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Proteomics combines morphological, physiological andbiochemical attributes to unravel the survival strategy ofAnabaena sp. PCC7120 under arsenic stress

Sarita Pandeya, Rashmi Raib, Lal Chand Raia,⁎aMolecular Biology Section, Laboratory of Algal Biology, Center of Advanced Study in Botany, Banaras Hindu University,Varanasi-221005, IndiabLaboratory of Morphogenesis, Center of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India

A R T I C L E I N F O

Abbreviations: PGK, phosphoglycerate kinasPPP, pentose phosphate pathway; CAT, catalase⁎ Corresponding author. Tel.: +91 542 6701110

E-mail addresses: lcrbhu15@gmail.com, lc

1874-3919/$ – see front matter © 2011 Elseviedoi:10.1016/j.jprot.2011.10.011

A B S T R A C T

Article history:Received 2 July 2011Accepted 17 October 2011Available online 25 October 2011

Proteomics in conjunction with morphological, physiological and biochemical variables hasbeen employed for the first time to unravel survival strategies of the diazotrophiccyanobacterium Anabaena sp. PCC7120 under Arsenic (As) stress. Significant reduction ingrowth, carbon fixation, nitrogenase activity and chlorophyll content after 1 day (1 d) andrecovery after 15 days (15 d) of As exposure indicates the acclimation of the test organismagainst As stress. The formation of akinete like structures is a novel observation neverreported before inAnabaena sp. PCC7120. Proteomic characterization using 2-DE showed aver-age 537, 422 and 439 spots in control, 1 and 15 d treatment respectively.MALDI-TOF and LC-MSof As-treated Anabaena revealed a total of 45 differentially expressed proteins, of which 13were novel (hypothetical) ones. Down-regulation of phosphoglycerate kinase (PGK), fructosebisphosphate aldolase II (FBA II), fructose 1,6 bisphosphatase (FBPase), transketolase (TK),and ATP synthase on day 1 and their significant recovery on the 15th day presumably main-tained the glycolysis, pentose phosphate pathway (PPP) and turnover rate of Calvin cycle,hence survival of the test organism.Up-regulation of catalase (CAT), peroxiredoxin (Prx), thior-edoxin (Trx) and oxidoreductase appears to protect the cells fromoxidative stress. Appreciableinduction in phytochelatin content (2.4 fold), GST activity (2.3 fold), and transcripts of phyto-chelatin synthase (5.0 fold), arsenate reductase (8.5 fold) and arsenite efflux genes — asr1102(5.0 fold), alr1097 (4.7 fold) reiterates their role in As sequestration and shielding of the organ-ism fromAs toxicity.While up-regulatedmetabolic and antioxidative defense proteins, phyto-chelatin and GST work synchronously, the ars genes play a central role in detoxification andsurvival ofAnabaena under As stress. The proposed hypotheticalmodel explains the interactionof metabolic proteins associated with the survival of Anabaena sp. PCC7120 under As stress.

© 2011 Elsevier B.V. All rights reserved.

Keywords:ArsenicAnabaena sp. PCC71202-DEMALDI-TOF/MSRT-PCRWestern blotting

1. Introduction

India is one of the largest countries of the world requiring ahuge amount of food production in limited agricultural land

e; FBA II, fructose bisphosph; Prx, peroxiredoxin; Trx, th; fax: +91 542 2368174.rai@bhu.ac.in (L.C. Rai).

r B.V. All rights reserved.

to feed its gigantic population. According to the estimates,the total rice production in India is about 148,260,000 metrictons, bringing the country second only to China (193,354,175metric tons) in world rice production [1]. Unfortunately, abiot-

ate aldolase II; FBPase, fructose 1,6 bisphosphatase; TK, transketolase;ioredoxin; pcs, phytochelatin synthase; GST, glutathione S-transferase

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ic stresses like salinity, drought, heat, pesticides, and heavymetals are negatively affecting crop productivity. Neverthe-less, the increasing groundwater contamination by arsenicand its use for irrigation as well as excessive use of chemicalfertilizers and metal-containing pesticides are not only jeop-ardizing the survival of microbial communities including cya-nobacteria inhabiting therein but leaving the soil still worstfor cultivation especially in South East Asian countries [2,3].

Cyanobacteria, the photosynthetic prokaryotes, contributesubstantially to the nitrogen economy and constitute a promi-nent component ofmicrobial population inwetland soils, espe-cially rice paddy fields [4] and play a vital role in themaintenance of soil fertility and rice productivity. The long evo-lutionaryhistory, 3.5 billion years, of cyanobacteria is a testimo-ny to their adaptive capability against environmental vagariesthereby contemplating them as an excellent model to studystress responses. Although a large number of cyanobacteria in-cluding species of Anabaena are known to fix nitrogen, the testcyanobacterium Anabaena sp. PCC7120 (hereafter Anabaena), isinteresting because of the availability of its full genome se-quence and tolerance to environmental stresses [5].

Cyanobacterial adaptation to stress is coupled with pro-found changes in proteome repertoire. Since proteins are di-rectly involved in stress responses, proteomic studies canunravel the possible relationships between protein abun-dance and stress acclimation. Over the past years, the proteo-mic approach has been applied to analyze the proteinsinvolved in stress responses in cyanobacteria, such as Cu [6],salinity [7,8], heat and UV-B [9–11], butachlor [12], and Fe star-vation [13]. However, As-induced proteomic changes in cya-nobacteria have remained unexplored as yet. Information onAs toxicity in cyanobacteria are confined to phosphate arse-nate interaction in Synechococcus leopoliensis, Synechococcus sp.[14,15], Anabaena variabilis [16], Anabaena PCC7120 [17], bioac-cumulation of As in Phormidium sp., Phormidium laminosum[18–20], and antioxidative enzymes of Anabaena doliolum [21],Phormidium laminosum [20], and the role of arsenic resistancegenes (ars operon) in Synechocystis [22].

Arsenic adversely affects the metabolic processes andleads to (i) oxidative stress through ROS generation therebydamaging proteins, lipids and nucleic acids [23,24], (ii) inhibi-tion of RubisCO [25], chlorophyll biosynthesis, photosyntheticpigments [21,26], photosynthesis [27], and (iii) inhibition ofATP [28] by uncoupling phosphorylation. Arsenic stressed or-ganisms have been reported to adopt to one of the followingstrategies: (i) competitive inhibition of arsenate uptake byphosphate [15], (ii) over induction of GSTs [29], (iii) biotransfor-mation of arsenic species [30], (iv) GSH and phytochelatin (PC)based sequestration of As [31–33], and (v) reduction of arse-nate into arsenite and its efflux from the cell through arsCand arsB genes respectively [34].

The information available on the ars genes of bacteria [34],yeast [35] and plants [36,37] are interesting but quite divergent.The ars genes in bacteria are in the form of an ars operon pre-sent both on the chromosome as well as plasmid. The ars oper-on is constituted by arsR, -B, and -C encoding a trans-actingrepressor, an arsenite efflux protein and an arsenate reductaserespectively. Two additional genes arsA and arsD encoding anarsenite-stimulated ATPase and a metalloid-responsive tran-scriptional repressor are also known. Notwithstanding above,

the ars genes of Synechocystis [22] are in the formof arsBHC oper-on containing three genes: arsB (arsenite efflux protein), arsHunknown protein and arsC (arsenate reductase) regulated bythe transcriptional repressor arsR. In silico analysis of the Ana-baena genome depicted the presence of a cluster of 4 genes(http://genome.kazusa.or.jp/cyanobase/Anabaena): asr1102(homologue of arsB), all1103 (transcriptional regulator arsR),alr1104 (hypothetical protein), and alr1105 (arsenate reductase);while three genes are arranged in one orientation, all1103 is di-vergently arranged, hence their operonic structure is differentfrom other ars operons. In addition to above an arsB homologuealr1097 (multidrug efflux protein) conceivably involved in Asstress management is also present upstream to asr1102. It isworthmentioning that apart from alr1103, 8 transcriptional reg-ulators (alr2766, alr1867, alr1044, alr0831, all5056, all3903, all3743,and all7621) belonging to theArsR family having 50.6–67.3% sim-ilarity with the arsR (sll1957) of Synechocystis are also present inthe genome of Anabaena. Furthermore, the catalytic residuesof arsenate reductase from Anabaena (CKHNSRRS) differ in re-spect to some amino acids from Staphylococcus (CTGNSCRS)and Synechocystis (CKRNSCRS).

In view of the above themajor gaps identified in relation tocyanobacteria and As stress include: (i) As toxicity has neverbeen addressed in N2-fixing cyanobacteria employing proteo-mics along with morphological, physiological, and biochemi-cal variables, (ii) complete lack of data on the role of ars andother genes like pcs, thioredoxin, peroxiredoxin, gst of Ana-baena in As toxicity management. The present study thereforecombines proteomics in tandem with morphological, physio-logical, and biochemical attributes, and selected stress re-sponsive genes to decipher the mechanism of As toxicity indiazotrophic cyanobacteria.

2. Materials and methods

2.1. Experimental setup and growth measurement

Anabaena was cultured in BG-11 medium [38] buffered withHEPES (0.5 g L−1) at 24±2 °C under 72 μmol photon m−2 s−1 PAR(photosynthetically active radiation) with a photoperiod of14:10 h (light:dark) at pH 7.5. A 40mMof As (Na2HAsO4·7H2O) se-lected for the experimentwas LC50 concentration, as determinedby the plate colony count method of Rai and Raizada [39]. Timedurations selected for this work were 1 d (short term) and 15 d(long term). Exponentially growing cells of Anabaena were trea-ted with 40mM As and the growth estimated by measuring theOD (optical density) of the culture at 750 nm inaUV–VIS spectro-photometer (Systronics, India) on every third day up to the 15thday. Chlorophyll a and phycocyanin were measured as per themethod of Bennett and Bogorad [40] by taking the absorbanceat 663 and 645 nm respectively. Microphotographs of periodic-schiff reagent stained [41] and unstained cells were taken byphase contrast microscope (Nikon E800). All the experimentswere done with three biological replicates.

2.2. Arsenic concentration measurement

Oven-dried cyanobacterial pellet (50 mg) was digested in 3:1HNO3 and H2O2 [42]. Total As was estimated by atomic

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absorption spectrophotometer (Perkin–Elmer; A Analyst 600)fitted with graphite furnace.

2.3. Biochemical assays

Oxidative damage of lipid was measured in terms of the totalcontent of 2-thiobarbituric acid reactive substances (TBA-RS)and expressed as equivalent of MDA (malondialdehyde)using themethod of Cakmak andHorst [43]. The total peroxidewas measured according to Sagisaka [44]. Superoxide radicalwas estimated by the method of Chaitanya and Naithani [45].For assay of superoxide dismutase (SOD), catalase (CAT), gluta-thione reductase (GR) and glutathione S-transferase (GST) cellextract was prepared by sonication in lysis buffer under icecold condition. The sonicated sample was centrifuged at15,000×g for 30 min at 4 °C and the resulting supernatantwas used for the assay of the antioxidant enzymes. SOD activ-ity was assayed by monitoring the inhibition of reduction ofNBT according to the method of Gianopolitis and Ries [46].CAT activity was determined by the method of Aebi [47].Enzyme activity was calculated from the initial rate ofthe enzyme using the extinction coefficient of H2O2 of40 mM−1 cm−1 at 240 nm. GR activity was assayed by followingNADPH oxidation at 340 nm in a UV-spectrophotometer as perthemethod of Schaedle and Bassham [48]. Activity of GST wasmeasured by themethod of Habig and Jacoby [49] by taking ab-sorbance at 340 nm for 6 min at 37 °C. Arsenate reductaseassay was done by the method of Anderson et al. [50]. Activitywas monitored by measuring the decrease in NADPH absor-bance at 340 nm and the NADPH oxidation was calculatedusing a molar extinction coefficient of 6200 M−1 cm−1. Prolinecontent of the cell pellet was measured following the methodof Bates et al. [51]. Total soluble protein was measured by themethod of Bradford [52]. GSH was estimated by 5,5′dithiobis-(2-nitrobenzoic acid) (DTNB)-glutathione reductase coupledassay as described by Anderson [53]. Total thiol content wasestimated according to themethod of Ellman [54]. Phytochela-tin content was measured according to Bhargava et al. [55].

2.4. Measurement of physiological parameters

Carbon fixation was measured by recording the uptake of 14Cfrom NaH14CO3 (sp. activity 18.59105 Bq) as described by Raiand Raizada [39].The rate of respiration was determined bymeasuring the total O2 consumed in the dark for a giventime period minus nonspecific O2 uptake [56]. The size of theATP pool was measured by the method as described by Lars-son and Olsson [57]. NADPH/NADH level of the cell extract inTris–Cl (pH 8.0) was measured by recording absorbance at340 nm [58]. Electron transport activities were measuredaccording to Tripathy and Mohanty [59]. The nitrogenase ac-tivity of the test organism was determined by an acetylene re-duction assay [60].

2.5. Protein extraction and 2-DE separation

Protein extraction was performed according to amodified pro-tocol of Wagner et al. [61]. Anabaena cells harvested by centri-fugation (10,000×g, 10 min.), suspended in 2 ml extractionbuffer containing 10 mM Tris–HCl (pH 8.0), 1.5 mM MgCl2 and

10 mM KCl, were ground under liquid nitrogen followed bycentrifugation at 12,000×g for 1 h. The supernatant was pre-cipitated with 10% TCA in acetone, left overnight and centri-fuged at 6000×g for 15 min. To recover protein pellet waswashed 3 to 4 times with ice cold acetone. The air dried pelletwas dissolved in solubilization buffer containing 7 M urea, 2 Mthiourea, 4% CHAPS, 40 mM DTT and 1.0% IPG buffer (4–7).Traces of bromophenol blue were added and centrifuged at19,000×g for 10 min. Sample entry was made through in-gelrehydration. A total of 300 μl of solubilization buffer contain-ing 250 μg protein sample was incubated with the dry IPGstrips (pH 4–7; 13 cm; GE healthcare, USA) at 20 °C for 16 h.The first dimension separation was conducted at 20 °C withan Ettan IPGphor system (GE Healthcare BIO-Science, USA).Focusing was performed in 7 steps: linear 30 V for 00.30 h,150 V for 2:00 h, 300 V for 00:40 h, 500 V for 4 h, gradient1000 V for 1 h, gradient 8000 V for 2 h and finally 8000 V for13:00 h. Focused IPG strips were then equilibrated by first in-cubating them in an equilibration solution (6 M urea, 30%glycerol, 2% SDS, 50 mM Tris–HCl, pH 8.8, and 1% DTT and atrace amount of bromophenol blue) for 15 min, followed by in-cubation in 2.5% iodoacetamide in the same equilibration so-lution instead of 1% DTT for 15 min. The strip was placed onthe top of 12.5% SDS-PAGE and sealed with 0.5% agarose. Elec-trophoresis was carried out at 10 mA/gel for 30 min, then25 mA/gel for 5 h using a Hoefer SE 600 apparatus (AmershamBiosciences, USA). The gel was stained with CBB R-250.

2.6. Analysis of data

The gels were scanned and protein spots were analyzed byusing PDQuest software version 7.1 (Bio-Rad). Three replicatesof CBB stained gels from three independent biological sampleswere used for analysis. After background subtraction and spotdetection, spots were matched. Spot intensities were calculat-ed and normalized by determining the relative intensity ofeach spot. Only spots with statistically significant (DMRT,p<0.05) and reproducible changes were considered, and theprotein spots with an abundance ratio of at least 1.5 foldwere selected as differentially expressed proteins. Forty fiveprotein spots showing significant and reproducible changeswere selected and subjected to MALDI-TOF and LC-MS ana-lyses followed by homology search using MASCOT on com-mercial basis from The Center for Genomic Application(TCGA), New Delhi, India. The protein databases employedwere, MSDB (The Mass Spectometry Protein Sequence Data-base) and NCBInr (The National Center for Biotechnology In-formation non-redundant). In brief, 1 μl of trypsinizedpeptide samples were mixed with matrix, namely α-cyano-4-hydroxy cinnamic acid (CHCA). Following drying, the peptideswere spotted on ground steel plate and subjected to BrukerUltra flex MALDI-TOF/TOF and 2D Nano LC-ESI-Trap (Agilent)for mass spectrometric identification. Data acquisition andanalysis were performed using flex control and flex analysis/biotools version 2.2 software, respectively (Bruker Daltonics).All spectra were smoothened, and all peptidemass fingerprintspectra were internally calibrated. Then, the measured trypticpeptidemasses were transferred throughMS BioTool programas inputs to search against the taxonomy of Bacteria inthe protein databases using MASCOT 2.0 software (Matrix

924 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 9 2 1 – 9 3 7

Science). The Peptide mass fingerprinting (PMF) and MS/MSion search were searched with the following MASCOT set-tings: taxonomy as Bacteria; peptide mass tolerance of±100 ppm for peptide mass fingerprinting and ±1.2 Da forMS/MS, monoisotopic mass, alkylation of cysteine by carba-midomethylation as a fixed modification, and oxidation ofmethionine as a variable modification. True probabilitybased scoring is the key to recognizing and avoiding false pos-itives. MASCOT software provides probability based scoring.For peptide mass fingerprinting and MS/MS ion search, statis-tically significant (p<0.05) matches were regarded as correcthits. The matches having a Mascot score higher than thethreshold score (51–74 in MSDB and 56–80 in NCBI databasesearches) are the selected proteins in this study. Further toavoid false positives, an additional in-house BLAST search atNCBI (http://www.ncbi.nlm.nih.gov) was done to reconfirmall the matches.

2.7. Total RNA isolation and primer design

Cyanobacterial pellet was ground in liquid nitrogen and RNAextracted in TRIzol reagent (Invitrogen Inc., CA, USA). Thegene specific primers were designed using the PRIMER 3 soft-ware. The sequences of the primers are given in (Supplemen-tary Table S1). RT-PCR of these genes was done as perBhargava et al. [6]. The intensities of the RT-PCR products onagarose gels were quantified with the Gel Doc 2000 systemusing Quantity one software (BioRad, USA).

2.8. Western blot analysis

SDS-PAGE was performed according to the method of Sam-brook and Russell [62]. 20 μg protein was separated by SDS-PAGE and electroblotted onto a PVDF membrane (MilliporeImmobilon-P) using a dual mini-electroblot system (PrecisionInstruments, Varanasi, India) as described previously [63].The immunoblots were photographed using a gel documenta-tion system (Bio-Rad, USA) and their quantitative densitomet-ric analysis was performed using the Quantity One Software(Bio-Rad, USA).

2.9. Statistical analysis

Values in figures and tables were expressed as means±SD.Statistical analysis was carried out with three biological repli-cates for proteomic and physiological analyses. The results ofthe spot intensities and physiological data were statisticallyanalyzed by one-way ANOVA and the Duncan's new multiplerange test (DMRT) to determine the significant differenceamong group means. A p value ≤0.05 was considered statisti-cally significant (SPSS for Windows, version 12.0).

3. Results and discussion

3.1. Growth behavior and intracellular As

In order to investigate how Anabaena responds to As stress, itsgrowth performance was examined in As supplemented cul-ture medium (Fig. 1A). Approximately 43% inhibition of

growth at 40 mM As after 24 h of treatment, demonstrates itshigh sensitivity to As stress. However, it showed an increaseon successive days attaining 68.7% recovery after 15 d of treat-ment as compared to the control. Interestingly, the intracellu-lar As that was 36% of the total As supplemented in themedium on day 1 subsequently dropped down to 23% on day7 and 6% on day 15, thus indicating its efflux from the cell.This was supported by data on an increase in As content inthe culture medium on day 7 and 15 (Fig. 1B). This decreasein intracellular As was vividly reflected in terms of the growthrecovery of the Anabaena (Fig. 1A). Arsenic produced a declineof 36.1% and 33.3% in chlorophyll a (Fig. 1C) and phycocyanin(Fig. 1D) contents respectively over the control on day 1. As-induced reduction in chlorophyll content may be due to theinhibition of δ-aminolevulinic acid dehydrogenase and proto-chlorophyllide reductase [64] as also known for A. doliolum[21], maize [65], red clover [26] and Pteris [66].

3.2. Morphological and physiological responses ofAnabaena to As stress

Survival under stress requires metabolic adjustments and ac-climation where morphological alterations could be the firstand the foremost visible response. To examine morphologicalchanges, if any in As-treated Anabaena, the phase contrast mi-croscopy of periodic Schiff stained untreated control and As-treated cells was carried out [41]. Arsenic treatment within48–72 h produced filament fragmentation, thickening, en-largement and vacuolation of cells, transformation of bluishgreen cells into yellow brown and dense pigmentation at oneside of the cell (Supplementary Fig. S1B, C). This increase incell size may be due to accumulation of proline to thwart As-induced osmotic imbalance. One of the most interesting ob-servations was the appearance of akinete like structures (Sup-plementary Fig. S1D) never reported before in Anabaena [38].This might be due to a 2.8 fold induction of the hypotheticalprotein alr4050 having 98% similarity with the akinete markerprotein of A. variabilis [67]. Thus As appears to generate astress that triggers the induction of akinete like structures inAnabaena. These morphological alterations were retained upto 7 days where after the akinete frequency went down andthe Anabaena cells resumed the shape of untreated controlcells (Supplementary Fig. S1A) thus indicating recovery fromAs stress (Supplementary Fig. S1D).

Table 1 compiles data on the PSI, PSII, whole chain, 14C up-take, NADPH and ATP contents and nitrogenase activity overtwo different time points, 1 d and 15 d. A significant inhibitionof all the physiological parameters on day1 (DMRT, p<0.05)followed by their recovery on day 15 was observed. PSI, PSII,whole chain, 14C uptake, ATP and NADPH contents and nitro-genase activity decreased to the tune of 19.3%, 32.4%, 36.5%,35.2%, 30.9%, 16.7%, and 92.8% respectively after 1 d As treat-ment thereby depicting their propensity to As stress. The low-ered PSII activity was manifested in the form of loweredcarbon fixation and ATP content leading to low energy stateof the cell. The decrease in carbon fixation could be attributedto As-induced down regulation of rubisco subunits [25]. Con-trary to 1 d treatment the PSI, PSII, whole chain, 14C uptake,ATP content, and nitrogenase activity of Anabaena treatedwith As for 15 d showed only 12.2%, 14.6%, 18.4%, 20.7%,

Fig. 1 – (A) Growth behavior of Anabaena in terms of absorbance at 750 nm; lines with solid circle control and lines withhollow circle, 40 mM As-treated control, (B) percentage of As inside the cell and in the culture medium, (C) chlorophyll a and(D) phycocyanin content at different days in control and 40 mM As-treated Anabaena. Bars indicate±SD.

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21.7% and 68.9% inhibition respectively thereby indicatingtheir recovery fromAs stress. Contrary to this the NADPH con-tent showed a significant increase of 25% over the untreatedcontrol cells. Appreciable increase in NADPH seems justifiedin view of its requirement by cellular enzymes during Asstress. Likewise the respiration registered approximately24.3% and 33.5% increase over the control on day 1 and 15 re-spectively. An escalated respiration may be a requirement ofthe cell to meet the energetic cost associated with the repairof cellular damage. However, an increased respiration and de-creased ATP pool on day 1 may be due to (i) high requirement

Table 1 – Physiological parameters of Anabaena sp. PCC 7120 un

Physiological parameters Contro

PS-I activity (μmol O2 consumed μg−1 protein h−1) 13.45±0.0PS-II activity (μmol O2 evolved μg protein−1 h−1) 9.53±0.1Whole chain (μmol O2 evolved μg protein−1 h−1) 19.04±0.014C uptake (CPM×103) 40.62±1.0μg ATP mg protein−1 9.69±0.1mM NADPH mg protein−1 0.12±0.0Respiration (μM O2 consumed mg protein−1 min−1) 8.27±0.0Nitrogenase activity (nmol C2H2 mg protein−1 h−1) 118.6±1.1

All values are mean±SD. Values with (+ and − signs) in parenthesis indicaDifferent alphabets show significantly different values (P<0.05, DMRT).

of ATP for cell repair and maintenance, and (ii) sensitivity ofthe ATP synthase complex to As [24].

3.3. Biochemical responses of Anabaena to As stress

Despite being a non-redox-active metalloid, intra-conversionof As from one ionic form to another, generates ROS thuscausing oxidative stress [68]. One major consequence of oxi-dative stress is lipid peroxidation [21,69,70] measured interms of MDA and peroxide contents. Compared to the con-trol, As produced 1.81 fold increase in MDA and 4.7 fold in

der 40 mM sodium arsenate treatment.

l 1 day 15 days

9a 10.86±0.11c (−19.3) 11.8±0.10b (−12.2)1a 6.44±0.12c (−32.4) 8.13±0.02b (−14.6)1a 12.09±0.015c (−36.5) 15.53±0.100b (−18.4)5a 26.31±.95c (−35.2) 32.21±0.10c (−20.7)3a 6.69±0.11c (−30.9) 7.58±0.01b (−21.7)1b 0.1±0.01c (−16.7) 0.15±0.01a (+25)2c 10.28±0.02b (+24.3) 11.04±0.02a (+33.5)0a 8.48±0.10c (−92.8) 36.8±1.1b (−68.9)

te percent increase or decrease, respectively, as compared to control.

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peroxide contents (Fig. 2A, B) on day 1, which subsequentlydecreased overtime thereby indicating alleviation of oxidativestress. To examine if this is due to upregulation of differentstress coping enzymes the activities of SOD, CAT, GST andGR were measured. SOD showed about 1.3 fold inhibitionafter 15 d (Fig. 2D). Insignificant superoxide anion formation(Fig. 2C) in one hand and increased H2O2 content on theother indicates photorespiration to be another source ofH2O2 production. A 3.5 fold increase in CAT activity on day 1(Fig. 2E) is attributed to its involvement in detoxification ofH2O2 which is in agreement with the results of Pteris [71]. Pro-line showed 2.9 fold induction (Fig. 2F) in content suggestingits role in protection of cell from As stress [21].

Above observations were further attested by assay ofGSH, total thiol, phytochelatin (PC) contents and GR activity(Fig. 3A–D). A 3.1 fold increase in the activity of GR (Fig. 3D)under As stress may help in regeneration of GSH from GSSGthereby restoring the GSH pool of the cell. Increased GR activ-ity has been found to enhance Cd tolerance in Brassica juncea[72], and As in rice [69]. Contrary to the known increase inthe level of GSH, a 1.7 fold decline in its pool was noticed[Fig. 3B]. Notwithstanding above, 1.48 and 2.27 fold increase

Fig. 2 – Effect of As on (A) lipid peroxidation, (B) hydrogen peroxidactivity, and (F) proline content. Bars indicate±SD.

in total thiol and phytochelatin contents respectively were ob-served after 15 d (Fig. 3A, C). GSH being precursor of PC mighthave been consumed during PC synthesis, hence decrease inits level [32] is quite expected. Phytochelatins possess stron-ger ROS scavenging ability than the GSH [73]. Increased phyto-cheltin content vis-à-vis phytochelatin synthase (encoded byalr0975) activity in the test organism was attested by a 5.0and 4.8 fold increase in transcript and immunoblot of phyto-chelatin synthase respectively (Fig. 4A, B). In view of our earli-er report on the role of pcs gene (alr0975) of Anabaena inconferring multiple stress tolerance in E. coli [74], its role incombating As stress appears quite logical.

Glutathione S-transferases are a pivotal component in thecell's defense system known to eliminate endogenous lipidand fatty acid peroxides thus alleviating oxidative stress andlipid peroxidation [75]. A 1.8 fold increase in enzymatic activ-ity (Fig. 4C) and 2.3 fold induction of GST as discernible fromWestern blotting on the 15th day (Fig. 4D) not only affirmedits protective role against As induced oxidative stress butwas supported by the reports of Ahsan et al. [69] and Nortonet al. [76]. Increased GST is also known to repair the As dam-aged proteins [77]; it shares around 25% sequence homology

e content, (C) superoxide radical content, (D) SOD, (E) catalase

Fig. 3 – Effect of As on (A) thiol, (B) GSH, (C) Phytochelatin contents, and (D) GR activity of As-treated Anabaena at different timepoints. Bars indicate±SD.

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with human GSTO1 and possesses a conserved region quitesimilar to the signature HC(X)5R motif, which is conserved inall the arsenate reductases investigated [78]. In view of this,it was hypothesized that GSTs might complement arsenatereductase under As stress. In silico analysis (http://www.ncbi.nlm.nih.gov/BLAST/) of the amino acid sequence of GSTshowed 45% similarity with the arsenate reductase of Ana-baena, thus its involvement in As detoxification is quitecircumspect.

3.4. MALDI-TOF MS and LC-MS identification andclassification of As-responsive proteins

To investigate the temporal changes in the protein profileduring As stress, 2-DE analysis of the soluble cytosolic pro-teins was carried out. An analysis of CBB R-250 stained 2-DEgels using PD Quest Version 7.1 software (Bio-Rad) showedan average of 537, 422 and 439 spots in control, 1 d and 15 dtreatment (Fig. 5). Supplementary Fig. S2A shows the Venn di-agram of the differentially expressed protein at different timepoints under As treatment. The overlap between a and b (a∩bzone) contains a set of 211 proteins altered after 1 d of Astreatment. Another subset as represented by b–a contains221 newly induced proteins following 1 d As-treatment. Ofthese, 130 showed transient appearance and 91 could endure15 d of As toxicity. Presumably, the former ones representearly stress responsive proteins whose abundance declinedas the stress conditions started stabilizing, while the latter

ones might be the acclimation proteins. Not surprisingly,these 91 proteins showed down regulation after 1 d followedby their recovery on the 15th day of As-treatment. These ob-servations imply that, instead of early damage these proteinscould manage the oxidative stress, thereby helping the organ-ism to adapt. Further, a third set of 184 proteins as repre-sented by (a∩b)–(a∩b∩c) were those induced only after 15 dof As treatment. These proteins are conceivably required tomaintain carbon pool, redox homeostasis and hence survivalof the test organism. Finally a fourth subset of 125 proteinsas represented by c–(a∩b) are those synthesized de novoafter 15 days of As treatment and presumed to offer toleranceto As stress.

Of the forty five differentially expressed protein spots se-lected for MALDI-TOF/MS and LC-MS analyses, 31 (above 1.5fold) represent the total number of upregulated proteins ob-served in the proteome of As-treated Anabaena sp, whereas14 proteins were arbitrarily selected from the category ofdown-regulated proteins (Table 2, Supplementary Figs. S3–S47). Examination of the electrophoretic pattern indicatedthat the inferred mass or iso-electric point values differeddue perhaps to post-translationalmodifications such as glyco-sylation, phosphorylation etc., or degradation leading tochange in the molecular weight and charge of proteins. Theproteins identified were classified into seven categories: ener-gy metabolism (photosynthesis, carbon fixation and respira-tion), ROS scavenging and defense, protein synthesis andfolding, amino acid metabolism, transcriptional regulators,

Fig. 4 – (A) RT-PCR of pcs gene and its relative intensity graph. 16S rRNA gene was used to check equal loading of RNA indifferent samples. M, marker; N, negative RT control; C, control; 1D, 7D, and 15D represents 1, 7, and 15 days of As treatment.(B) Western blot of PCS protein after treatment with 40 mM As at different time points. Protein separated by SDS-PAGE,electroblotted onto a PVDF membrane, and cross-reacted with anti-PCS antibody. Effect of As on (C) GST activity, (D) Westernblot of GST protein. Protein separated by SDS-PAGE, electroblotted onto a PVDF membrane and cross-reacted with anti-GSTantibody. C, control; 1D, 3D, 7D, and 15D represents 1, 3, 7, and 15 days of As treatment.

928 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 9 2 1 – 9 3 7

cofactor synthesis, and hypothetical proteins. The largestfunctional categorywas constituted of proteins (Supplementa-ry Fig. S2B) involved in energy metabolism (38%) followed byhypothetical proteins (32%). It is worthwhile to mention thatnovel (hypothetical) proteins with such a high number havenever been reported in proteomic data sets [24,25,69; Supple-mentary Table S2]. These proteins are likely to provide new in-sights about the defense of cyanobacteria from As stress.

3.4.1. Energy metabolism (photosynthesis, carbon fixationand respiration)Fig. 6 and Table 2 compile data on 11 downregulated proteinse.g. transketolase (TK; spot 2), fructose bis-phosphate aldolaseI (FBA I; spot 29), fructose bis-phosphatase (FBPase; spot 27),ATP synthase (spot 26), phosphoglycerate kinase (PGK; spot28, 32), phycocyanin α chain(spot 8), phycocyanin β chain(spot 38), Mn stabilizing protein (MSP; spot 21), δ-amino levuli-nic acid dehydratase (ALAD; spot 36), uroporphyrinogen dec-aroxylase (URO-D; spot 40) and the hypothetical proteinall3076 (similar to photosystem II oxygen-evolving complexprotein PsbP; spot 24) after 1 d of As treatment followed bytheir recovery on the subsequent days. This is an explicit evi-dence of the transient adaptive strategy of the test organismagainst As stress. TK is an amphibolic universally required en-zyme for PPP for NADPH production and Calvin cycle. Its down

regulation may be due to (i) high sensitivity to accumulatedH2O2 [79] in the cells after 1 d of As treatment (Fig. 3B), and(ii) competitive inhibition of TK catalyzed reactions by As[80]. Recovery of TK after 1 d, when H2O2 level went down, in-creased the metabolic flux through PPP generating glyceralde-hyde 3-phosphate (GAP) to be utilized in the glycolyticpathway for ATP generation.

FBA I (spots 29,35) and FBA II (spot 20) are important en-zymes of glycolysis and Calvin cycle. FBAI is functionally re-dundant and approximately 90% of the total activity isascribed to FBA II in the Calvin cycle of Synechocystis sp.PCC6803 [81]. FBA II reversibly catalyzes the conversion offructose 1, 6-bis phosphate (FBP) to GAP and dihydroxy ace-tone 3-phosphate (DHAP). During stress, GAP and FBP maybe converted to glucose 6-phosphate for reentry into the PPPfor NADPH synthesis. Furthermore, FBA can lead to RUBP re-generation [82]. In the light of the reports of Mishra et al. [63]one is tempted to presume that enhanced expression of FBAI and a 2.1 fold induction of FBA II, maintained the glycolysis,PPP and turnover rate of Calvin cycle. The latter two performmost of the carbohydrate metabolism in cyanobacteriawhich possess an incomplete Krebs cycle. FBPase increasesthe synthesis of fructose 6-phosphate and glucose. So theupregulation of FBPase also indicates improved carbon fixa-tion as compared to 1 d which is attested by an increased

Fig. 5 – The 2-DE images of cytosolic protein extracts fromAnabaena (A) control; (B) exposed to 40 mMAs for 1 and (C) 15 days. Allgels were run in triplicate. Proteins were extracted and separated by 2-DE and visualized by CBB staining. UntreatedAnabaenawas used as control. The protein (250 μg) was applied to pH 4–7 IPG dry strips with 12.5% linear vertical SDS-PAGE asthe second dimension. The arrows with numbers on 2-DE gel indicate the changed protein, which are further identified byMALDI-TOF and LC-MS. Themolecularmassmarker (Sigma) and pI are indicated on the left side and above the gels respectively.

929J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 9 2 1 – 9 3 7

carbon fixation, NADPH/NADH production and improved cellgrowth (Table 1).

Phosphoglycerate kinase is a monomeric two domain en-zyme involved in the Calvin cycle and energy conservingphase of glycolysis. PGK's primary function is to generateATP and increase the respiration rate under stressful condi-tion. Thus, the observed overexpression of PGK (spots 28,32)and ATP synthase (spot 26) may be responsible for enhancedATP synthesis required for energy and maintenance of theCalvin cycle. Two other up-regulated proteins ALAD andURO-D, members of the tetrapyrrole biosynthetic pathway, re-quired for chlorophyll biosynthesis were increased by 1.7 and1.9 fold respectively on 15 d. The induction of these proteinsclearly delineates the tolerance of the chlorophyll biosynthe-sis machinery as reflected by enhanced chlorophyll contentson 15th d (Fig. 1C) under As stress. This also finds supportfrom the induction of these two proteins in Spirulina upontemperature downshift [83].

Enhanced expression of the hypothetical protein all3076(spot 24), phycocyanin α chain (spots 8, 15,41,44), phycocyaninβ chain (spots 31,38) and Mn stabilizing protein precursor(spot 21) was observed after an initial As shock. Oxygen-evolving proteins and MSP play crucial roles in photosynthe-sis by controlling O2 evolution and maintaining the stability

of photosystem II (PSII) in cyanobacteria [84]. MSP stabilizesthe Mn cluster, modulates Ca2+ and Cl− requirement for O2

evolution [85] and allows the water-oxidizing center to oper-ate at its best efficiency [86].

3.4.2. ROS scavenging and defense enzymesAbiotic stresses induce production of ROS, which not onlydamage cellular compartments but act as a signaling mole-cule for stress [87]. Organisms regulate ROS level throughcomplex mechanisms such as scavenging through antioxi-dant proteins such as Mn-SOD, Fe-SOD, oxidoreductase,all3090/Mn containing catalase, peroxiredoxins, thioredoxinsand AhpC/TSA. Surprisingly, the down regulation of the activ-ities as well as abundance of both the isoforms of SODwas ob-served after the 1st and 15th days. The decline in SOD activitymay be due to its sensitivity to high H2O2 content in the cell[88]. Our results find support from the work of Abercrombieet al. [89] in Arabidopsis and Gunes et al. [90] in chickpea plantssubjected to As stress. The observed high H2O2 (Fig. 3B) may bedue to photorespiration where glycolate is converted intoglyoxylate with the aid of oxygenase [91]. This prompts us tobelieve operation of glycolate metabolism in As stressed Ana-baena (Fig. 8), as reported for A. doliolum [8] and AnabaenaPCC7120 [92] under salt stress.

Table 2 – Arsenic-induced differentially expressed proteins in Anabaena sp. PCC 7120 identified by MALDI-TOF/LC-MS.

Spots Identified Protein Mr (kDa)/pI Theor. k

Mr (kDa)/pI Exper. l

Accessionno. m

No. ofpeptidesmatched

Mowsescore

Expression n

C 1 d 15 d

Energy metabolism (photosynthesis, carbon fixation and respiration)2 Transketolase 72.311/5.9 65.9/6.1 AI2223 28 244 155.0±3.5a 66.0±4.3c 96.0±2.9b

8 Phycocyanin α chain 17.504/7.7 18/5.94 A29674 7 90 195.0±7.5a 96.0±4.6c 181.0±3.6b

15 Phycocyanin α chain 17.504/7.7 18/ 7.0 A29674 8 127 109±12c 328.0±15.8a 257.0±17.0b

41 Phycocyanin α chain 17.504/6.82 24/6.8 giI17228025 6 131 30.0±2.7c 39.0±3.2b 54.0±4.8a

44 Phycocyanin α chain 17.504/6.82 8/6.1 giI17228025 6 191 16.0±1.4c 50.0±4.8b 101.0±12.9a

31 Phycocyanin β chain 18.546/5.0 57/5.0 giI38894 1 63 21.0±3.7c 56.0±4.9b 107.0±9.3a

38 Phycocyanin β chain 18.546/5.0 35/5.8 giI38894 1 107 103.0±8.8b 80.0±2.3b 188.0±23.0a

20 Fructose bis phosphatealdolase II

38.764/5.4 36/4.1 AC2376 9 313 20.0±1.2c 25.2±1.9b 42.7±2.5a

29 Fructose bis phosphatealdolase I

38.76/5.4 37/5.56 giI17232055 12 77 260.0±14.2a 178.0±15.2c 214.0±17b

35 Fructose bis phosphatealdolase I

38.764/5.49 36/4.3 giI7387539 1 96 20.0±4.6c 54.0±3.9b 112.0±8.0a

21 Mn stabilizing proteinprecursor

29.848/4.94 30/4.78 AG2287 7 183 19.7±1.8b 14.3±1.6c 30.3±1.4a

26 ATP synthase subunit A 54.4/5.0 53/5.12 giI17227501 19 99 279.0±14.7a 155.0±18.2b 243.0±20.9a

27 Fructose 1,6-bisphosphatase II 37.05/4.9 45/5.12 giI17228536 17 93 211.0±13.7a 128.0±17.4c 168.0±15b

28 Phosphoglycerate kinase 42.5/5.0 38/5.2 giI17231623 15 120 148.0±10.3b 133.0±10.9b 217.0±15.8a

32 Phosphoglycerate kinase 42.5/5.0 37/4.2 giI17231623 8 389 99.0±8.3b 77.0±6.4c 144.0±8.0a

34 Delta aminolevulinic aciddehydratase

35.792/5.22 31/4.7 giI17231872 2 79 68.0±1.2b 53.0±3.8c 117.0±7.8a

40 Uroporphyrinogendecarboxylase

38.795/6.33 38/6.8 giI17231401 8 229 31.0±2.8b 34.0±3.6b 59.0±3.1a

ROS scavenging and defense5 AhpC/TSA family protein 23.7/5.0 28/5.4 AD2356 14 114 216.3±5.0a 177.0±5.9b 143.6±5.5c

6 Superoxide dismutase 30.6/6.3 30/6.41 AF1815 10 140 186.0±6.0a 119.3±5.5b 106.0±5.5c

7 Iron superoxide dismutase 22.4/5.2 25/5.58 AC2173 16 155 188.0±4.5a 105.0±5b 110.3±4.5b

12 Oxidoreductase 30.9/5.3 36/5.58 AF2453 15 126 173.0±17.0b 319.0±17.0a 192.0±21.0b

37 Peroxiredoxin 22.731/4.87 23/4.8 giI17232133 2 127 36.0±6.2c 71.0±7.7b 108.0±8.4a

45 Thioredoxin 11.827/5.23 10/5.3 giI17227548 27 412 59.0±4.3c 132.0±9.9b 166.0±11.0a

Protein synthesis and folding22 Translation elongation

factor Ts34.425/4.94 29/4.97 AG2404 5 189 141.0±17.0b 105.0±15.8b 472.0±21.7a

33 Elongation factor Tu 44.84/5.25 45/5.0 giI17231829 3 78 100.0±11.2b 62.0±8.8c 158.0±13.4a

1 DnaK type molecularchaperone

68.037/4.7 66.0/4.69 AH2023 28 201 232.0±6.0a 135.0±6.5c 208.0±6.0b

Amino acid metabolism4 Dihydropicolinate reductase 29.617/5.1 28/5.39 AG2123 14 100 215.0±5.0a 146.0±4.0c 181.7±5.5b

39 Cysteine synthase 34.035/6.0 30/6.0 giI13473875 7 127 40.0±5.4b 61.0±4.2a 69.0±7.3a

Transcriptional regulator17 Anti-sigma-factor antagonist

(STAS) domain protein.14.542/5.76 14/5.87 Q3M4S7

ANAVT11 197 6.9±0.8c 23.7±1.0a 10.3±1.3b

43 Transcriptional regulator 12.978/5.62 14/5.2 giI17229162 1 74 23.0±1.9c 52.0±6.9b 65.0±3.8a

Cofactor synthesis30 Pyridoxine 5′-phosphate

synthase OS26.087/5.3 28/5.52 PDXJ-ANASP 3 58 188.0±11.6a 111.0±16.8b 138.0±14.2b

Hypothetical proteins3 alr0803 43.571/5.6 43/5.7 AI1906 16 179 156.0±3.6b 228.0±4.0a 120.0±4.9c

42 alr0803 43.571/5.61 21/5.5 giI17228298 3 94 30.0±2.7b 52.0±6.4a 54.0±7.9a

9 alr0806 17.685/4.3 29/4.12 AD1907 11 112 191.0±7.2a 108.0±4.0c 163.0±3.0b

10 alr4050 37.237/5.0 32/5.2 AC23312 18 125 209.0±5.0c 589.3±5.5a 300.7±6.0b

11 alr3199 39.7 09/5.0 38/5.35 AH2205 27 221 201.3±10.0c 598.0±15.0a 424.7±17.0b

13 alr3090 25.649/5.1 27/5.27 AC2192 14 109 145.0±17.1b 338.0±22.5a 178.0±12.0b

14 all4894 14.572/6.7 24/5.63 AF2417 12 173 129.0±23.0b 260.0±27.0a 149.0±21.0b

16 all1009 14.167/9.4 14.2/6.95 AF1932 33 253 3.1±0.05b 34.1±2.0a 3.4±0.08b

18 alr4550 60.65/4.76 52/4.6 AF2374 12 469 4.0±0.7b 4.8±0.7b 44.5±0.7a

930 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 9 2 1 – 9 3 7

Table 2 (continued)

Spots Identified Protein Mr (kDa)/pI Theor. k

Mr (kDa)/pI Exper. l

Accessionno. m

No. ofpeptidesmatched

Mowsescore

Expression n

C 1 d 15 d

19 all4499 58.642/5.02 55/5.0 AC2368 4 91 49.0±3.9b 48.0±2.7b 201±5.9a

23 all1864 27.14/4.6 26/4.7 AB2039 9 109 76.0±4.9b 47.0±2.7c 154.0±2.1a

24 all3076 27.46/6.4 20/5.53 AE2190 9 122 37.2±1.9a 22.0±1.7b 34.1±2.1a

25 alr3904 25.898/5.5 29/5.7 AI2293 19 200 58.0±2.1a 32.0±2.6b 29.0±2.8b

36 alr4171 35.265/8.1 27/4.2 giI17231663 1 86 62.0±6.7b 80.0±3.6b 116.0±14.7a

k Theoretical mass (kDa) and pI of identified proteins.l Experimental mass (kDa) and pI of identified proteins.m Database accession number NCBInr and MSDB.n Relative abundance of protein spots of Anabaena following exposure to arsenic at different time points. Values are mean±SD. Differentalphabets show significantly different values (P<0.05, DMRT).

Hypothetical proteins

931J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 9 2 1 – 9 3 7

In viewof the significant accumulation of H2O2 after 1 d, theH2O2 detoxifying enzymes e.g., AhpC, catalase, oxidoredutase,peroxiredoxin and thioredoxin were observed in the proteomemap of Anabaena. A 2.3 fold upregulation of hypothetical pro-tein all3090 (spot 13) and 1.8 fold of oxidoreductase (spot 12)after 1 d followed by their down regulation on 15th day wasrecorded (Fig. 5, Supplementary S48). The overexpression ofall3090 (homologous to catalase) and oxidoreductase impliedprotection of cell fromoxidative stress as also reported for des-iccation stress [93]. In view of the fact that AhpC is a scavengerof low level of intracellular H2O2 while catalase does so at highintracellular H2O2 [94], it is likely that high intracellular H2O2

Fig. 6 – Expression pattern of selected proteins. C, control; 1and 15 represent the days on which As treatment was givento Anabaena. Numbers represent the spots no. on 2DE gel.Proteins showing significant and reproducible changes weresubjected to MALDI-TOF/MS and LC-MS analysis. Details aregiven in Table 2.

inhibits AhpC leading to its down regulation on differentdays. Under above situation, up-regulation of CAT and oxido-reductase may ensure H2O2 detoxification when AhpC fails toexpress. Two of the five identified proteins, peroxiredoxin(Prx; spot 37) and thioredoxin (Trx; spot 45), were maximallyupregulated on 15 d (3.0 and 2.8 folds respectively) (Fig. 6).This is supported by our earlier data on the downregulationof AhpC and upregulation of Prx in A. doliolum under heatstress [10]. Prx has multiple roles like (i) reduction of H2O2

and organic hydroperoxides [95], (ii) acting as a molecularchaperone similar to HSPs [96], and (iii) modulation of cell sig-naling. Upregulation of Prx has also been reported in maize[24] and Scystosiphon gracilis [97] in response to As and copperstress respectively. Thus Prx has the potential to protect Ana-baena from As stress.

Thioredoxin efficiently reduces intramolecular disulfidebridges on target proteins [98] and maintains cellular process-es like ribonucleotide reduction and suppression of apoptosis,supplies reducing equivalents to the antioxidant systems [99]and regulates biosynthesis of hydrophobic amino acids inChlamydomonas reinharditii [100]. Thus an upregulated (2.8fold) Trx may be envisioned to manage oxidative stress im-posed by As on the test cyanobacterium. This proposition isalso supported by the upregulation of Trx under salt stressin Hordeum vulgare [101] and heat stress in Populas [102].

3.4.3. Protein biosynthesis and foldingOf the three proteins of this category where DnaK (spot 1)showed down regulation, the EF-Ts (spot 22) and EF-Tu (spot33) depicted 3.3 and 1.6 fold accumulation respectively after15 d. DnaK, known as Hsp70 in E. coli is temporally overex-pressed in cells exposed to stress and protects from protein de-naturation [103]. Downregulation of DnaK after 1 d of Astreatment may be due to a dramatic decrease in the intracellu-lar ATP level, rendering DnaK's N terminal ATPase domain nu-cleotide depleted and thus inactive. Upon return to non-stressed conditions the DnaK gets upregulated [104]. EF-Ts me-diates regeneration of EF-Tu–GDP complex which facilitatesentry of aminoacyl-tRNA into the ribosome enabling proteinsynthesis. This finds support from the work of Heim et al.[105] where overproduction of EF-Ts in Enterococcuswas respon-sible for protein synthesis. EF-Tu has a dual function in proteinsynthesis and stress response [106]. Under stressful conditions

932 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 9 2 1 – 9 3 7

EF-Tu functions like DnaK [107,108]. Our results suggest thatoverexpressed EF-Tu might offer tolerance against As stress asalso known for maize [109] and E. coli under heat stress [110],Pseudomonas fluorescens under cobalt stress [108] and A. doliolumunder salt stress [8].

3.4.4. Amino acid and cofactor biosynthesisArsenic was found to affect the abundance of three amino acidbiosynthesis enzymes (i) dihydropicolinate reductase (DHPR;spot 4), an enzyme involved in the biosynthesis of diaminopi-melic acid. Its inactivation after 1 d and a slight upregulationon 15 d indicates improved lysine biosynthesis and growth ofthe test organism. Similarly, the expression level of cysteinesynthase (CS: spot 39) was markedly upregulated (1.7 fold)under arsenic stress on 15 d. Cysteine is a biosynthetic precur-sor of GSH, a reducing tri peptide thiol implicated inmaintain-ing intracellular redox status and associated in xenobioticdetoxification [111]. Thus, over expression of CS following Astreatment is to meet the high demand of cysteine during bio-synthesis of cysteine-rich polypeptides like metallothioneins,phytochelatins or GSH required for As detoxification. This ob-servation finds support from the reports of Requejo and Tena

Fig. 7 – Effect of As on (A) arsenate reductase activity, (B) RT-PCRarsenite transporter (alr1097), and (D) hypothetical protein genecheck equal loading of RNA in different samples. M, marker; N, nand 15 days of As treatment.

[28] in maize and Ahsan et al. [69] in rice against As stress.Spot no. 36 showed 99.7% homology with L-asparaginase ofA. variabilis which is involved not only in asparagine catabo-lism but also in degradation of proteins and cyanophycin[112]. Since plants require more nitrogen under stress the am-monia produced by L-asparaginase may be used for proteinsynthesis. In view of above a 1.9 fold induction and putativefunction of L-asparaginases in response to As stress in Ana-baena seems justified. Since As repressed the nitrogenase ac-tivity (Table 1) the nitrogen depleted cells of Anabaena mustobtain nitrogen through alternative routes like conversion ofasparagine into ammonia by L-asparaginase. Alternatively, L-asparaginase may increase the amount of free amino acidswhich may abet in maintaining osmolarity of the cell. Recov-ery of pyridoxine 5-phosphate synthase (spot 30) on day 15indicates accumulation of pyridoxine 5-phosphate, a catalyti-cally active form of the vitamin B6, required for the activity ofdifferent enzymes.

3.4.5. Transcription regulatory proteinsSpot 43 showing a 2.8 fold induction on 15 d was identified asa transcriptional regulator. Another protein (spot 17) with a

of arsenate reductase gene (alr1105), (C) multidrug resistantasr1102 at different time points. 16S rRNA gene was used toegative RT control; C, control; 1D, 7D, and 15D represents 1,7,

933J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 9 2 1 – 9 3 7

1.5 fold induction at the same time showed 99% homologywith anti sigma factor antagonist of A. variabilis thus may beaccredited as an anti-sigma factor anatagonist (http://www.ncbi.nlm.nih.gov/BLAST/). Transcription regulatory proteinsin Anabaena are the multitasking proteins involved in a varie-ty of functions e.g. synthesis of functional hydrogenase andnitrogenase [113]. Upregulation of both of these proteinsmay be speculated to regulate the expression of As stress re-sponsive genes.

3.4.6. Hypothetical proteinsDespite similarities to proteins from other organisms, thefunction of hypothetical proteins is mostly unknown. TheBLASTP (http://www.ncbi.nlm.nih.gov/BLAST/) was used toscore the homology of the hypothetical proteins in the data-base. In this study, thirteen hypothetical proteins includingalr0803 (spots 3,42), alr0806 (spot 9), alr4050 (spot 10), alr3199(spot 11), alr3090 (spot 13), all4894 (spot 14), all1009 (spot 16),alr4550 (spot 18), all4499 (spot 19), all1864 (spot 23), all3076(spot 24), alr3904 (spot 25), and alr4171 (spot 36) were found(Supplementary Table S3). The kinetics of hypothetical pro-tein alr0803 depicted 1.5 fold accumulation after 1 d and de-cline on 15 d and showed homology to signal transductionhistidine kinases. Bhargava et al. [6] have reported involve-ment of alr0803 in Cu homeostasis in Cu-acclimated A. dolio-lum. In view of this, alr0803 may conceivably function as

Fig. 8 – Hypothetical mechanistic model depicting the survival starrows indicate enhanced enzyme activity and protein expressio

sensor of As stress and help in As homeostasis of the cells. Itis pertinent to mention that the hypothetical proteinsalr0803, alr4050, alr3199, all4894, all1009 are also known tobe induced under desiccation. Induction of these proteins on1 d and down regulation on the 15th day is not only a lucid in-dication of their transient behavior but development of desic-cation like situation in the cell. The upregulation of alr4050(2.8 fold) a homologue of akinete marker protein [67] andalr3199 (3.0 fold) a homologue of HHE cation binding proteinfinds support from the transcript of their homologues in UVstressed Nostoc commune [114] and dehydration stressed Nostocsp. strain HK-01 [115]. Induction of all4894, a fasciclin super-family protein involved in cell adhesion and interaction withthe extracellular matrix [116], may be responsible for aggrega-tion of cyanobacterial filaments to avoid As exposure. An 11.0fold induction of all1009 on day 1 in the As-treated cells findssupport from Anabaena under drought when sigJ sigma factorwas over expressed [117]. Maximum induction of these hypo-thetical proteins on 1 d supports As induced desiccation in-side the cell. alr4550 and all4499 depicted 11.0 and 4.1 foldinduction respectively on the 15th day (Table 2) and possessedrespectively 97.9% and 94.9% homology with the S-layerregion-like protein of A. variabilis ATCC 29413 (SupplementaryTable S3). In Bacillus spharicus JG-A12 this layer binds uraniumand other heavymetals [118]. Furthermore, these two proteinsshow homology with the outer membrane porin OprB

rategy of Anabaena sp. PCC 7120 under As stress. Upwardn.

934 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 9 2 1 – 9 3 7

(carbohydrate selective porin) of Pseudomonas aeruginosa andare the most abundant outer membrane proteins in hetero-cysts [119]. Therefore, these proteins presumably sequesterAs and transport carbohydrate from vegetative cells toheterocyst.

3.5. Role of ars genes in survival of Anabaena under Asstress

Morphological, physiological, biochemical and molecular dataclearly indicate acclimation and recovery of Anabaena againstAs stress. Arsenic detoxification known in several bacteria in-cluding Escherichia coli, Staphylococcus aureus and the cyanobac-terium Synechocystis sp. [22] is regulated by ars genes [120].Anabaena was found to grow at 4.0 fold higher concentrationof As compared to Pseudomonas putida [121] and Anabaenacylindrica [122]. To obtain information on the fate of As in Ana-baena the following experiments were done (i) measurementof intracellular As content, (ii) activity assay and transcriptanalysis of arsenate reductase, and (iii) transcript analysis oftwo putative arsenite efflux genes asr1102 and alr1097 whichshow respectively 87.1% and 82% similarity (http://www.ncbi.nlm.nih.gov/BLAST/) to the known arsenite efflux genesof Synechocystis PCC6803. The intracellular arsenic contentand the arsenate reductase activity showed completely con-trasting behavior (Figs. 1B, 7A). An approximately 8.5 fold in-crease in arsenate reductase activity (Fig. 7A) was supportedby an 8.1 fold increase in its transcript level on the 15th day(Fig. 7B). Arsenate reductase is known to use glutaredoxin,glutathione or thioredoxin as reductant for its activity [123].A 3.0 fold induction of thioredoxin on 2-DE gel and its time de-pendent increase strongly advocates it to be the source of re-ductant for this enzyme. Transcript analysis of two putativeefflux genes e.g. asr1102 and alr1097 also showed time depen-dent induction attaining 5.0 and 4.7 folds respectively on the15th day (Fig. 7C, D). Results from these experiments sug-gested that detoxification was brought about by reduction ofAs(V) to As(III) and efflux by the above putative efflux genesthus relieving the test organism from the As stress (Fig. 8).

4. Conclusions

The present study is the first to employ multiple approachesincluding proteomic, morphological, physiological, biochemi-cal, and induction of arsenic resistance and arsenite effluxgenes, phytochelatin and oxidative stress responsive proteinsto comprehend the survival of the nitrogen fixing cyanobacte-rium Anabaena under As stress. The hypothetical model(Fig. 8) provides an overview of the metabolic adjustment ofthe cell under As stress.

Acknowledgments

Sarita Pandey and Rashmi Rai thank the CSIR for SRF and L.C.Rai to DST for J.C. Bose National Fellowship and UGC, NewDelhi for financial support and the Department of Botany,Banaras Hindu University, Varanasi for facilities.

Appendix A. Supplementary data

Supplementary data to this article can be found online atdoi:10.1016/j.jprot.2011.10.011.

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