Comparative proteomics unveils cross species variations in Anabaena under salt stress

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JPROT-01662; No of Pages 17

Comparative proteomics unveils cross speciesvariations in Anabaena under salt stress

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Snigdha Rai, Chhavi Agrawal, Alok Kumar Shrivastava, Prashant Kumar Singh, L.C. Rai⁎

Molecular Biology Section, Laboratory of Algal Biology, Centre of Advanced Study in Botany, Banaras Hindu University,Varanasi 221005, India
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⁎ Corresponding author. Tel.: +91 542 6701110E-mail addresses: [email protected], lc

1874-3919/$ – see front matter © 2014 Elseviehttp://dx.doi.org/10.1016/j.jprot.2013.12.020

Please cite this article as: Rai S, et al, Comp(2014), http://dx.doi.org/10.1016/j.jprot.201

A B S T R A C T

Article history:Received 21 May 2013Accepted 12 December 2013

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The present study compares protein diversity within three Anabaena species (Anabaenadoliolum, Anabaena sp. PCC 7120 and Anabaena L31). 2-DE based analysis of 256 protein spotsin control and 1, 3, 5, and 7 days of salt treatment resulted into 96 proteins arching acrossfourteen functional categories were assigned to biochemical pathways using KOBAS 2.0.While 52.34% of the evaluated protein spots were common across three species, theremaining 47.66% fraction mainly comprised of the hypothetical and unknown proteins.PSORTb, CDD, Motifscan and Pfam revealed function and subcellular localization for 27 ofthe 31 hypothetical and unknown proteins. The differences in high salt tolerance (LC50) ofA. doliolum over A. L31 was reflected by (i) many fold accumulation (as spot volumes) ofAlr3090, Alr0803, peptidyl prolyl cis-trans isomerase andmodulator of DNA gyrase proteins,and (ii) a better photosynthesis and energy homeostasis as indicated through photosystemactivity, respiration, ATP and NADPH contents. Some common noteworthy salt effectsinclude (i) photosystem damage, (ii) DNA damage repair, (iii) upregulated protein synthesis,(iv) enhanced sulphur metabolism, and (v) upregulated pentose phosphate pathway. 34 ofthe identified protein spots are novel entries to the Anabaena salt proteome. This studyreveals the existence of separate strategies even within species to combat stress.

Biological significanceThis study for the first time enumerates protein diversity in three Anabaena species employingtheir presence/absence and relative abundance. Proteomics integrated with physiology andbioinformatics deciphers differential salt tolerance among the studied species and is the first ofits kind to predict the function of hypothetical and unknown proteins. Salt-induced proteomicalterations clearly demonstrate significantmetabolic shifts and existence of separatemolecularphenomeamong the species investigated. Thismay be responsible for niche specificity limitingtheir application as biofertilizer. Of the 96 identified proteins, a large chunk are new entries tothe Anabaena salt proteome while some protein genesmay be used as potential candidates forengineering salt tolerant cyanobacteria.

© 2014 Elsevier B.V. All rights reserved.

Keywords:AnabaenaSalt stressComparative proteomePhysiologyBioinformaticsTwo-dimensionalgel electrophoresis

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1. Introduction

Salinity adversely affects 19.5% of the irrigated agricultural landsworld over and poses a serious threat to world crop productivity

; fax: +91 542 2368174/[email protected] (L.C. Rai).

r B.V. All rights reserved.

arative proteomics unve3.12.020

[1,2]. High salt concentrations in cells cause crucial changes inion and water homeostasis leading to arrested growth, damageat the molecular level and even death [3]. Salt-induced effectshave extensively been studied in selected relatively salt tolerant

01124.

ils cross species variations inAnabaena under salt stress, J Prot

http://dx.doi.org/10.1016/j.jprot.2013.12.020

mailto:[email protected]

mailto:[email protected]



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cyanobacteria like Synechocystis PCC 6803 and Euhalothece sp.[4–7]. This resulted in a general understanding of salt toleranceand its effects particularly on photosynthesis and respiration [8].Heterocystous filamentous cyanobacteria are of particular inter-est due to their N2-fixing potential. They constitute dominantpaddymicroflora and are excellent biofertilizers augmenting riceproductivity [9]. Prolonged water logging in paddy fields addsto the risk of salt stress both on rice and the cyanobacterialpopulation therein. Hence investigating salt stress impacts inthese N2 fixers not only provides a snapshot of the effects inagriculturally relevant microbes but a likely effect in higherplants also.

Anabaena sp. are native to the tropical conditions and fixapproximately 20–30 kg N/ha/season [10–12]. While differentgeographical isolates (species) of Anabaena display appreciableniche specificity delimiting their proliferation in altered ecolog-ical environments, the genera exhibit wide distribution across arange of salinity [13]. A preferredmodel for plant based studies,Anabaena displays remarkable evolutionary advances like celldifferentiation, pattern formation, intercellular communication,regulated gene rearrangement andexpression [14,15]. An in-silicoanalysis demonstrated that Anabaena sp. PCC 7120 harbors ahost ofmultidomain proteins with no orthologs in other speciesof cyanobacteria and plants [16]. A systematic and comprehen-sive investigation of their stress response is therefore necessaryto understand their adaptation and survival strategies againstsalinity.

Salinity related effects in Anabaena include disturbed ionicbalance, dysfunction of plasma membrane, production of ROS,damage of vital processes such as photosynthesis, nitrogenfixation, altered mRNA expression and protein profile [17].Some marked acclimation strategies include (i) controlled ionuptake and ion discrimination, (ii) active efflux of intracellularsodium, (iii) accumulation of compatible solutes and stressproteins [18,19], and (iv) tuning of bio-energetic processes tocope up with the shattered homeostasis [20].

Salt-induced alterations in the cellular machinery resultfrommodification in gene expression [21] whichmay lead to theaccumulationof certainmetabolites andappearance/disappear-ance or increase/decrease in abundance of cellular proteins.According to Sahi et al. [21], salt stress response is a quantitativetrait altering expression of specific and housekeeping genes.Understanding protein dynamics under salinity appears areliable approach for the evaluation of the ongoing molecularevents [22]. ‘Proteomics’ provides a platform to examine envi-ronmental influences, genetic manipulation, stress mediatedadaptations and strain variability [23]. Two-dimensional gelelectrophoresis coupled with mass spectrometry is a powerfultool for qualitative and quantitative assessment of proteomicchanges [22].

Although there exist a considerable number of proteomicinvestigations in cyanobacteria such as Synechocystis PCC 6803[24–26], Trichodesmium erythraeum IMS101 [27], and Spirulinaplatensis [28] analyzing responses under a set of conditions, stressand for biotechnological purposes, there is no informationavailable on species specific responses in cyanobacteria ingeneral and Anabaena species in particular in response to astress.While a study onMicrocystis aeruginosa toxic andnon-toxicisolates brings out variations with respect to microcystinbiosynthesis [29,30], Prochlorococcus ecotypes displayed marked

Please cite this article as: Rai S, et al, Comparative proteomics unve(2014), http://dx.doi.org/10.1016/j.jprot.2013.12.020

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differences in their protein expression in response to environ-mental phosphate concentrations [31]. Exploring inter-speciesdifferences could be interesting in view of in-silico data oncyanobacterial genome where 20–30% proteins are speciesspecific due to conceivable horizontal gene transfer [32]. In areport, Koonin and Wolf [33] talk of ‘molecular phenome’especially in prokaryotes where protein expression and abun-dance are considered as constraints affecting robustness andplasticity of biological systems hencemay differ within the sameset of orthologous genes. Also, it is important to address as towhat imparts relative tolerance/sensitivity within Anabaenaspecies under stress. While the morphology based comparisonof Anabaena crassa and Anabaena circinalis affirmed phenotypicplasticity [34], it is not helpful per se for other forms and calls formolecular analysis. TheSDS-PAGEproteinprofile of salt sensitiveAnabaena L31 and salt tolerant Anabaena torulosa revealed nichespecificity and differential response of proteins to salt stress [7].Thus a detailed proteomic analysis appears to hold potential inidentifying proteins differentially expressed under stresses andstrategy in place for such differences. The present study ex-amines 2-DE based diversity and relative abundance of proteinsin three Anabaena species subjected to salt stress. Of theAnabaena spp selected for the present study while A. PCC 7120(http://genomekazusa.or.jp/Cyanobase/anabaena) is fully se-quenced, A. doliolum and A. L31 are environmental isolates. 16SrDNA based phylogenetic assessment affirmed that the threespecies are different from each other (Supplementary 1 inAppendix A). A significant difference in their salt LC50 valuesprompted us to carry out comparative a proteomic study.A. doliolum with an LC50 of 150 mM for NaCl was taken as salttolerant and A. L31 with an LC50 of 62 mM as salt sensitivespecies [7]. This study was anticipated to address the following(i) whether protein diversity exists across species with respect tosalt stress, (ii) if so, whether relative abundance of commonproteins and presence/absence of species specific proteins hasany role in salt tolerance, (iii) if the proteomic data showcorrelation with the physiological attributes, and (iv) whichmajor and novel proteins are responsible for cell survival undersalt stress. This study is the first to provide data on large scaleinter-species comparative proteomics in diazotrophic cyano-bacteria under stress. It not only categorizes protein underdifferent functional categories but also attempts to correlate theobserved proteomic changes with physiology and incorporatesbioinformatics to predict to some extent the function of hy-pothetical and unknown proteins. It further highlights that(i) changes in relative expression and presence/absence ofspecies specific proteins could be a reason for inter-speciesdifferences in tolerance/sensitivities under salt stress, (ii) thelocal isolates inhabiting varied geographical areas and nichespecificity might have significant differences and hence cannotbe represented by fewmodel organisms.

2. Materials and method

2.1. Organisms, growth conditions andphylogenetic assessment

The three Anabaena sp. (i) Anabaena doliolum, (ii) Anabaena PCC7120, and (iii) Anabaena L31 were grown in BG-11N− medium


http://genomekazusa.or.jp/Cyanobase/anabaena


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(without NaNO3) buffered with Tris/HCl at 25 ± 2 °C under daylight fluorescent tubes emitting 72 μmol photon m−2 s−1 PAR(photosynthetically active radiation) light intensity with aphotoperiod of 14:10 h at pH 7.5. The cultures were shaken byhand 2–3 times daily for aeration. In order to examine thecloseness of the three species a phylogenetic tree based on16S rDNA was constructed using neighbor joining method.

2.2. Experimental design and growth measurement

Three biological replicates were used for comparing theproteomes in control and 1, 3, 5, 7 days of salt treatment(Fig. 1, Supplementary 1 in Appendix A). The “control” gels ofeach Anabaena sp. in the text refer to the cultures without salttreatment at day 0. In order to assure that the changes inprotein profile are because of salt treatment and not thecontinued incubation of batch cultures, Anabaena cells with-out salt were run in parallel with the salt treated samples (i.e.“control at respective days”) and compared at respective days.A 5.0 M stock solution of NaCl was used for salt treatment andthe LC50 dosage for each Anabaena sp. was determined by theplate colony count method [35]. Exponentially growing cells ofthe three Anabaena species were treated with their respectiveLC50 doses of salt and the growth estimated bymeasuring O.D.of the culture at 750 nm in a UV–VIS spectrophotometer(Systronics, India) on every third day up to fifteen days.

2.3. Measurement of physiological parameters

The rate of respiration (defined as O2-consumption in dark)was determined by measuring total O2 consumed in the darkfor a given time period minus non-specific O2 uptake [36]. Thesize of the ATP pool was measured by the method asdescribed by Larsson and Olsson [37]. NADPH/NADH level ofthe cell extract in Tris-Cl (pH 8.0) was measured by recordingabsorbance at 340 nm [38]. Electron transport activities(Photosystem I, Photosystem II) were measured according toTripathy and Mohanty [39].

2.4. Protein extraction and quantification

Anabaena cells harvested by centrifugation (10,000 ×g, 10 min)were suspended in 2 ml extraction buffer containing 10 mMTris–HCl (pH 8.0), 1.5 mM MgCl2 and 10 mM KCl in the presenceof 10 μM PMSF (Sigma), ground in liquid nitrogen followed bycentrifugation at 12,000 ×g for 1 h. The supernatant (500 μl) wascleanedusing 2-DE cleanupkit (GEHealthcareBio-sciences,USA)and precipitated proteins were solubilized in buffer containing7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT and 1.0% IPGbuffer (4–7 pH). The protein concentration was measured usingthe method of Bradford [40].

2.5. 2-DE separation of protein

Traces of bromophenol blue were added to the protein sampleand centrifuged at 19,000 ×g for 10 min. A total of 300 μl ofsolubilization buffer containing 250 μg protein samples wasapplied to the immobilized pH gradient (IPG) strips (pH 4–7;13 cm; GE healthcare, USA) and the strips were covered withmineral oil for 16 h at room temperature. The first dimension


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separationwas conductedat 20 °Cwith anEttan IPGphor system(GE Healthcare, USA). Focusing was performed in 7 steps: 30 Vfor 00.30 h, 150 V for 2:00 h, 300 V for 00:40 h, 500 V for 4:00 h,gradient 1000 V for 1:00 h, gradient 8000 V for 2:00 h and finally8000 V for 13:00 h. Focused IPG strips were equilibrated by firstincubating in an equilibration solution (6 M urea, 30% glycerol,2% SDS, 50 mMTris–HCl pH 8.8, and 1%DTT and a trace amountof bromophenol blue) for 20 min followed by incubation in 2.5%iodoacetamide in the same equilibration solution instead of 1%DTT for 20 min. The strip was placed on the top of 12.0%SDS-PAGE and sealed with 0.5% agarose. Electrophoresis wascarried out at 10 mA/gel for 30 min followed by 25 mA/gel for5 h using a Hoefer SE 600 apparatus (Amersham Biosciences,USA). The gelswere stainedwith Coomassie Brilliant Blue R-250.

2.6. Analysis of gels

2-DE gel image analysis software PDQuest™ Basic version 8.0.1(Bio-Rad) was used for gel analysis. After background subtrac-tion and spot detection, spots were matched. Spot quantifica-tion in the control and salt treated gels was done by measuringspot volumes (intensity × mm2). 256 spots having statisticallysignificant (DMRT, p < 0.05) and reproducible changes wereconsidered and the proteins with an abundance ratio of at least1.5 fold were taken as differentially expressed proteins. Theprotein spots of interest were manually excised from the geland processed for MALDI-TOF MS/MS identification.

2.7. Protein identification by MALDI-TOF MS/MS

Samples for MALDI-TOF analysis were prepared as per theprotocol of Bruker Daltonics [41]. 100 ml of wash solution(1:1 ratio of acetonitrile and 100 mM ammonium bi carbonate)was added to excised spots and incubated for 30 min fordestaining. It was further incubated in absolute acetonitrile for3 min and pellets were air dried. Reduction of the protein spotswas done using 10 mM DTT dissolved in 100 mM NH4HCO3 for1 h at 50 °C. The samples were further alkylated using 55 mMiodoactamide in 100 mM NH4HCO3 for 45 min in dark atroom temperature. The gel pieces were dehydrated with 100%acetonitrile, air dried and covered with 20 μl of 25 μg/mlsequencing grade trypsin solution (Promega Gold, USA) andincubated at 37 °C. The polypeptides were eluted from the gelpiece in 2.5% trifloro acetic acid (TFA) in 50% acetonitrile byvortexing for 15 min. Extracted peptides were vacuum concen-trated and 1 μl of trypsinized peptide samples was mixed withmatrix, CHCA (2.5 mg/ml in 50% acetonitrile) on the target plate(384 well stainless steel, Bruker Daltonics, Germany) andsubjected to Auto flex speed MALDI-TOF for mass spectrometricidentification. Data acquisition and analysis (peak list) wereperformed using flex analysis and biotools versions 3.3 and 3.2respectively (Bruker, Daltonics). All spectra were smoothenedand internally calibrated. The human keratin and trypsin inautodigest peptide ions were excluded. The number of misscleavage permitted was 1. The measured tryptic peptide masseswere transferred throughMSBioTool programas inputs to searchagainst the taxonomy of other bacteria in the databases usingMASCOT 2.0 software (Matrix Science). The protein databaseemployed was, NCBInr (The National Center for BiotechnologyInformation non-redundant). The MS/MS ion search were



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performed with the following MASCOT settings: taxonomy asother bacteria; peptide mass tolerance of 200 ppm for precursorions and ±1.2 Da for fragment ions, peptide charge setting as +1,fixed modification such as carbamidomethyl on cysteine,variable modification of oxidation on methionine residue.Proteins with a mascot score >100 were considered significant(p < 0.05) for identification. 21 proteins with mascot score in therange of 75–100 and ≥5% sequence coverage were considered aspositive identification. False discovery rate (FDR) calculationswere done using reverse decoy database, taxonomic group as“other bacteria” to ensure an FDR of not more than 5%. For fewprotein spots, FDR upto 25% has also been accepted as positiveindication considering the significant alteration in their relativeabundance and non available genome sequences for the localisolates A. doliolum and Anabaena L31.

2.8. Bioinformatic predictions

The identified proteins were divided into fourteen functionalcategories as per Cyanobase (http://genomekazusa.or.jp/Cyanobase/anabaena) and their biochemical pathways werepredicted using KOBAS 2.0 software (http://Kobas.cbi.pku.edu.in) which assigned proteins to the known pathways in theKEGG database (http://www.genome.jp/kegg/pathway.html).Proteins having no information in the KEGG database, weresearched in GO (http://www.geneontology.org/). PSORT-Bversion 3.0.2 was used to categorize the proteins as cytoplas-mic, inner membrane, periplasmic, and outer membranetaking into account the N terminus signal sequence and thehydrophobic regions in the peptide.

Proteins contain one or more functional and conservedregions called domains. An understanding of these can provideinsights into thepossible functions ofnovel proteins. A variety ofin-silico parameters were used to decipher the physicochemicaland functional attributes of hypothetical and unknown proteinsin the experimental datasets. This includes the ConservedDomainDatabase (CDD) ofNCBI's interface for fast identificationof conserved domains in protein sequences via RPS-BLAST, thePfam database (Pfam 26.0) to predict the protein domains andfamilies, MotifScan for finding all known motifs that occur in asequence, ExPASy ProtParam to compute the physicochemical

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Fig. 2 – Growth behavior of (A) Anabaena doliolum (B) Anabaena sp750 nm.


properties like molecular weight and theoretical pI of the saltstress responsive proteins.

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3. Results

3.1. Differences in the LC50 values of the three species

A significant difference in LC50 dosage of NaCl (measured interms of percent survival) was observed for three species ofAnabaena (Supplementary 1 in Appendix A). This being 62, 100and 150 mM for A. L31,A. PCC 7120 andA. doliolum respectively.These differences are clearly visible in the growth performanceof the three species at their LC50 doses measured in terms ofabsorbance at 750 nm (Fig. 2).

3.2. Physiological responses of Anabaena sp. to salt stress

Cyanobacterial thylakoid accommodates photosynthetic andrespiratory electron transport chains resembling a complex andbranched network. The two processes under high salt concen-tration are tightly regulated for a balanced cellular energetics[42]. To assess the changes in photosynthetic and respiratoryelectron transport under salt stress photosystem I (PSI) andphotosystem II (PSII) were measured at 1, 3, 5 and 7 days oftreatment (Table 1). In A. doliolum and A. PCC 7120, a significantdecrease in PSI was observed on day 1 of treatment whichgradually recovered on subsequent days while in A. L31 asignificant decrease was observed on all days of salt treatment.In contrast to this the PSII activity appeared highly salt sensitiveas reflected by a continuous decrease over time in all thetreatments; this being maximum in A. L31 and minimum inA. doliolum. All the three species registered enhanced but nearlyequal amount of oxygen consumption (Table 1). Data pertainingto NADPH and ATP revealed a large increase in salt treatedAnabaena sp. as compared to the control. An increase in NADPHof upto 5.44, 2.0 and 1.83 fold was observed inA. doliolum,A. PCC7120 and A. L31 respectively. While ATP content in A. doliolumand A. PCC 7120 showed 1.33–1.91and 1.08–1.45 fold increaseafter salt treatment, the A. L31 depicted an initial increasefollowed by a decrease on day 7 of treatment.

. PCC 7120 and (C) Anabaena L31 in terms of absorbance at




http://Kobas.cbi.pku.edu.in

http://Kobas.cbi.pku.edu.in

http://www.genome.jp/kegg/pathway.html

http://www.geneontology.org/


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Table 1t1:1 – Physiological variables of Anabaena sp. under salt (NaCl) treatment. All values are mean ± SD. Values with + and −t1:2 signs in parenthesis indicate fold increase or decrease, respectively, as compared to control. Different alphabets showt1:3 significantly different values (P < 0.05, Duncan’s multiple range test).t1:4t1:5 Anabaena

sp.Days PS-I activity

(μmol O2 consumedμg−1 protein h−1)

PS-II activity(μmol O2 evolved μg

protein−1 h−1)

Respiration(μM O2 consumed mg

protein−1 min−1)

NADPH(mM NADPH mg

protein−1)

ATP assay(ng ATP mgprotein−1)

t1:6 A. doliolum Control 15.0 ± 0.32b 6.8 ± 0.02a 11.0 ± 0.01d 0.16 ± 0.05e 37.5 ± 0.8e

t1:7 1Day 5.70 ± 0.11d

(−2.63)5.00 ± 0.06b

(−1.36)12.6 ± 0.21c

(+1.15)0.34 ± 0.1d

(+2.13)50.0 ± 1.5d

(+1.33)t1:8 3Day 12.4 ± 0.10c

(−1.21)2.20 ± 0.22d

(−3.09)14.5 ± 0.08b

(+1.32)0.38 ± 0.04c

(+2.38)57.5 ± 2.9c

(+1.53)t1:9 5Day 14.20 ± 0.5b

(−1.06)2.80 ± 0.31c

(−2.43)15.1 ± 0.80a

(+1.37)0.84 ± 0.05b

(+5.25)71.5 ± 6.3a

(+1.91)t1:10 7Day 22.9 ± 0.64a

(+1.53)2.11 ± 0.05c

(−3.22)15.9 ± 0.15a

(+1.45)0.87 ± 0.23a

(+5.44)70.0 ± 5.4b

(+1.87)t1:11 A. PCC 7120 Control 10.0 ± 0.25a 9.00 ± 0.05a 10.0 ± 0.03d 0.10 ± 0.03d 39.0 ± 2.5d

t1:12 1Day 6.20 ± 0.07c

(−1.61)3.79 ± 0.02c

(−2.37)12.6 ± 0.08c

(+1.26)0.12 ± 0.07c

(+1.20)42.0 ± 5.1c

(+1.08)t1:13 3Day 3.90 ± 0.10d

(−2.56)3.20 ± 0.19c

(−2.81)13.8 ± 0.08b

(+1.38)0.17 ± 0.06b

(+1.70)47.5 ± 3.3b

(+1.22)t1:14 5Day 6.00 ± 0.22c

(−1.67)4.30 ± 0.04b

(−2.09)15.0 ± 0.06a

(+1.5)0.20 ± 0.02a

(+2.00)56.5 ± 0.9a

(+1.45)t1:15 7Day 8.00 ± 0.05b

(−1.25)2.37 ± 0.13d

(−3.8)16.6 ± 0.23a

(+1.66)0.12 ± 0.04c

(+1.20)42.5 ± 1.1c

(+1.09)t1:16 A. L31 Control 8.60 ± 0.07a 11.0 ± 0.34a 9.10 ± 0.52d 0.06 ± 0.01d 28.5 ± 1.1c

t1:17 1Day 2.00 ± 0.11c

(−4.30)2.00 ± 0.02b

(−5.5)11.7 ± 0.24d

(+1.29)0.07 ± 0.03c

(+1.17)36.5 ± 0.9b

(+1.28)t1:18 3Day 1.60 ± 0.03d

(−5.38)1.50 ± 0.10c

(−7.33)13.3 ± 0.11c

(+1.46)0.09 ± 0.01b

(+1.50)43.0 ± 3.0a

(+1.51)t1:19 5Day 2.80 ± 0.01b

(−3.07)1.40 ± 0.04c

(−7.86)15.5 ± 0.05b

(+1.70)0.11 ± 0.04a

(+1.83)33.5 ± 2.4b

(+1.18)t1:20 7Day 2.43 ± 0.04c

(−3.54)2.00 ± 0.06b

(−5.5)17.6 ± 0.07a

(+1.93)0.09 ± 0.01b

(+1.50)27.1 ± 0.8c

(−1.05)


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RRE3.3. Protein expression profiles and pathway analysis

Salt-induced changes in the protein profiles ofA. doliolum,A. PCC7120 andA. L31 at 1, 3, 5 and 7 days of treatmentwere examinedby2-DE (IEF-SDS-PAGE) followedbyMALDI-TOF-MS/MSanalysis.The gels were run in biological triplicates and the averagenumber of spots in control and treated gels was between 597and 753, 551 and 699 and 661 and 736 in the three speciesrespectively (Table 3, Supplementary 1 in Appendix A). The gelswith most significant differences over time (control, 1 day and5 days) have been displayed as Figs. 3, 5 and 7 in the text whilegels of 3 and 7 day treatment along with control on respectivedays are shown in Figs. 11, 12 and 13 of the Supplementary 1 inAppendix A. Relative abundance of proteins, measured as spotvolumes for control and treatment gels are shown in Figs. 4, 6and 8. A total of 256 protein spots with significant andreproducible changes were divided into 7 categories e.g. proteinspots present in all the three species (category1), protein spotsidentified in each species alone (category 2 for A. doliolum, 3 forA. PCC 7120 and 4 for A. L31) and protein spots common to anytwo species (category 5 for A. doliolum and A. PCC 7120, 6 for A.doliolum and A. L31 and 7 for A. PCC 7120 and A. L31). Of these,134 spots corresponding to 29 proteins accounting 52.34% of thetotal were present in all the three species (category 1) henceconsidered as “common proteins”. Groups of 13, 10 and 19proteins were identified only in A. doliolum, A. PCC 7120 andA. L31 respectively (Table 2).


On the basis of the cellular function, the proteins werefurther divided into fourteen categories (according to cyanobase-Anabaena) namely photosynthesis and respiration (A); energymetabolism (B); oxidative stress defense (C); transcription (D);translation and chaperon function (E); amino acid metabolism(F); purines, pyrimidines, nucleosides and nucleotides (G), cofac-tor biosynthesis, prosthetic groups and carriers (H); regulatoryfunction (I); fatty acid, phospholipid and sterol metabolism (J);transport and binding (K); others (L); hypothetical (M) andunknown proteins (N). The details of the identified proteins areavailable in Table 4 (Supplementary 1 in Appendix A). Functionaldistribution of the identified proteins has been shown in the piechart (Fig. 9, Supplementary 1 in Appendix A). While 20.48%and 21.18% protein spots in A. doliolum and A. PCC 7120 werecomprised of the hypothetical proteins, about 23.75% proteinspots in A. L31 belonged to photosynthesis and respiration.

3.4. Photosynthesis and respiration

Seventeen proteins showing homology with RBCL, FNR, Ch.AFd, ATPsyn (inner membrane protein), GAPDH, PRCLP, OCP,PSII-OEC (inner membrane protein), PSI FeS, CCM, PCβ, PCα,MSP, APCα, APCβ, PEC and FDX were identified. Among thethree species, an appreciable variation in protein expression ofA. L31was observedwhere PCα and PCβ, PRCLP, APCα, APCβ andPEC belonging to photosynthetic antenna proteins showedupregulation with respect to control. Flavodoxin which



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Fig. 3 – The 2-DE images of total cytosolic protein extract from Anabaena doliolum under control, 1Day and 5 Day of salttreatment (150 mM NaCl). For 3 and 7 day 2-DE gel images please refer to the Supplementary 1 in Appendix A.


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functions in electron transfer in place of ferridoxin under stressshowedmore than 3 fold induction. About 15.66% and 12.94% ofproteins in A. doliolum and A.PCC 7120 constituted photo-synthesis and energymetabolism. OCP (alr4783 and all3149 inA. doliolum and A. L31) a photoprotectant acting as an energydissipater via state charge transfer [43] showed altered expres-sion in the two species while FNR, catalyzing electron transferbetween ferridoxin and NADP+/NADPHwas significantly upreg-ulated in A. doliolum and A. PCC 7120. Salt treatment caused anincrease in the relative abundance of ATPsyn. inA. doliolum andA. PCC 7120, the spots in A. L31 showed transient expression.GAPDH associated with glycolysis and Calvin cycle showedaccumulation in A. doliolum and A. PCC 7120.

3.5. Energy metabolism

Ten proteins i.e. PGK, TK, FBPald., G6PI, 6PGlu.D (a periplasmicprotein), PD, enolase, FBPase, G6PD and GSAT are known tobelong to glycolysis, pentose phosphate pathway, calvin cycleand porphyrin/chlorophyll metabolism respectively. WhilePGK, TK and FBPase registered 2–3 fold upregulation with


respect to control, 6PGlu.D declinedonprolonged salt treatmentand GSAT showed initial accumulation followed by a decreaseon days 5 and 7. Likewise enolase showed accumulation; spotscorresponding G6PD displayed a decrease in abundance inA. L31. Proteins FBPald and PD in general showed maximumaccumulation on 5th day and a decline on the 7th day of salttreatment. G6PI catalyzing the second step of glycolysis showedtransient expression in the three species. While this proteindemonstrated a gradual increase to the tune of 8.76 fold onday 5, it was down regulated on day 7 in salt treated cells ofA. doliolum. Contrary to this, more than 6 fold down regulationwas observed on day 1 of salt treatment in A. PCC 7120 whichimproved on subsequent days. G6PI in A. L31 did not depictsignificant difference on days 1 and 3 of treatment while adecrease was observed on days 5 and 7.

3.6. Oxidative stress defense proteins

Oxidative stress is an imbalance between the systemicmanifestations of reactive oxygen species and biologicalsystem's ability to detoxify the reactive intermediates. Salt



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mediated changes in the osmoticum and ionic inequity canalso cause production of reactive oxygen species and oxida-tive stress. Salt exposed Anabaena cells registered changes in 9proteins Prx, alr3090 (similar to catalase), superoxide dismut-ase (SOD A and SOD B), GR, OR and AhpC/TSA family proteins.Of these, alr3090, SODA, SODB and GR belong to the Haliwell–

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Fig. 4 – Changes in expression of the identified proteins in A. doltreatment (150 mM NaCl) measured as spot volumes (mean ± SD


Asada pathway. While a general increase in these enzymesprovides evidence to the operation of an active ROS scaveng-ing under salt stress, some variations in the expression levelof these proteins in the three species were recorded. While asimilar pattern of accumulation (2–8 fold) was observed forPrx, SOD, GR and alr3090, the AhpC/TSA in A. L31 displayed a

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iolum under Control, day 1, day 3, day 5 and day 7 of salt) using PDQuest.



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Fig. 5 – The 2-DE images of total cytosolic protein extract from Anabaena PCC 7120 under control, 1 day and 5 day of salttreatment (100 mM NaCl). For 3 and 7 day 2-DE gel images please refer to the Supplementary 1 in Appendix A.


UNCOdecrease on day 1 followed by a gradual increase in expression

on subsequent days. Spot nos. D73, P73 and L53 showing 3–8fold increase over control corresponds to similar proteinoxidoreductase (corresponding gene alr5182), a class ofenzyme that catalyzes oxidoreduction reactions. Many foldincrease in oxidoreductase in the three species suggests anotable alteration in redox status in response to high saltconcentration.

3.7. Transcription, translation, folding and aminoacid metabolism

Thirty seven spots corresponding to twelve proteins were thoseinvolved in transcription, translation, chaperone activity andamino acidmetabolism. Among themolecular chaperons, DnaKandGroEL displayed 2–6 fold increase in salt treated cells. PPIaseidentified as alr0577 (spots P75, L71) and all4287 (spots D31, P82,L49) are molecular chaperones catalyzing the cis-trans isomer-ization of proline imidic peptide bonds in oligopeptides andaccelerate folding of proteins [6]. Except P82, registering 1.9 fold


decrease, all PPIase spots exhibited 1.5 to 7.5 fold accumulationon salt treated gels. Hsp1 identified only inA. doliolum (spot D60)revealed 2.5 fold increase in salt treated cells. PNPase, found inbacterial degradosomes, involved in RNA processing and tightlyregulated by secondary structural elements of RNA (IPR012162)displayed increase in treated cells.

Thirteen spots corresponding to five proteins—30sRPs1,30sRPs6, RBPD, RBPE and EFTs are collectively involved inribosome assembly, post transcriptional processing and trans-lation. These protein spots demonstrated appreciable increaseof 1.5 to 4.5 fold under salt stress. Another protein EFTu wasidentified only in A. L31 (L8, L9) where L8 showed a gradualincrease up to 5.8 fold, spot L9 displayed an increase of 2 fold inrelative abundance on day 5 which reached the level of controlon day 7. Four spots of GS and three spots of PGD involved inmetabolism of glycine, serine, alanine, aspartate, glutamateand threonine were upregulated (2–3 fold) in all the threespecies. M.DNAgy and PS are new entries to the salt proteome.M.DNAgy resembles peptidase of unknown catalytic function(KO3592) modulating activity of DNA gyrase (PubMed: 8604133)



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Fig. 6 – Changes in the expression of the identified proteins in A. PCC 7120 under Control, day 1, day 3, day 5 and day 7 of salttreatment (100 mM NaCl) measured as spot volumes (mean ± SD) using PDQuest.


and possibly involved in secretion (PubMed: 2082149). Itshowed 1.5 to 2 fold increase on initial days of salt treatmentin A. doliolum and A. PCC 7120. PS is reported to be involved insynthesis of non-ribosomal peptides depicted an increase inrelative abundance upon salt treatment.


3.8. Regulatory, transport and binding proteins

The two component response regulator (RR) registered anincrease of up to 3 fold under salt treatment. Two spots (D59and P87) corresponding to the ABC transporter depicted 1.72–



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Fig. 7 – The 2-DE images of total cytosolic protein extract from Anabaena L31under control, 1 day and 5 days of salt treatment(62 mM NaCl). For 3 and 7 day 2-DE gel images please refer to the Supplementary 1 in Appendix A.


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undergoing salt stress. Decrease of OMP identified in A. L31under salt stress may be due to the negative effects onmembrane fluidity [17].

3.9. Proteins involved in other metabolisms

Three proteins showing homology with PBP, DHO and NDKbelonged to the purine and pyrimidine metabolism andmanifested a general induction in expression in salt treatedAnabaena proteome. NDKs are enzymes that catalyze theexchange of phosphate groups between different nucleosidediphosphates. This suggests that salt alters the nucleic acidcomposition of the cells leading to DNA damage.

ACPphos. belonging to pantothenate and CoA metabolism,showed differential expression in the three species. While thisprotein was found to accumulate in A. doliolum and A. PCC 7120(spotsD35 and P35), a gradual decrease of 3.5 foldwas evident inA. L31 (spot L68). Similarly two spots of PBP exhibiting increase


in salt treated A. doliolum and A. PCC 7120 were associated withvitamin B6 (pyridoxal phosphate) metabolism and function ascofactor inmany reactions of amino acidmetabolism, includingtrans-amination, deamination, and decarboxylation. It is worthstating that the protein UROD, a member of tetrapyrrolbiosynthetic pathway required for chlorophyll biosynthesisshowing induction in A. PCC 7120 under salt finds supportfrom the studies in Anabaena and Spirulina [43,28]. Two proteinspots (P46, P83) in A. PCC 7120 having 2.46 to 5.74 fold inductionwere identified as probable glutathione S transferase (GST) andfunction in cellular defense against oxidative stress [44]. Anaccumulated GST is believed to suffice for GSH requirement forthe conjugation of toxic membrane lipid peroxidation productsproduced during salt stress [45].

3.10. Hypothetical, unknown and other proteins

Twenty three, eight and eight proteins respectively belongedto the hypothetical, unknown and other category. In-silico



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Fig. 8 – Changes in expression of the identified proteins in A. L31 under control, day 1, day 3, day 5 and day 7 of salt treatment(62 mM NaCl) measured as spot volumes (mean ± SD) using PDQuest.


Uapproaches such as CDD, Pfam, MotifScan and PSORTb wereused to find out their function and subcellular localization(Table 5, Supplementary 1 in Appendix A). Of these, spotscorresponding genes alr0803, alr3090, all4051, all4050 andalr3199 were found to be common across the three species.Among these alr0803 and alr3090 upregulated under salt werepredicted to belong to signal transduction histidine kinase andMn catalase. all4050, all4051 and all5315 showing induction inpresence of salt contain PRC barrel domain (pfam05239). Spotscorresponding alr3199 displayed a remarkable increase of 12fold inA. doliolum, 2.5–11.5 fold inA. PCC 7120 and 3.5–6 fold inA.L31; it is hemerythrin superfamily protein with DNase andnickase activity [46]. alr4979 exhibits similarity to hypothetical


DUF3386 family protein (pfam 11866). Salt-induced increase inspots corresponding all4894, all3797 and all4647 belong to betaIgH3 fasciclin family [pfam02469] and considered to function incell wall/membrane biogenesis [COG2335]. In contrast, analysisof some protein spots demonstrated similarity with more thanone functional protein, for example all3324 and all3325 (1.5–3.0fold increase inA. L31 and 1.45–3.2 fold decrease inA. PCC 7120)showed linkage to beta lactamase, histidinol dehydrogenaseand cysteinyl tRNA synthetase.

Spots corresponding alr5335 and alr7634 (identified inA. doliolum) contain putative ferredoxin domains; alr0765,alr0882 and alr3904 (identified in A. PCC 7120) contain CP12(pfam02672), uspA and beta lactamase domains respectively.



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Table 2t2:1 – Salt-induced differentially expressed proteins in the three Anabaena sp. identified by MALDI-TOF/MC-MS. Thet2:2 acronyms used, the corresponding spots and the Accession no. of the identified protein spots are provided. Spots identifiedt2:3 in A. doliolum, A. PCC 7120 and A. L31 have been designated as D, P and L respectively.t2:4t2:5 Proteins identified in all the three species.

(common proteins)Accession No. Acronym Spot no.

t2:6 1. Ferridoxin NADP reductase gi|17231613 FNR D10, D38,D49, P10, P38, L38, L61t2:7 2. ATP synthase beta subunit gi|17232531 ATPsyn. D18, D32, P32, L58, L59t2:8 3. Phycobilisome rod core linker protein gi|17228030 PRCLP D22, P21, P22, L37t2:9 4. Phosphoglycerate kinase gi|17229234

gi|17231623PGK D4, P4

D45, P45, L10, L11t2:10 5. Transketolase gi|17230836 TK D16, D19, D34, P16, P34, L1, L3t2:11 6. FBPaldolase gi|17232055 FBPald. D25, P14, P25, L21t2:12 7. Glucose 6-P isomerase gi|17228545 G6PI D33, P33, L73t2:13 8. Glutamate-1semialdehyde aminotransferase gi|161353744

gi|17132360gi|257096616

GSAT D43P43, P72L25

t2:14 9. 6 phosphogluconate dehydrogenase gi|17232767gi|17229094gi|1722909

6-PGluD D51, P26, L36L72L81

t2:15 10. Pyruvate dehydrogenase gi|17227618 PD D53, P53, L22t2:16 11. Fructose 1,6-bisphosphatase gi|17228536 FBPase D82, P24, L7t2:17 12. Peroxiredoxin gi|17232133

gi|17227548Prx D13, D27, D28, D81, P13, P27,

L40, L76 P55,t2:18 13. Oxidoreductase gi|17232674 OR D73, P73, L53t2:19 14. DnaK gi|17229234 D1, P1, L14, L46t2:20 15. GroEL gi|17229388

gi|17231154D2, P2, L15D3, D40, P3

t2:21 16. Elongation factorTs gi|17232283 EFTs D39, P39, P85, L45t2:22 17. Peptidyl prolyl cis-trans isomerase gi|17231779

gi|17228073PPIase D31, P82, L49

P75, L71t2:23 18. Glutamine synthetase gi|6646836

gi|17229820GS D6, P6

P18, L60t2:24 19. Phosphoglycerate dehydrogenase gi|17229382 PGD D83, P77, L2t2:25 20. Dihydroorotase gi|17229795 DHO D62, P80, L35t2:26 21. Two component response regulator gi|17231314 RR D30, P30, L31t2:27 22. Acyl carrier protein phosphodiesterase gi|17229597 ACPphos. D35, P35, L68t2:28 23. Nutrient stress induced DNA binding protein gi|17231300 Nut.DBP D67, P28, P48, L41, L42t2:29 24. Cystathione beta synthase gi|17231908 CS D65, P65, L62t2:30 25. Alkyl hydroperoxide reductase gi|17231896 AhpC D36, P36, L80t2:31 26. alr0803 gi|17228298 D7, D14, P7, P78,P79, L34t2:32 27. all4050 gi|17231542 D11, D47, D68, P47, P63, P71, L32, L79t2:33 28. all4051 gi|17231543 D41, P84, L56, P76t2:34 29. alr3090 gi|17230582 D75, D77, P15, P70, L27, L28, L29t2:35

t2:36 Proteins identified in A. doliolum and A. PCC7120t2:37 1. Glyceraldehyde 3 p dehydrogenase gi|17232554 GADPH D9, D37, P37t2:38 2. chain A ferridoxin gi|17231640 ChA Fd D23, P23t2:39 3. Similar to photosystem II oxygen evolving

complex 23 K proteingi|17230568 PSII-OEC D70, P56

t2:40 4. Superoxide dismutaseA gi|17227566 SODA D76, P31t2:41 5. Superoxide dismutaseB gi|17230430 SODB D29, D52, P29, P52t2:42 6. Polyribonucleotide Nucleotidyltransferase gi|17231888 PNPase D17, P17t2:43 7. Modulator of DNA gyrase gi|17232710 M.DNAgy D44, P44t2:44 8. 30s ribosomal protein S1 gi|17227632

gi|1723229430sRPs1 D66, D71, D72, P11

P49t2:45 9. Bifunctional purine biosynthesis protein gi|17230585 PBP D26, P81t2:46 10. Pyridoxal phosphate biosynthetic protein gi|17227714 PDX D63, P54t2:47 11. ABC transporter ATP binding protein gi|17229985 ABCtrans D59, P87t2:48 12. Putative heterocyst to vegetative cell connection

proteingi|17229095 HVCCP D5, P5

t2:49 13.alr4979 gi|17228298gi|17232471

D20P69

t2:50 14. all4894 gi|17232386 D50, D58, P74t2:51 15. all5315 gi|17232807 D64, P88t2:52 16. alr3199 gi|17230691 D8, D12, P12t2:53 17. alr0806 gi|17228301 D54, P58, P59


Please cite this article as: Rai S, et al, Comparative proteomics unveils cross species variations inAnabaena under salt stress, J Prot(2014), http://dx.doi.org/10.1016/j.jprot.2013.12.020


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t2:54 Table 2 (continued)

t2:55 Proteins identified in all the three species.(common proteins)

Accession No. Acronym Spot no.

t2:56 Proteins identified in A. doliolum and A. L31t2:57 1. Ribulose bis phosphate carboxylase large subunit gi|17229016 RBCL D15, L4, L47t2:58 2. Orange carotenoid protein gi|17232275

gi|17230641OCP D46, L44

t2:59

t2:60 Proteins identified in A. PCC 7120 and A. L31t2:61 1. RNA binding protein D gi|17232175 RBPD P66, L65t2:62 2. RNA binding protein E gi|17230269 RBPE P61, L77t2:63 3. Pterin 4 alpha carbinolamine dehydratase gi|17232041 PCD P68, L63, L64t2:64 4. Phycocyanin beta gi|38894 PCβ P41, L13t2:65 5. all3324 gi|17230816 P62, L69t2:66 6. all3325 gi|17230817 P86, L84t2:67

t2:68 Proteins identified specifically in A. doliolumt2:69 1. Glutathione reductase gi|17232460 GR D56t2:70 2. Heat Shock Protein 1 gi|17229301 HSP1 D60t2:71 3. Glutamate 1 semialdehyde amino transferase gi|17232595 DAPAT D78t2:72 4. Carbon dioxide concentrating mechanism protein gi|17228363 CCM D79t2:73 5. alr5335 gi|17232827 D57t2:74 6. alr7634 gi|17158770 D61t2:75 7. all3398 gi|17230890 D69t2:76 8. all3797 gi|17231289 D74t2:77 9. all4647 gi|17232139 D80t2:78 10. all5091 gi|113954759 D21t2:79 11. alr0267 gi|17227763 D24t2:80 12. all4782 gi|17232274 D48t2:81 13. all1530 gi|17229022 D55t2:82

t2:83 Proteins identified specifically in A. PCC 7120t2:84 1. Uroporphyrinogen decarboxylase gi|17231401 UROD P20t2:85 2. Phycocyanin alpha chain gi|17228025 PCα P64t2:86 3. PSI FeS subunitVII, core rkn center protein gi|17230955 PSI-FeS P67t2:87 4. 30s ribosomal protein S6 gi|17232294 30sRPs6 P49t2:88 5. Nucleotide diphosphate kinase gi|17230894 NDK P42t2:89 6. Probable glutathione S transferase gi|17228619 GST P46, P83t2:90 7. alr0765 gi|17130110 P19t2:91 8. alr3904 gi|17231396 P57t2:92 9. alr0882 gi|17228377 P89t2:93 10. all1009 gi|17228504 P50t2:94

t2:95 Proteins identified specifically in A. L31t2:96 1. Flavodoxin gi|2117434 Fd L74t2:97 2. PSII manganese-stabilzing protein gi|39055 MSP L20t2:98 3. Allophycocyanin alpha gi|17227517 APCα L48, L57, L82t2:99 4. Allophycocyanin beta gi|17229819 APCβ L50, L70t2:100 5. phycoerythrocyanin alpha gi|17228020 PEC L55t2:101 6. Enolase gi|17231030 L12t2:102 7. DNA-directed RNA polymerase subunit beta gi|148240396 RNApoly. L18t2:103 8. Elongation factor Tu gi|17231829 EFTu L8, L9t2:104 9. Similar to peptide synthase gi|17231549 PS L30t2:105 10. Aldoketoreductase gi|17229808 AKR L52t2:106 11. Alpha Glucano transferase gi|17228370 αGT L51t2:107 12. Porin outer membrane protein gi|17228329 OMP L6t2:108 13. N-acyl neuraminate 9 phosphate gi|116071335 NNPS L23t2:109 14. alr3813 gi|17231305 L54t2:110 15. asl0060 gi|75908874 L66t2:111 16. alr4171 giI17231663 L67t2:112 17. alr3277 gi|17230769 L78t2:113 18. alr1850 gi|17229342 L39t2:114 19. all2110 gi|17229602 L19


Please cite this article as: Rai S, et al, Comparative proteomics unveils cross species variations inAnabaena under salt stress, J Prot(2014), http://dx.doi.org/10.1016/j.jprot.2013.12.020


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Similarily spots identified in A. L31 show similarity to Vonwillibrand factor (all2110), CBS domain protein (alr3813), ketolase(alr1850), L-asparaginase II (alr4171) and late competence devel-opment protein (alr3277). However, asl0060 (Pfam_fs: DUF427) didnot show match to any known proteins in the database. Asignificant down regulation of 2.6–7.5 fold was observed forall2110, alr3813, asl0060. Spot corresponding alr3277 increased by2 fold, alr1850 showed 4.9 fold induction and alr4171 registeredinitial increase followedby adecrease of 4.3 fold under salt stress.

Among the eight unknown protein spots corresponding—all3199 (haemerythrin superfamily), all5091 (homolog of actin),all1530 (transaldolase group protein), all1009 (CBS domaincontaining protein), all0872 (signal transduction protein contain-ing NACHT domain), alr0806 (CAB/ELIP/HLIP family protein) andalr0267 (α-chain integrins) showed accumulation under salttreatment while all4782 (with no recognized functional similar-ity) registered 2.6 fold decrease in the presence of salt stress.

Protein spots corresponding to HVCCP, Nut.DBP, CBS, NNPS,PCD,DAPAT,αGT,AKRconstituted the categoryof other proteins.Among these CBS and Nut.DBP were common to all the threespecies, DAPATwas identified inA. doliolum, NNPS, αGT and AKRin A. L31. Two spots (D5, P5) matching with HVCCP showingaccumulation under salt stress aremicroplasmodesmata proteinrequired for connecting heterocyst to the vegetative cell.Nut.DBP, a member of ferritin like superfamily protein Dps [47]showed a transient expression in A. doliolum and A. PCC 7120registering a decrease on day 1 followed by a gradual increase upto 2 fold and thereafter a decrease on seventh day of treatment.Spots corresponding to sulphurmetabolism protein CBS showedaccumulation on all treatment days in A. doliolum and A. PCC7120 (spot no. D65, P65) while an increase followed by a decreasewas observed in A. L31 (spot L62). It is involved in catalyzing thefirst step of the trans-sulfurylation pathway from homocysteineto cystathione. DAPAT, involved in the final steps of lysinebiosynthesis pathway showed decline in A. doliolum under salttreatment. The protein NNPS identified in A. L31 revealedhomology with N-acyl neuraminate 9 phosphate synthase ofSynechococcus and registered 3.3 fold increase in treated cells.While αGT showed accumulation, AKR was initially increasedupto 2.5 fold and then declined to 2.2 fold on day 7 in salt treatedA. L31.
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UNC4. Discussion

High salt concentration inside cells decreases protein hydro-phobicity [48], changes native conformation of proteins anddisrupts catalytic activity of enzymes [49]. The observedpresence/absence and relative abundance of 256 proteinspots in the three Anabaena species under salt along withLC50 dose, physiological variables (PSI, PSII and respiration)and biochemical parameters (ATP and NADPH) clearly pointstoward better salt management in A. doliolum as compared toA. PCC 7120 and A. L31. It requires mentioning that while thenumber of spots investigated does not represent the entireprotein in Anabaena, the spots selected for analysis were thosedisplaying maximum variation in the gels of control andsalt treatment thus exhibiting salt-induced proteomicchanges in Anabaena spp. Of the 83, 89 and 84 identifiedspots in A. doliolum, A. PCC 7120 and A. L31, 52.34% were


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common to the 3 Anabaena species showing difference in theirrelative expression (Figs. 4, 6 and 8). The remaining number ofspots found either in one or any two of the species pointstowards inter-species differences in salt responsive proteins.In addition, on calculating the number of protein spots in eachspecies showing significant decline on prolonged saltexposure (7th day of treatment), a maximum of 33 spotswere from A. L31 covering 26.19% of the total against 6 and 8spots in A. doliolum and A. PCC 7120 respectively. Thisindicated the maximum protein instability in A. L31 desig-nating it as most salt sensitive species among the three. Fig.10 provides an overview of protein diversity (Supplementary 1in Appendix A).

4.1. Photosynthetic apparatus under salt stress

Salinity in general is reported to inhibit photosynthesis andcarbon pool [50–52]. While PSI activity in A. doliolum and A. PCC7120 showed improvement after initial days of salt exposure ascompared to the control (7th day), a continuous decrease in PSIwas recorded inA. L31. PSII activity in contrast showed a gradualdecline in all the three studied Anabaena species. Decrease inabundance of PSII-OEC and MSP (photosytem II proteins) andincrease in PSI-FeS (photosytem I protein) are in accordancewith the physiological findings. The upregulated PSI might beenvisioned to support the damaged PSII and maintain NADPHpool in the cells [53]while inhibition of both PSI and PSII inA. L31reflects amuch severe damage to its photosyntheticmachinery.In addition (i) down regulation of OCP (a photoprotectantdissipating excess energy of LHC) inA. doliolum and upregulationin A. L31, (ii) variation in expression of phycobilisomecorecomplex proteins-APC and PEC, and (iii) increase in PRCLP(phycobilisome complexanchor protein) indicate salt-induceddamage of the light harvesting complex and antenna proteins.Accumulation of flavodoxin in salt treated A. L31and FNR(enzyme catalyzing electron transfer between ferredoxin/flavodoxin and NADP+) in A. PCC 7120 and A. L31 may be arequirement for efficient electron transfer and hence NADPHproduction under salt stress [53–55]. However, the observeddown regulation of FNR in A. doliolum needs further investiga-tion. A common increase in UROD (protein involved in biosyn-thesis of pigment proteins), GSAT (an isomerase participating inporphyrin and chlorophyll metabolism employing pyridoxalphosphate as a cofactor) and PBP (enzyme for synthesis ofpyridoxal phosphate) signifies up regulated pigment synthesismachinery under salt stress [56].

4.2. DNA damage and protein synthesis under salt stress

While little is known about DNA damage under salt stress,reports on nuclear deformation in barley [57] and alteration ofDNA in cotton [58] under high salt seem reasonable becausethe ROS, formed as a secondary effect of salinity, may affectboth sugar and basemoieties in DNA [59]. The observations ofthe present study: (i) increase in proteins-DHO and PBP(linked to purine/pyrimidine synthesis), (ii) rise in the ATP(in A. doliolum and A. PCC 7120) and ATP synth., and (iii)upregulation of NDK (enzymemediating synthesis of nucleotidesby using ATP) clearly indicate use of energy pool and increasedsynthesis of nucleotides for DNA damage repair. Furthermore,



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increase of protein spots corresponding alr3199 (DNAse/nickase),alr3940 (a dps family protein protecting DNA from oxidativedamage, IPR023188), Nut.DBP (protecting chromosomal DNAfrom damage under nutrient-limited and oxidative stress condi-tions) and M.DNA gy. (modulating activity of DNA gyrase) mightbe a coordinated stratagem to combat DNA damage under saltstress.

Salt stress in Anabaena is known to increase proteinsynthesis and refolding of denatured proteins. Proteinsshowing appreciable changes include (i) subunits of ribosomeassembly (30S RPs1 in A. doliolum and A. PCC 7120; 30S RPs6 inA. PCC 7120), (ii) elongation factors (EfTu in A. L31; EfTs in A.doliolum and A. PCC 7120), (iii) post transcriptional regulators(RNA binding proteins D and E in A. PCC 7120 and A. L31), and(iv) molecular chaperons HSP1, DnaK and GroEL. Whileaccumulation of chaperons may indicate accumulation ofunfolded and non-functional proteins [60,61] under saltstress, a general increase in the proteins of transcription andtranslation conceivably compensates the denatured proteinsand helps the synthesis of new stress proteins. Accumulationof RBPD and RBPE in A. PCC 7120 and decline inA. L31 suggestsdifferences in the post transcriptional processing of the twoAnabaena species. The identified PPIase, PNPase and PS arenew proteins to Anabaena salt proteome. PPIase is a molecularchaperone [62] associated in folding of periplasmic proteins;its maximum upregulation in A. doliolum followed by A. PCC7120 and A. L31suggests its decisive role in differentialresponse of the 3 species under salt stress. While PNPase is aprokaryotic degradosome (Pubmed: 11796219), PS is known tobe involved in non-ribosomal peptide synthesis.

4.3. Other metabolic processes under salt stress

Proteins such as G-6PI, FBPase II, FBP aldolase, GAPDH, PGK andenolase are associated with the second, third, forth, sixth,seventh and ninth steps of glycolysis while PD determines thefate of pyruvate to acetyl coenzyme, interlinking glycolysis withthe TCA cycle. In A. doliolum, all identified enzymes except G6PIand FBPase showed an increase on all days of salt treatmentas compared to control. In A. PCC 7120, enzymes like PGK,FBPaldolase, G6PI were slightly decreased on day 1 of saltexposure, but a general increase in all glycolytic enzymes wasvisualized in treated cells than the control. Similarly the listedenzymes in salt treated A. L31 recorded a general increase overcontrol. The above findings attest an enhanced respiration inAnabaena under high salt. Induction of TK and NADPH suggestsoperation of pentose phosphate pathway in cells under saltstress. Stoichiometrically, the Calvin–Benson cycle costsNADPHand pentose phosphate pathway (PPP) employs the sameenzymes to convert hexose and pentose phosphate resultinginproduction ofNADPH for production of ribose-5-phosphate asa precursor for nucleotide synthesis. Induction of other pentosephosphate pathway enzymes—6PGluD and G6Pdehyd furthersupports the above view.

A significant alteration inORandAKRproteins togetherwithrecorded increase in GR, SOD PRX, CAT and GST indicates thatsalt stress upsets the cellular redox status of Anabaena andproduces oxidative stress [63]. While increase in DAPAT, PGly.DandGS indicates enhancedaminoacidmetabolism, appreciableincrease in CBS (catalyzing trans-sulfuration) along with PPB


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possibly will improve sulphur metabolism. The CBS pathwayusing pyridoxal phosphate as cofactor is a pre-requisite forbiogenesis of FeS clusters in proteins like ferredoxins, rieskeproteins, photosystem I, sulfite reductase, nitrite reductase,nitrogenase, glutamate synthase, aconitase, succinate dehy-drogenase and ferredoxin/thioredoxin reductase [64].

4.4. Hypothetical and unknown proteins under salt stress

This study registered 20 hypothetical proteins wherein onlyalr0803, all4050, all4051, alr3090 and alr3199 were foundcommon to all the species. Some of the protein spots likealr0803 and alr3199 displayed large accumulation under saltdeclaring them most essential proteins for the system. Largescale bacteria genome sequencing projects reveal that theseuncharacterized proteins constitute approx. 30–40% of totalsystem proteins [65,66]. This indicates that proteins withunassigned functionality are important players for cell survivaland response and tips-off towards an immense yet undisclosedgene reservoir worth biotechnological exploitation.

ED P5. Conclusion

Taking recourse to the integrated physiological, proteomicand bioinformatic approaches, it becomes evident that saltstress negatively affects photosynthesis, respiration, redoxstatus, oxidative stress, protein synthesis, sulphur metabo-lism and DNA linked processes. The existence of separatestrategy even within species to combat stress is evident fromdifferences in the proteome of the three Anabaena speciesstudied. Such diversity within genera finds support fromMicrocystis aeruginosa [29] and Prochlorococcus [67]. The bettersalt tolerance of A. doliolum in comparison to A. PCC 7120 andA. L31 could be linked to maximum induction of (i) ATP pool,(ii) NADPH level and pentose phosphate pathway, (iii) efficientphotosystem damage repair, (iv) better antioxidative systemand, (v) maximum expression of alr0803, PPIase, M.DNA gy.,alr3090 and alr3199 proteins. The observed variations clearlydemonstrate the existence of separate molecular adjustmentsi.e. “molecular phenome” [33] and supports the concept of 20–30% protein diversity [32] in prokaryotes. Hence, single systemstudies cannot suffice for the species of even the same genera.This reflects an adaptation of the organisms to diverseenvironmental niches [29]. This study recommends compar-ative proteomics over single species for a candid understand-ing of the strategies in place for abiotic stress management.

Acknowledgments

L.C. Rai is thankful to theScienceandEngineeringResearchBoardfor project and DST New Delhi for J. C. Bose National Fellowship.Snigdha Rai thanks the University Grants Commission for SRF,Chhavi Agrawal, Alok Kumar Shrivastava and Prashant KumarSingh thank the Department of Biotechnology, Council ofScientific and Industrial Research and Indian Council of Agricul-tural Research, New Delhi for JRF and SRF. We thank the Headand the ProgrammeCoordinator CAS in Botany and ISLS BanarasHindu University, Varanasi, India for MALDI-TOF/MS analysis.


pmid:11796219


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Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.jprot.2013.12.020.

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Comparative proteomics unveils cross species variations in Anabaena under salt stress

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