Phytohormones as regulators of heavy metal biosorption and toxicity in green alga Chlorella vulgaris (Chlorophyceae)

Phytohormones as regulators of heavy metal biosorption and toxicity in green alga Chlorella vulgaris (Chlorophyceae)
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  Research article Phytohormones as regulators of heavy metal biosorption and toxicity in greenalga  Chlorella vulgaris  (Chlorophyceae) Alicja Piotrowska-Niczyporuk a , * , Andrzej Bajguz a , El _ zbieta Zambrzycka b , Beata Godlewska- _ Zy 1 kiewicz b a University of Bialystok, Institute of Biology, Department of Plant Biochemistry and Toxicology, Swierkowa 20 B, 15-950 Bialystok, Poland b University of Bialystok, Institute of Chemistry, Department of Analytical Chemistry, Hurtowa 1, 15-399 Bialystok, Poland a r t i c l e i n f o  Article history: Received 26 August 2011Accepted 23 November 2011Available online 1 December 2011 Keywords: AntioxidantsCadmiumCopperLeadPhytohormonesROS a b s t r a c t The present study was undertaken to test the in fl uence of exogenously applied phytohormones: auxins(IAA, IBA, NAA, PAA), cytokinins (BA, CPPU, DPU, 2iP, Kin, TDZ,  Z  ), gibberellin (GA 3 ), jasmonic acid (JA) aswell as polyamine - spermidine (Spd) upon the growth and metabolism of green microalga  Chlorellavulgaris  (Chlorophyceae) exposed to heavy metal (Cd, Cu, Pb) stress. The inhibitory effect of heavy metalson algal growth, metabolite accumulation and enzymatic as well as non-enzymatic antioxidant systemwas arranged in the following order: Cd  >  Pb  >  Cu. Exogenously applied phytohormones modify thephytotoxicity of heavy metals.Auxins, cytokinins, gibberellin and spermidine (Spd) can alleviate stress symptoms by inhibiting heavymetal biosorption, restoring algal growth and primary metabolite level. Moreover, these phytohormonesand polyamine stimulate antioxidant enzymes ’  (superoxide dismutase, ascorbate peroxidase, catalase)activities and ascorbate as well as glutathione accumulation by producing increased antioxidant capacityin cells growing under abiotic stress. Increased activity of antioxidant enzymes reduced oxidative stressexpressed by lipid peroxidation and hydrogen peroxide level. In contrast JA enhanced heavy metaltoxicity leading to increase in metal biosorption and ROS generation. The decrease in cell number,chlorophylls, carotenoids, monosaccharides, soluble proteins, ascorbate and glutathione content as wellas antioxidant enzyme activity was also obtained in response to JA and heavy metals. Determining thestress markers (lipid peroxidation, hydrogen peroxide) and antioxidants ’  level as well as antioxidantenzyme activity in cells is important for understanding the metal-speci fi c mechanisms of toxicity andthat these associated novel endpoints may be useful metrics for accurately predicting toxicity. The datasuggest that phytohormones and polyamine play an important role in the  C. vulgaris  responding toabiotic stressor and algal adaptation ability to metal contamination of aquatic environment.   2011 Elsevier Masson SAS. All rights reserved. 1. Introduction Aquatic environment is often exposed to various pollutantsincluding heavy metals owing to increasing industrial and/oragricultural wastes. The danger of heavy metal pollution is due toits ability to circulatewithin aquatic and near-shore ecosystems fora prolonged length of time [1]. Green unicellular freshwater algae with high metabolism rates play the basic role in the primaryproduction and concentration of heavy metals [2,3]. As compared to some microbial biomasses, such as fungi, heavy metal bio-sorptioncapacityofgreenalgaeprovedtobethehighestbecauseof the algal cell wall, which is composed of a  fi ber-like structure andamorphous embedding matrix of various polysaccharides [2]. Forexample, heavy metals have been effectively removed using greenmicroalgae, such as  Chlorella vulgaris ,  Chlorella kesslerii ,  Scede-nesmusquadricauda and S. incrassatules [3 e 5].Inthisrespect,studyof the biosorption of cadmium (Cd), copper (Cu), and lead (Pb) by C. vulgaris  (Chlorophyceae) as a crucial component of naturalphytocenoses seems to be extremely important. Moreover, exoge-nously applied phytohormones and plant growth regulators canmodify the algal biosorption of heavy metals from contaminatedwater body [6 e 9].  Abbreviations:  APX, ascorbate peroxidase; BA,  N  6  -benzyladenine; CAT, catalase;Cd, cadmium; CPPU,  N  -(2-chloro-4-pyridyl)- N  9-phenylurea (forchlorophenuron);Cu, copper; DPU,  N,N  ’  -diphenylurea; DTT, dithiothreitol; GA 3 , gibberellic acid; IAA,indole-3-acetic acid; IBA, indole-3-butyric acid; 2iP, 2-isopentenyladenine; JA, jasmonic acid; Kin, kinetin; MDA, malondialdehyde; NAA, 1-naphthaleneaceticacid; NBT, nitroblue tetrazolium; NEM,  N  -ethylmaleimide; PAA, phenylacetic acid;Pb, lead; PVP, polyvinylpyrrolidone; ROS, reactive oxygen species; SOD, superoxidedismutase; Spd, spermidine; TBA, thiobarbituric acid; TCA, trichloroacetic acid;TDZ, 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron); Z,  trans -zeatin. *  Corresponding author. E-mail address: (A. Piotrowska-Niczyporuk). Contents lists available at SciVerse ScienceDirect Plant Physiology and Biochemistry journal homepage: 0981-9428/$  e  see front matter    2011 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.plaphy.2011.11.009 Plant Physiology and Biochemistry 52 (2012) 52 e 65  Heavy metals can cause adverse effects on the growth, celldivision, photosynthesis, and destruction of primary metabolites inalgal cultures [10 e 12]. Moreover, Cd, Cu and Pb are stronglyphytotoxic, partly because of the generation of reactive oxygenspecies (ROS) that react with lipids, proteins, photosyntheticpigments and nucleic acids causing lipid peroxidation, membranedamage, metabolite degradation, inactivation of enzymes and celldeath [13]. On the other hand, aquatic plants have evolved enzy- matic (superoxide dismutase, catalase, ascorbate peroxidase) andnon-enzymatic (ascorbate, glutathione) antioxidant mechanism toprevent the oxidative stress [14,15]. Survival in aquatic environ- ment contaminated by heavy metals may also depend on the algalability to generate and transit signals that adjust the metabolismaccordingly. Therefore, the search for signal molecules mediatingstress tolerance is an important step towards the better under-standing how lower plants respond to pollutants. Phytohormonesare active members of the signal cascade involved in the inductionof plant stress response. For example, abiotic stress results in bothalerted levels of plant hormones and decreased plant growth [16].The decreased auxin, cytokinin and gibberellic acid and increasedabscisic or jasmonic acid contents are often observed response inplants subjected to environmental stresses [16,17]. Several other compounds may also alleviate stress symptoms of environmentalstress [8,18]. An alternative strategy to ameliorate abiotic stress factors could therefore, to use exogenous application of phytohor-mones and plant growth regulators.This study was undertaken to determine whether phytohor-mones and plant growth regulator may contribute to ameliorationor intensi fi cation of negative impacts owing to heavy metal excess.For this reason, the effect of phytohormones, such as auxins (IAA,IBA, NAA, PAA), cytokinins (BA, CPPU, DPU, 2iP, Kin, TDZ,  Z  ),gibberellin (GA 3 ), jasmonic acid (JA) as well as plant growth regu-lator  e  polyamine (spermidine, Spd) on metal (Cd, Cu, Pb) bio-sorption, growth and biochemical changes in  C. vulgaris  exposed toabiotic environmental stress, was examined. The object of thepresent study was to test the hypothesis that the involvement of antioxidant system in the biochemical detoxi fi cation of Cd, Cu andPb in  C. vulgaris  culture is mediated byexogenously applied auxins,cytokinins, gibberellin, jasmonic acid, and polyamine. Obtainedresults may be important for better understanding of the planthormones role in biochemical adaptation of   C. vulgaris  to stressconditions present in polluted freshwater ecosystems. 2. Materials and methods  2.1. Plant material, culture conditions and treatments The green algae  C. vulgaris  (Chlorophyceae) were grown inmodi fi ed Knop ’ s medium (pH 6.8) under the conditions of 50  m mol m  2 s  1 light intensity and 16:8-h light/dark cycle at25(  1)   C [6,7,9,17]. Synchronization of the culture was controlled by studying cell division and the diagrams of cell size distribution.Growth of cultures was initiated by introduction of inoculumscontaining about 10 6 algal cells.IntheexperimenttheeffectofCd,Cu,andPbattheconcentration100  m M was analyzed. Concentration of heavy metals was selectedbased on our previous experiments [6,7] as the most toxic for algalgrowthandmetabolitecontent.Inordertothatappropriateamount Fig.1.  The effect of cytokinins (BA, Z, Kin, 2iP, DPU, CPPU, TDZ), auxins (IAA, NAA, PAA, IBA), gibberellin (GA 3 ), jasmonic acid (JA) and spermidine (Spd) in combination with heavymetals (Cd, Cu, Pb) on the number of cells of   C. vulgaris . Data are the means of four independent experiments  SD. Treatment with at least one letter the same are not signi fi cantlydifferent according to Duncan ’ s test.  A. Piotrowska-Niczyporuk et al. / Plant Physiology and Biochemistry 52 (2012) 52 e 65  53  of their nitrate forms: Cd(NO 3 ) 2 $ 4H 2 O, Pb(NO 3 ) 2 , Cu(NO 3 ) 2 $ 3H 2 Owas dissolved in distilled water and then added in right concentra-tion to Erlenmeyer  fl asks with Knop ’ s medium (250 mL). Moreover,the interactions of heavy metals with phytohormones (auxins,cytokinins, gibberellin, and jasmonic acid) and plant growth regu-lator (polyamine) were studied after 24 h of cultivation.The concentrations of phytohormones and polyamine wereselected based on the previous experiments. Urea-type cytokinins: N,N  ’  -diphenylurea (DPU), forchlorophenuron (CPPU) and thidia-zuron (TDZ) at 1  m M and adenine-type cytokinins: 2-isopentenyladenine (2iP), kinetin (Kin) and  N  6  -benzyladenine(BA) at 0.1  m M and  trans -zeatin (Z) at 0.01  m M were the mosteffectiveininductionof  C.vulgaris culturegrowth[19].Auxins,such indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA) and phenylacetic acid (PAA) arecharacterized by the highest biological activity at concentration50  m M [20]. Gibberellin (GA 3 ) stimulated algal growth at 10  m M [9]. Jasmonic acid (JA) was characterized by stimulatory activity in C. vulgaris  culture at 0.1  m M [21]. Among polyamines, spermidine (Spd) was chosen as the most active in algal culture, especially at100 m M[22]. Allchemical compounds,whichhave been usedin the studies, were purchased from Sigma e Aldrich Co., USA.  2.2. Determination of heavy metals in C. vulgaris cells Todeterminetheconcentrationofheavymetalsinalgae,culturesof   C. vulgaris  were centrifuged for 10 min at 10,000 g. The algalsuspensionwasdriedat105  Cfor12handmineralizedin65%nitricacid. Concentrations of Cd, Cu and Pb in prepared samples weredeterminedby fl ameatomicabsorptionspectrometryusingaSolaarM6 (Thermo Electron Corporation, UK) spectrometer with deute-rium background correction system. The absorbances of Cd, Cu andPb were measured in air-acetylene  fl ame with 0.5 nm spectralbandpass at wavelengths  l  ¼  228.8 nm,  l  ¼  324.7 nm, and l ¼ 217.0 nm, respectively [7].  2.3. Determination of cell number, proteins, monosaccharides and photosynthetic pigments The number of cells was determined by direct counting of cellsin the growth medium using a Bürker chamber [20 e 22]. TheconcentrationofproteininalgalcellswasdeterminedfollowingtheBradford[23]method,usingbovineserumalbuminasthestandard.The monosaccharide content was estimated according to theSomogyi [24] method. Wellburn [25] method was used for the determination of the content of photosynthetic pigments.  2.4. Stress markers determination Lipid peroxidationwas determined by measuring the amount of total MDA [26]. Algal cells were harvested by centrifugation at 10,000 g for 10 min and the resulting pellet was treated with 0.25%(w/v) TBA in 10% (w/v) TCA. After heating at 95   C for 30 min, themixture was cooled and centrifuged. The absorbance of thesupernatant at 532 nm was recorded and corrected for unspeci fi cturbidity by subtracting the value at 600 nm. The level of hydrogen Fig. 2.  The effect of cytokinins (BA, Z, Kin, 2iP, DPU, CPPU, TDZ), auxins (IAA, NAA, PAA, IBA), gibberellin (GA 3 ), jasmonic acid (JA) and spermidine (Spd) in combination with heavymetals (Cd, Cu, Pb) on the protein content in  C. vulgaris  cells. Data are the means of four independent experiments    SD. Treatment with at least one letter the same are notsigni fi cantly different according to Duncan ’ s test.  A. Piotrowska-Niczyporuk et al. / Plant Physiology and Biochemistry 52 (2012) 52 e 65 54  peroxide in  C. vulgaris  cells was measured spectrophotometricallyat 390 nm by reaction with KI [27].  2.5. Determination of antioxidants For extraction of total ascorbate,  C. vulgaris  cells were harvestedby fi ltrationandquickly homogenized inliquidN 2 and5%(w/v)TCA[28].Thehomogenatewascentrifugedfor5minat15,600g(4  C)andthe supernatant was assayed for the ascorbate content in a reactionmixturewith10mMDTT,0.2Mphosphatebuffer(pH7.4),0.5%NEM,10%TCA,42%H 3 PO 4 ,4%2,2 0 -dipyridyl,and3%FeCl 3 .Determinationof glutathione was essentially as described [29]. Glutathione wasextracted from algal cells in extracting buffer (2% sulfosalicylic acid,1 mM Na 2 EDTA, and 0.15% ascorbate) and homogenized. Thehomogenate was centrifuged at 12,000 g for 5 min. An aliquot of supernatant was then used for the measurement of the glutathionecontent byglutathione assay kit (Sigma e Aldrich Co., USA).  2.6. Determination of the antioxidant enzymes activities The antioxidant enzymes were extracted in 50 mM phosphatebuffer, pH 7.0, containing 1 mM EDTA, 0.05% (v/v) Triton X-100, 2%(w/v) PVP, and 1 mM ascorbic acid. Superoxide dismutase (SOD)activity of   C. vulgaris  was determined by measuring the inhibitionof photochemical reduction of NBT at 560 nm as suggested byBeauchamp and Fridovich [30]. One unit of SOD (per mg protein)was de fi ned as the amount causing 50% inhibition of the photo-chemicalreductionofNBT.Catalase(CAT)activitywasestimatedbyrecording the decrease in absorbance of H 2 O 2  at 240 nm [31]. Oneunit of CATactivity (U) was assumed as the amount of enzyme thatdecomposes1 m molofH 2 O 2 permgofsolubleproteinperminuteat30   C. The method given by Nakano and Asada [32] was followedfor determining ascorbate peroxidase (APX) activity of   C. vulgaris .The enzyme activity (U) was calculated as the amount of theenzyme that oxidizes 1  m mol of ascorbate consumed per mg of soluble protein per min at 30   C.  2.7. Replication and statistical analysis Eachtreatmentconsistedof4replicatesandeachexperimentwascarriedoutatleasttwiceatdifferenttimes.Thedatawereanalyzedbyone-wayanalysisofvariance(ANOVA)andthemeanswereseparatedusing Duncan ’ s multiple-range test (Statistica 6, StatSoft, USA). Thelevel of signi fi cance in all comparisons was  p < 0.05. 3. Results  3.1. The number of cells The decrease in cell number by 21%, 36% and 53% was observedin response to Cu, Pb and Cd, respectively (Fig.1). Cytokinins mixedwith heavy metals induced the highest increase in the cell number.During stress conditions,  C. vulgaris  exhibited sensitivity on cyto-kinins in the following order of their stimulating properties:DPU  >  CPPU  >  TDZ  >  Z  >  BA  >  Kin > 2iP. Auxins (IAA, NAA, PAA,IBA), displayed weaker biological activity under stress conditions. Fig. 3.  The effect of cytokinins (BA, Z, Kin, 2iP, DPU, CPPU, TDZ), auxins (IAA, NAA, PAA, IBA), gibberellin (GA 3 ), jasmonic acid (JA) and spermidine (Spd) in combination with heavymetals (Cd, Cu, Pb) on the chlorophyll  a  content in  C. vulgaris  cells. Data are the means of four independent experiments  SD. Treatment with at least one letter the same are notsigni fi cantly different according to Duncan ’ s test.  A. Piotrowska-Niczyporuk et al. / Plant Physiology and Biochemistry 52 (2012) 52 e 65  55  GA 3  in combinations with heavy metals increased cell number by7 e 21%. The stimulatory effect on the growth was also observed incase of Spd in combination with heavy metals in relation to theculture treated with heavy metals solely. On the other hand, JAstrongly enhanced the inhibitory in fl uence of the heavy metal onalgal growth leading to 23 e 60% decrease in the cell number.  3.2. The accumulation of heavy metals in the cells of C. vulgaris Thebioaccumulationofheavymetalsin C.vulgaris cellsisshowninTable1.PhytohormonesandSpdcanmodifythebiosorptionofCd,Cu and Pb from the medium. The highest inhibitoryeffect on heavymetals ’  accumulation was observed in algal cultures treated withcytokinins,especiallyDPU.Amongauxins,IAAwascharacterizedbythehighestinhibitoryeffectonmetalbiosorption.Theapplicationof GA 3 hadalsonegativeeffectonmetalbiosorptionbyalgalcellsfromnutrient medium. The inhibitoryeffect of phytohormones on metalbioaccumulation was arranged in the following order: DPU  > CPPU  >  TDZ  >  Z > GA 3 > BA  >  Kin > 2iP  >  IAA  >  NAA  >  PAA > IBA.InhibitorypropertieswerealsoobservedincaseofSpdtreatment.Incontrast, JA stimulated heavy metals ’  uptake.  3.3. Protein content in C. vulgaris cells Heavymetalsstressdeclinedproteincontentinalgalculturesby14 e 51% (Fig. 2).  C. vulgaris  cells treated with heavy metals andcytokinins were characterized by 11 e 85% restoration of proteincontent. The inhibitory effect was also suppressed by the coappli-cationofauxins,especiallyIAA(5 e 8%).ThecombinationofGA 3 andheavy metals stimulated 6 e 14% increase in protein level. Spdshowed the lowest biological activity in algal cells exposed to Cd, Fig. 4.  The effect of cytokinins (BA, Z, Kin, 2iP, DPU, CPPU, TDZ), auxins (IAA, NAA, PAA, IBA), gibberellin (GA 3 ), jasmonic acid (JA) and spermidine (Spd) in combination with heavymetals (Cd, Cu, Pb) on the chlorophyll  b  content in  C. vulgaris  cells. Data are the means of four independent experiments  SD. Treatment with at least one letter the same are notsigni fi cantly different according to Duncan ’ s test.  Table 1 Theeffectofcytokinins(BA,  Z  ,Kin,2iP,DPU,CPPU,TDZ),auxins(IAA,NAA,PAA, IBA),gibberellin (GA 3 ), jasmonic acid (JA) and spermidine (Spd) in combination with Cd,Cu or Pb on the heavy metal content in  C. vulgaris . Data are the means of fourindependentexperiments  SD.Treatmentwithatleastoneletter(a e  j)thesamearenot signi fi cantly different according to Duncan ’ s test.Heavy metals,phytohormonesand polyamineCd (fg cell  1 ) Cu (fg cell  1 ) Pb (fg cell  1 )Heavy metals 2213.6 (  5.6) a 1013.5 (  10.9) b 1645.9 (  16.6) c BA þ heavy metals 1988.7 (  11.3) d 599.9 (  11.1) e 1176.9 (  10.1) b Z þ heavy metals 1795.1 (  10.3) c 588.3 (  8.1) e 1048.5 (  9.9) b Kin þ heavy metals 2015.8 (  10.9) a 368.9 (  8.3) e 1400.6 (  14.5) c 2iP þ heavy metals 2097.5 (  15.1) a 799.8 (  7.7) e 1556.8 (  13.7) c DPU þ heavy metals 1427.1 (  14.6) c 603.8 (  12.5) e 827.5 (  9.5) e CPPU þ heavy metals 1559.9 (  5.8) c 550.2 (  5.9) e 997.4 (  8.2) e TDZ þ heavy metals 1677.3 (  13.4) c 677.5 (  7.9) e 1169.1 (  14.9) b IAA þ heavy metals 2124.8 (  9.3) a 834.1 (  11.5) e 1436.8 (  16.2) c NAA þ heavy metals 2270.1 (  11.5) a 947.9 (  11.3) e 1640.2 (  9.8) c PAA þ heavy metals 2304.8 (  20.8) f  1045.4 (  18.7) b 1660.8 (  19.3) c IBA þ heavy metals 2500.6 (  19.9) g 1123.1 (  14.5) b 1845.5 (  20.1) d GA 3  þ heavy metals 1920.5 (  19.7) d 728.6 (  12.1) e 1195.1 (  11.1) b  JA þ heavy metals 3801.7 (  13.9) i 1344.7 (  13.4) c 2107.4 (  20.9) a Spd þ heavy metals 2257.6 (  8.7) a 1098.8 (  9.3) b 1499.6 (  15.5) c Control 0  j 0  j 0  j  A. Piotrowska-Niczyporuk et al. / Plant Physiology and Biochemistry 52 (2012) 52 e 65 56
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