were adopted. To prepare cell extracts forenzyme assays, 1 g of cells was suspended in phosphate buffer(0.1 mol � L�1, pH 7.8), mixed with an equal volume of seasand, and then subjected to disruption with a bead beater.The cell-free extract was obtained after centrifugation(10,000g 9 10 min). The protein content of each extract wasquantified by the modified Lowry method (Bollag and Edel-stein 1991), using BSA (Merck) as a standard.

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A SOD-assay kit (BioVision, San Francisco, CA, USA) wasused to quantify SOD activity, following the protocol given bythe manufactory’s manual. The absorbance at 450 nm wasread. Activity of POD was assayed by measuring the oxida-tion of guaiacol at 470 nm (Chance and Maehly 1955). Oneunit (U) of POD activity was defined as the increase inabsorbance at 470 nm for 1 min per mg protein.

Quantification of proline. For quantitative determination ofintracellular proline content, the methods of Bates et al.(1973) were adopted. The cells were suspended in 5 mL of3% sulfosalicylic acid, mixed with an equal volume of seasand, and subjected to disruption with a bead beater. Thesupernatant obtained after centrifugation (10,000g 9 10 min)was mixed with 2 mL acetic acid and 2 mL of ninhydrinreagent in a boiling water bath for 60 min. The reaction wasterminated by an ice bath, extracted with toluene by vigorousshaking, and the extract was placed in the dark for 10 min.Absorbance at 520 nm of the resulting organic layer wasmeasured. A standard curve for proline was prepared withL-proline (Merck).

Statistical analysis. All experiments were conducted at leastthree times, with triplicate measurements for each treatment.The data obtained from each experiment were subjected tomultivariate analysis, using SYSTAT (version 12; Systat Soft-ware Inc., Richmond CA, USA). Pearson correlation and mul-tiple regression were analyzed to compensate for covariancefor all of the studied strains with different concentrations ofPEG (n = 112). The forward stepwise selection procedureswere used to select the most representative variables, and tryto compare the difference between cyanobacteria (n = 28)and chlorophytes (n = 84). Differences were considered to besignificant at P < 0.05 level.

RESULTS

RWC. In response to drought stress, various val-ues of RWC were measured for four species studied.For C. reinhardtii, C. vulgaris, and K. flaccidum, arapid loss of cellular water was measured during thefirst 60 min (Fig. 1), showing high dehydration rate.Compared to these three chlorophytes, L. boryanawas relatively resistant to dehydration, with WL aslow as 30% at the same observation time.

Differences in drought resistance among theorganisms were observed based on the dehydrationtime, that is, the reduction of RWC from 100% to0%. For C. reinhardtii, C. vulgaris, K. flaccidum, andL. boryana, the dehydration time was 60, 90, 90, and150 min, respectively. According to these results,the order of resistance to dehydration was C. rein-hardtii < K. flaccidum ≤ C. vulgaris < L. boryana.Tolerance to drought stress. The four studied species

grew well under the condition without droughtstress, though there existed a small difference ingrowth rate between them (cf. growth curves inFig. S2 in the Supporting Information). To ascer-tain the suitability of using chl-a as a measure ofbiomass, the ratio of chl-a to dry weight was checkedduring treatment with drought stress. The ratiossharply decreased over the course of the incubationduring the first day of treatment and remainedpractically constant in subsequent days up to a week(Fig. S3 in the Supporting Information). It dis-played no significant difference in ratios among thetreatments with various concentrations of PEG(Pearson correlation, P < 0.05). The mean values(lg chl-a � mg�1 dry weight) obtained in thisstudy were 10.23 � 3.07 for L. boryana, 20.52 � 4.84for C. vulgaris, 12.51 � 2.24 for C. reinhardtii, and5.53 � 2.06 for K. flaccidum. The difference in thegrowth rate between species became pronouncedunder drought stress induced by various concentra-tions of PEG-6000. The average growth rates mea-sured after the first 2 weeks of exposure show thatC. reinhardtii was very sensitive to the treatments,showing significant inhibition of growth at all PEGconcentrations tested and ceased to grow at PEGconcentrations >20% (�0.69 Mpa; Fig. 2). K. flacci-dum could not grow at PEG concentrations >30%(�1.03 Mpa). However, it still was able to grow atPEG>20%. L. boryana was less susceptible than thesetwo species, though a total inhibition was observed

FIG. 1. Time course of changes of relative water content(RWC) in four studied species. Values are mean � SE, n = 3.

FIG. 2. Average growth rates measured within one week as afunction of treated polyethylene glycol (PEG) concentrations forfour studied species. Values are means of three replicate experi-ments.

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at PEG >40% (�1.76 Mpa). In contrast, C. vulgariswas resistant to PEG treatments, compared with theother three species. Treatment with PEG resulted incertain degrees of growth inhibition of this speciesduring the first week of exposure, but completeinhibition did not occur even with PEG up to 40%.Moreover, the inhibited cells began to grow after1 week of treatment. As a result, an elevation ofgrowth rates was observed. Among the four organ-isms, C. vulgaris exhibited the highest tolerance todrought stress.Lipid peroxidation. Cellular MDA contents in the

four studied species did not differ from each othersignificantly prior to addition of PEG (Pearson cor-relation, P > 0.05; Fig. 3A). A remarkable increasein MDA content was observed after 1 day of treat-ment with 25% PEG, which was more significantthan treatment with 15% PEG. Subsequently, theMDA concentration gradually declined until theend of the incubation period. The highest MDAconcentration occurred in K. flaccidum and the low-est concentrations were in C. reinhardtii and L. borya-na; the MDA concentration was not significantlydifferent between these two species (Pearson corre-lation, P > 0.05).SOD and POD activities. In non-stress (control)

conditions, the four species had different SOD and

POD activities. Under drought stress induced by25% PEG, the SOD and POD activity increased(Fig. 3, B and C). The dynamics of SOD activitywere nearly the same among the four organisms.SOD activity increased in the first 2 d and thendeclined. In contrast to SOD, the dynamics of PODactivity varied among the organisms. For C. vulgarisand L. boryana, the maximum activity was reachedin the third day of incubation, while for K. flaccidumand C. reinhardtii, maximum activity was achieved inthe first day. C. vulgaris had the highest activity, fol-lowed in descending order by L. boryana, C. rein-hardtii, and K. flaccidum. The differences in PODactivity among the species were significant(P < 0.05).Intracellular proline level. Proline levels in treated

cells were related to the PEG concentrations treated(Fig. S4 in the Supporting Information). An eleva-tion of proline levels was observed when cells weretreated with 25% PEG (Fig. 4A), with variabledynamics among the species. L. boryana had higherintracellular proline levels during the first 2 days oftreatment with 25% PEG, whereas the highest levelsin C. vulgaris occurred after 3 days. Significantly,the elevation in proline levels were higher (i.e., 5-to10-fold) in these two drought-tolerant species, whencompared to the non-tolerant species (2- to 3-fold),namely K. flaccidum and C. reinhardtii. For L. boryanaand K. flaccidum, the highest proline level wasreached in the second day of incubation and thenthe level declined, whereas increasing proline levelswere observed for C. vulgaris and C. reinhardtii untilthe end of incubation (day 7; Fig. 4A).Protein content. After treatment with PEG, protein

content in L. boryana cells increased considerably,while in C. vulgaris, the concentration increasedonly slightly (Fig. 4B). In contrast, the protein con-tent in C. reinhardtii and K. flaccidum declined withincubation time. The magnitude of increased pro-tein content was positively correlated with thedegree of tolerance to drought stress induced byPEG.Carotenoids and phycobilin content. Under 15%

PEG, the changes in intracellular carotenoids con-tent in C. reinhardtii (from 2.7 [the control] to3.3 mg � g�1 dr.wt.) and in K. flaccidum (from 2.1 to2.5 mg � g�1 dr.wt.) were quite low, when comparedwith C. vulgaris (from 4.2 to 8.8 mg � g�1 dr.wt.)and L. boryana (from 2.2 to 5.9 mg � g�1 dr.wt.).Apparently, a remarkable increase up to 2- to 3-foldoccurred in the latter two species. Under 25% PEG,the carotenoids content in L. boryana increasedfrom 2.2 to 7.1 mg � g�1 dr.wt., while in other threespecies declined with increasing incubation time(Fig. 4C).The concentrations of PC and APC in L. boryana

cells decreased during treatment with 25% PEG,from 45.6 to 33.7 mg � g�1 and from 33.6 to26.2 mg � g�1 dr.wt., respectively. Under treatmentwith 15% PEG, changes in the concentrations of

FIG. 3. Changes in the intracellular contents of (A) malondial-dehye (MDA), (B) superoxide dismutase (SOD), and (C) peroxi-dase (POD) in Chlorella vulgaris (open circle), Chlamydomonasreinhardtii (filled circle), Klebsormidium flaccidum (filled triangle),and Leptolyngbya. boryana (open triangle) cells under treatmentwith 25% PEG. Values are means � SE, n = 3.

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PC and APC were insignificant, from 45.6 to43.7 mg � g�1 and from 33.6 to 34.2 mg � g�1 dr.wt.,respectively. The ratio of PC/APC also decreased inevery treatment (Fig. 5) due to a faster decline ofPC than APC.Statistical analysis. The results of Pearson correla-

tion analysis showed that the cellular content of chl-a was correlated positively with that of carotenoids,and negatively with MDA and proline (P < 0.001;Table 1; Fig. S4). Positive correlation coefficients

were also obtained between carotenoids and SOD,carotenoids and POD, MDA and proline, SOD andPOD, SOD and protein, and POD and protein.A multiple linear regression analysis was per-

formed using chl-a as a dependent variable. Theequations in Table 2 show the relation of the bio-mass displayed as chl-a and the variables of SOD,MDA, proline, and protein (R2 = 0.386, theVIF = 1.75–2.77) for all the four species studied.When the data of cyanobacterium was excluded,similar results (R2 = 0.414, VIF = 1.48–2.76) andvariables were obtained (Table 2), showing a multi-collinearity of the data from this organism. Prolinewas the only variable which significantly correlated(P < 0.001, n = 60) with chl-a in multiple linearregression analysis.To take into account the influence of indepen-

dence on variables, a forward stepwise regressionwas used to further analyze covariance. Table 3shows that for both chlorophytes and cyanobacte-rium, proline, carotenoids, SOD, and MDA can beused as a measure of the tolerance to drought stresswith significance. However, MDA is insignificant forcyanobacterium (L. boryana).

DISCUSSION

Under drought stress conditions, algae and cyano-bacteria exhibit some changes in photosyntheticelectron transport that would lead to the formationof free radicals, including ROS. Subsequently, per-oxidation is mediated by ROS that readily attackunsaturated fatty acids, yielding lipid hydroperox-ides and alkoxyl and peroxyl radicals. The radicals,then, would initiate chain reactions in the mem-branes and result in change and disruption of lipidstructure, membrane organization, integrity, andpermeability (Molinari et al. 2007, Qian et al.2009). This process of peroxidation would lead tothe production of carbonyl compounds such asMDA (Aziz and Larher 1998). The results of ourstudy show that treatment with drought stress hasgiven rise to an enhancement of MDA levels in stud-ied cells. The elevated MDA levels are remarkable atthe first day of treatment, but decline rapidly there-after. Moreover, the elevated MDA levels vary withthe species studied and does not exhibit any correla-tion with the degree of tolerance to drought stress.Thus, the elevation in intracellular MDA levelsbelongs to a short-term response of cells to stress.According to this, change in intracellular MDA lev-els is an indication of cellular damage, rather thanan indication of tolerance to drought stress.In response to stress, cells tend to accumulate a

considerable amount of proline intracellularly. Pro-line is a compatible compound which could act as anosmo-regulant (Wu et al. 1998, Molinari et al. 2004),a redox buffer (Hare and Cress 1997), a protectant ofenzymes and proteins (Nikolopoulos and Manetas1991, Delauney and Verma 1993), a regulator of

FIG. 4. Changes in the intracellular contents of (A) proline,(B) protein, and (C) b-carotene in Chlorella vulgaris (open circle),Chlamydomonas reinhardtii (filled circle), Klebsormidium flaccidum(filled triangle), and Leptolyngbya boryana (open triangle) cellsunder treatment with 25% PEG. Values are means � SE, n = 3.

FIG. 5. Effects of treatment with various concentrations ofpolyethylene glycol (PEG) on the ratios of phycocyanin/allophyc-ocyanin (PC/APC) of Leptolyngbya boryana cells. Values aremeans � SE, n = 3.

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cytosolic acidity (Venekemp 1989), a scavenger offree radicals (Matysik et al. 2002), and a membranestabilizer by interactions with phospholipids(Wu et al. 1995). Although some authors questionedthe cause–effect relationships between proline andstress (Cordovilla et al. 1996, Schat et al. 1997, Qianet al. 2001), this study shows that proline levels inPEG-treated cells are positively correlated with thetreated doses and the drought-tolerant species accu-mulates more proline than non-tolerant ones, similarto the reports by Delauney et al. (1993) and Molinariet al. (2007) for higher plants. Based on these, pro-line accumulation could be a response of soil algaeand cyanobacteria to drought stress and be correlatedwith the tolerance to such a stress.

Tripathi and Gaur (2004) found in Scenedesmussp. that the accumulation of proline was triggeredby ROS. The enhanced proline levels were consid-ered to provide protection against damage by ROS(Molinari et al. 2007). Our results also showed thatthe enhanced intracellular proline levels were posi-tively correlated with the levels of SOD and POD.These two enzymes were known to be involved inthe Halliwell-Asada detoxification pathway (Aroraet al. 2002). It is not surprising that the activities ofeither enzyme are enhanced simultaneously inresponse to drought stress. However, the enhancedactivities of SOD by drought stress were one orderhigher than POD in this study (cf. Fig. 3). Possibly,this might be due to that SOD is a major and

TABLE 1. Pearson linear correlation coefficients between the measured parameters in all four studied species treated withdrought stress of 0%–25% PEG.

Variable Chl-a Carotene MDA SOD POD Proline

Carotene 0.540***MDA �0.443*** �0.245**SOD �0.018 0.712*** 0.022POD �0.021 0.668*** 0.245** 0.865***Proline �0.599*** �0.222* 0.526*** 0.318*** 0.409***Protein �0.145 0.478*** �0.097 0.711*** 0.666*** 0.444***

Chl-a, chlorophyll-a; MDA, malondialdehye; SOD, superoxide dismutase; POD, peroxidase; PEG, polyethylene glycol.The significance level: ***P < 0.001; **P < 0.01; *P < 0.05 for n = 112.

TABLE 2. Results of multiple linear regression analysis on four studied species treated with drought stress of 0%–25% PEG.

ParameterChlorophytaa + Cyanobacteriumb (n = 112) Chlorophyta only (n = 84)

Chl-a = 1208.41 + 4.81 9 SOD � 4.04 9 MDA � 3.83 9 Pro-line � 1.137 9 Protein

Chl-a = 1147.62 + 4.94 9 Protein � 4.60 9 Pro-line � 3.31 9 MDA + 3.16 9 SOD

Variable SE t-value P VIF SE t-value P VIF

Constant 74.77 16.16 <0.001 – 152.04 7.55 <0.001 –MDA 2.45 �1.65 0.101 1.75 2.55 �1.30 0.198 1.63SOD 2.53 1.90 0.060 2.08 3.23 0.98 0.332 2.28Proline 0.75 �5.09 <0.001 2.12 0.97 �4.67 <0.001 1.48Protein 2.34 �0.49 0.627 2.77 4.83 1.02 0.310 2.76R2 0.386 0.414

Chl-a, chlorophyll-a; MDA, malondialdehye; SOD, superoxide dismutase; R2, square of multiple correlation coefficient; PEG,polyethylene glycol, VIF, variance inflation factor.

aChlorophyta include C. reinhardtii, C. vulgaris, and K. flaccidum.bCyanobacterium= L. boryana.

TABLE 3. Results of forward stepwise regression analysis for four studied species treated with drought stress of 0%–25%PEG. Dependent variable was the growth rate measured in terms of chlorophyll-a.

Chlorophytaa + Cyanobacterium (n = 112) Chlorophyta (n = 84) Cyanobacterium (n = 28)

Variable Proline* b-Carotene*** MDACarotene*** SOD*** APC/Chl-a***SOD*** MDA* ProteinMDA*

R2 0.638 0.590 0.878

Chl-a, chlorophyll-a; MDA, malondialdehye; SOD, superoxide dismutase; APC, allophycocyanin; R2, square of multiple correla-tion coefficient; PEG, polyethylene glycol.The significance level: ***P < 0.001; **P < 0.01; *P < 0.05.aChlorophyta include C. reinhardtii, C. vulgaris, and K. flaccidum; Cyanobacterium= L. boryana.

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primary scavenger of O2�, and the H2O2 reaction

product is subsequently disassembled to H2O andO2 by POD (Asada 1992, 1999, Zhang and Kirkham1994). Namely, the enzyme activity is expressed dif-ferently. In response to drought stress, the elevationin SOD activity is a primary and major effect.

In this study, initial activities (i.e., in the absenceof drought stress) of SOD and POD were signifi-cantly higher in the drought-tolerant species, C. vul-garis and L. boryana (Pearson correlation, P < 0.05;Fig. 3B). Furthermore, the enhanced levels ofenzyme activity following PEG treatment wereremarkably higher in both species compared to thenon-tolerant species, namely C. reinhardtii andK. flaccidum (Pearson correlation, P < 0.05; Fig. 3, Band C). Thus, the activities of SOD and POD incells are correlated with levels of tolerance todrought stress in the studied species, similar to theup-regulation in antioxidant capacity reported forother algae and cyanobacteria (Mallick and Mohn2000, Coll�en and Davison 2001, Abd El-Baky et al.2004).

Carotenoids are known to act as effective quench-ers of singlet O2 and chlorophyll triplets (Salgueroet al. 2003, Ledford and Niyogi 2005). According toOsmond et al. (1997), carotenoids may play a rolein protecting cells against ROS by reacting with lipidperoxidation products to terminate chain reactions,by scavenging singlet O2 and dissipating the energyas heat, by reacting with triplet or excited chloro-phyll molecules to prevent formation of singlet O2,or by dissipating excess excitation energy throughthe xanthophyll cycle. Some microalgae and cyano-bacteria tend to accumulate carotenoids whengrown under specific conditions that limit growth,such as nutrient imbalance, oxidative stress, intenseillumination, and high osmotic pressure (Salgueroet al. 2003, Pisal and Lele 2004, Lohscheider et al.2011). In this study, accumulation of carotenoidswas observed in drought-tolerant species L. boryanaand C. vulgaris, but not in non-tolerant species(C. reinhardtii & K. flaccidum). Moreover, thedynamic of carotenoid content during PEG treat-ment was less pronounced in C. vulgaris than inL. boryana, showing a coincidence with the order ofresistance to dehydration. Therefore, accumulationof carotenoids might play a role in drought stressand be associated with drought tolerance in thestudied soil algae and cyanobacterium.

The phycobiliproteins (PBP) including PC andAPC are attached to thylakoid membranes incyanobacterial cells (Grossman et al. 1993). Understress conditions, the composition of PBP might vary(Reuter and Muller 1993). In this study, a declineof the PC/APC ratio was observed in L. boryana dur-ing treatment with PEG, implying not only PC wasmore susceptible than APC, but this might beascribed to the inhibition of pigment synthesis. Theexternal localization of PC on intracellular thylakoidmembrane might be one of the possible reasons for

the greater sensitivity, due to more exposedness tothe action of stress (Prasad et al. 2005).In response to stress conditions, a decrease (Jusu

et al. 2004) or an increase (Assche et al. 1988) incellular protein content has been reported for dif-ferent organisms. In this study, the protein contentof stressed cells, particularly of L. boryana, increasedin response to drought stress induced by PEG, show-ing a positive correlation between elevated proteincontent and the degree of tolerance to droughtstress. It is assumed that the elevated protein con-tent might be of stress proteins or closely correlatedto this group of proteins.Under PEG treatment, the cyanobacterium L. bor-

yana displayed a relatively higher tolerance than thechlorophytes. Other than the metabolic characteris-tics of this species, the tolerance might be attributedat least partially to the presence of a mucilaginousenvelope composed of EPS entangled in filamen-tous structure. EPS in the envelope would serve as amatrix for the immobilization of other componentsthat may protect the cell walls from damage duringswelling and shrinkage associated with droughtstress (Caiola et al. 1996). Other than this, EPSwould prevent cells from losing water to certaindegree. This is particularly important for the BSCgrowing at the soils with low water-holding capacity,like the locality from which the studied strains areisolated. Thus, as indicated by Adhikary (1998), thepresence of EPS in cyanobacteria might play animportant role in drought tolerance. This is consid-ered one of the reasons why L. boryana displayshigher tolerance to drought stress than other threespecies studied.Chl-a is commonly used as a proxy for relative

biomass (Kalchev et al. 1996, Kahlert and Pettersson2002). But, chl-a contents might vary from speciesto species (Boyer et al. 2009) and be changedwith environmental conditions, such as irradiance(Falkowski and Laroche 1991), nutrient limitations(Latasa and Berdalet 1994, Todd et al. 2008) andthe physiological status (Brunet et al. 1996). In thisstudy, we have checked the ratios of chl-a to dryweight of the studied species under water stress.The results showed that the contents of chl-a instressed cells are correlated highly with biomass overthe time studied. This suggests that the chl-a esti-mated growth rate for deducing the tendency of tol-erance should be compatible to those on the basisof biomass.Conclusion. The four studied organisms displayed

various degrees of tolerance to desiccation. Droughtstress induced the enhancement of the activities ofsome free radical scavenging enzymes and the intra-cellular levels of proline and a lipid degradationcompound. It is confirmed that the levels ofproline, carotenoids, and the activities of SOD arethe best representatives for reflecting the toleranceto drought stress in soil algae and cyanobacteria.Our results suggest that both the cyanobacterium

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Supporting Information

Additional Supporting Information may befound in the online version of this article at thepublisher’s web site:

Figure S1. The isolated soil cyanobacteriumand algae from Haoyan Mountain for this study.(A) Leptolyngbya boryana, (B) Chlamydomonas rein-hardtii, (C) Chlorella vulgaris, (D) Klebsormidiumflaccidum; bar = 10 µm.

Figure S2. The growth curves in terms of chlo-rophyll a concentrations of Chlamydomonas rein-hardtii, C. vulgaris, Klebsormidium flaccidum, andLeptolyngbya boryana under the culture conditionswithout PEG treatment. Values are means � SE,n = 3.

Figure S3. Changes in the ratios of chlorophylla/dry weight of Chlamydomonas reinhardtii, C. vul-garis, Klebsormidium flaccidum, and Leptolyngbya bor-yana over time under the culture conditionswithout PEG treatment. Values are means � SE,n = 3.

Figure S4. The regression analysis of averagecontents of Chl-a, MDA, and proline in all ofstudied strains treated with different concentra-tions of PEG. Values are means � SE.

TOLERANCE OF SOIL ALGAE 139


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