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J O U RN A L OF P ROT EO M IC S 9 6 ( 2 01 4 ) 2 7 1 –2 90

Available online at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/jprot

Comparative proteomics reveals association of early accumulated proteins in conferring butachlor tolerance in three N2-fixing Anabaena spp. Chhavi Agrawala , Sonia Sena , Shilpi Singha , Snigdha Raia , Prashant Kumar Singha , Vinay Kumar Singhb , L.C. Rai a,⁎ a

Molecular Biology Section, Laboratory of Algal Biology, Center of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India b Centre for Bioinformatics, School of Biotechnology, Banaras Hindu University, Varanasi 221005, India

AR TIC LE I N FO

ABS TR ACT

Article history:

Butachlor an extensively used rice field herbicide negatively affects the cyanobacterial

Received 12 July 2013

proliferation, yet the molecular mechanism underlying its toxicity in diazotrophic

Accepted 15 November 2013

cyanobacteria is largely unknown. The present study focuses on the comparative proteomics to decode the molecular basis of butachlor toxicity/tolerance in three Anabaena species e.g. Anabaena sp. PCC 7120, Anabaena doliolum and Anabaena L31. 75

Keywords:

differentially expressed proteins from each Anabaena sp. included those involved in

Anabaena

photosynthesis, C, N and protein metabolism, redox homeostasis, and signal transduction.

Butachlor

While early accumulated proteins related to photosynthesis (atpA, atpB), carbon metabo-

2DE

lism (glpx, fba and prk), protein folding (groEL, PPIase), regulation (orrA) and other function

MALDI–TOF MS/MS

(OR, akr) appeared crucial for tolerance of Anabaena L31, the late accumulated proteins in

Early and late accumulated proteins

Anabaena 7120 presumably offer acclimation during prolonged exposure to butachlor. Contrary to the above, a multitude of down-accumulated proteins vis-a-vis metabolisms augment sensitivity of A. doliolum to butachlor. A cluster of high abundant proteins (atpA, groEL, OR, AGTase, Alr0803, Alr0806, Alr3090, Alr3199, All4050 and All4051) common across the three species may be taken as markers for butachlor tolerance and deserve exploitation for stress management and transgenic development. Biological significance Cyanobacteria offer an eco-friendly alternative to chemical fertilizers for increasing productivity, especially in rice cultivation. This study is the first to compare the proteome of three diazotrophic cyanobacteria subjected to butachlor, a pre-emergent herbicide extensively used in rice paddy. Changes in protein dynamics over time along with physiological and biochemical attributes clearly provide a comprehensive overview on differential tolerance of Anabaena species to butachlor. Molecular docking further added a new dimension in identification of potential protein candidates for butachlor stress management in cyanobacteria. This study strongly recommends combined application of Anabaena spp. A. L31 and A. PCC7120 as biofertilizer in paddy fields undergoing butachlor treatment. © 2013 Elsevier B.V. All rights reserved.

⁎ Corresponding author. 1874-3919/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2013.11.015

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1.

J O U RN A L OF P ROTE O M IC S 9 6 ( 2 01 4 ) 2 7 1 –29 0

Introduction

The increasing world population projected to reach up to 9 billion by the year of 2050 and shrinking agriculture land call for boosting agriculture productivity. Two broad options to satisfy this demand are (1) use of high yielding crop varieties (HYVs), and (2) judicious use of agrochemicals. Modern agriculture practices are resorting to increasing use of agrochemicals for augmenting productivity but compelling to compromise with their hazardous impacts on environment and non-target organisms. In view of this, deploying nitrogen fixing microorganisms as biofertilizers has emerged as eco-friendly option to increase crop yield [1–4]. Among the free living nitrogen fixers, cyanobacteria assume significance because they are the largest and most widely distributed photosynthetic diazotrophs contributing to the C and N economy of paddy field soils [5–8]. Approximately 75% of the total algal flora in Indian rice fields is constituted by cyanobacteria [9] the prominent ones being species of Anabaena, Aulosira, Cylindrospermum, Gloeotrichia, Nostoc, Rivularia and others [6]. These agriculturally important diazotrophs are often challenged by a variety of abiotic stresses such as salinity, drought, pesticides, metals and metalloids, ultraviolet radiation, high temperature and others. Herbicides are extremely detrimental to cyanobacteria [10] which share many of the physiological properties of higher plants including the site of herbicide action [11]. Butachlor (N-butoxymethyl-2-chloro-2′,6′-diethylacetanilide) is an extensively used pre-emergent rice field herbicide to control unwanted weeds. Its recommended field dose ranges from 10 μM to 150 μM [12,13] and the LC50 concentrations of butachlor (25, 32 and 36 μM) selected for the present study fall within the above recommended dose. It belongs to a chloroacetanilide class of herbicide infamous as general inhibitor targeting elongases [14] of very long chain fatty acid biosynthesis (VLCFAs) [15,16]. In addition to lipid biosynthesis, butachlor adversely affects various other metabolic processes and redox homeostasis. It is known to induce oxidative stress and genotoxicity in fresh water fish [17–19], frog tadpole [20,21] and Alium [22], negatively affect growth and nitrogen fixation of phototrophic nonsulfur bacteria [23], photosynthesis, protein, RNA, and lipid synthesis of isolated leaf cells of Phaseolus vulgaris L. [24], alleviate glutathione and glutathione associated enzymes in butachlor tolerant plants [25]. It imparts toxicity and mutagenicity in Nostoc muscorum [26], inhibits growth, nitrogen fixation and photosynthesis of many cyanobacterial species including Anabaena doliolum and Nostoc [13,27–29]. Recent years have witnessed the emergence of proteomics as a powerful tool to mirror the changes and unravel the toxicity and acclimation strategies of organisms confronted by abiotic [30,31] and biotic stresses [32]. Proteomics holds out a platform to examine genetic manipulation, environmental adaptations and strain variability [33]. Two dimensional gel electrophoresis coupled with mass spectrometry is an acclaimed tool for qualitative and quantitative assessment of proteomic changes [34]. Cyanobacterial adaptation to stress is coupled with profound changes in proteome repertoire. Over the past years, proteomics has been employed to examine the cyanobacterial response to stresses like heavy metals [35,36], salinity [37–39], heat and UV-B [40–43], Fe starvation [44], and arsenic [45].

Although some efforts have gone into to address butachlor toxicity on selected physiological and biochemical attributes of cyanobacteria [27,29], its impact on cyanobacterial proteomics has remained neglected except the sole report on Aulosira fertilissima [46]. Taking recourse to the above the present study was designed to analyze the proteome of three Anabaena spp.: Anabaena sp. PCC7120 (Anabaena 7120 hereafter), A. doliolum and Anabaena L31 to address the following questions: (i) how does three Anabaena spp. respond to butachlor stress? (ii) which proteins are specifically responsible for sensitivity/tolerance of Anabaena to butachlor? and (iii) if proteins under question are species specific or stress specific. To fine tune our understanding of the molecular basis of butachlor toxicity/tolerance in cyanobacteria, molecular docking of some highly over expressed proteins (known and hypothetical ones) with butachlor was done. A larger goal of this study was to bring into being a holistic view if plants in general and cyanobacteria in particular would provide a unified response or species specific response to stresses and to fish out some butachlor tolerant proteins for the development of transgenic cyanobacterial diazotrophs capable of proliferation in rice paddy fields.

2.

Materials and method

2.1.

Organisms and growth conditions

All three Anabaena spp., Anabaena 7120, A. doliolum and Anabaena L31 were cultivated photoautotrophically under sterile condition in BG11 medium (N2-fixing condition) buffered with Tris/HCl at 25 ± 2 °C under day light fluorescent tubes emitting 72 μmol photon m−2 s−1 PAR (photosynthetically active radiation) light intensity with a photoperiod of 14:10 h at pH 7.5. The cultures were shaken manually 2–3 times daily for aeration.

2.2.

Experimental setup and treatment

The LC50 concentration of butachlor was determined by the plate colony count method [47]. A 0.2 M stock solution of butachlor (commercial grade, EC50%) was prepared in double distilled water and sterilized by passing through the Millipore membrane filter (0.22 μm). Exponentially growing cells of Anabaena 7120, A. doliolum and Anabaena L31 treated with their respective LC50 doses of butachlor were collected at four time points (1, 3, 5, and 7 days). Cells never exposed to butachlor were used as control. Three biological replicates of each Anabaena sp. were used for comparing their proteomes at control, 1, 3, 5 and 7 days of butachlor treatment (Fig. S1; supplementary information).

2.3.

Physiological parameters

Electron transport activities were measured according to the method of Tripathy and Mohanty [48]. The rate of respiration (defined as O2-consumption in dark) was determined by measuring total O2 consumed in the dark for a given time period minus nonspecific O2 uptake [49]. The size of ATP pool was measured by the method of Larson and Olsson [50]. NADPH/NADH level of the cell extract in Tris–Cl (pH 8.0) was measured by recording absorbance at 340 nm [51].

J O U RN A L OF P ROT EO M IC S 9 6 ( 2 01 4 ) 2 7 1 –2 90

2.4.

Assay of enzyme activity and total thiol content

Glutathione reductase (GR) activity was assayed by following NADPH oxidation at 340 nm in a UV/Vis-spectrophotometer as per the method of Schaedle and Bassham [52]. Activity of GST was measured by the method of Habig and Jacoby [53] using 1 mM L-chloro-2,4-dinitrobenzene and 2 mM-GSH by recording absorbance at 340 nm for 5 min at 37 °C. Total thiol content was estimated according to the method of Ellman [55].

2.5.

Protein extraction and 2-DE separation

Protein extraction was performed according to a protocol of Pandey et al. [45]. Growth of the cultures were followed to late-exponential phase where O.D.750 nm = 0.70. Anabaena cells harvested and ground under liquid nitrogen were centrifuged at 12,000 ×g for 45 min. The supernatant containing cytosolic proteins was precipitated with 2DE clean up kit (GE Healthcare Bio-sciences, USA) following the manufacturer's instructions. The precipitated proteins were solubilized in buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT and 1.0% IPG buffer (4–7). The protein concentration was measured according to the method of Bradford using BSA (bovine serum albumin) as a standard (2 mg ml−1) [54]. For IEF traces of bromophenol blue was added to the sample and centrifuged at 19,000 ×g for 10 min. A total of 350 μl of solubilization buffer containing ~800 μg protein sample was incubated with the dry IPG gel strips (pH 4–7 linear gradients 13 cm; GE healthcare, USA) at RT for 12–16 h. Focusing was performed (Ettan IPGphor system, GE Healthcare Bio-science) at 20 °C 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, gradient 1000 V for 1 h, gradient 8000 V for 2 h and finally 8000 V for 13 h. After focusing, the strips were equilibrated for 15 min by first incubating them in an equilibration solution (6 M urea, 30%v/v glycerol, 2% w/v SDS, 50 mM Tris–HCl, pH 8.8 and a trace of bromophenol blue) having 1% w/v DTT, followed by 15 min incubation with 2.5% w/v iodoacetamide in the same equilibration solution instead of 1% DTT. The equilibrated strips were transferred to 12% SDSpolyacrylamide gels, separated in the second dimension at 10 mA for 30 min and then 25 mA for 5 h (Hoefer SE 600 apparatus, Amersham Biosciences, USA). The gels were stained with coomassie brilliant blue R-250 (CBB).

2.6.

Analysis of data

The gels were scanned and protein spots were analyzed by using PDQuest software version 7.1 (Bio-Rad) that include spot detection, quantification, background subtraction, and spot matching between multiple gels. Missing values imputation was done by K-Nearest Neighbor (KNN) algorithm [MATLAB]. After missing values imputation, total spot intensity per gel was used to normalize spot intensities (% of individual spot intensity/Σ% spot intensity of each gel) to compensate for variations between gel replicates. These data sets were also log transformed to reduce the spot volume-spot deviation dependency. The relative spot volumes corresponding to the control and treated samples at the selected time points (three biological replicates for each sample) were compared using

273

Student's t-test. P values less than 0.05 were considered statistically significant. The protein spots with an abundance ratio of more than 1.5 fold at least at one time point were subjected to MALDI–TOF/MS analysis followed by homology search using MASCOT.

2.7. Protein identification by peptide mass fingerprinting and MALDI–TOF MS/MS For identification of differentially expressed proteins, 2-DE spots were excised, washed at least 3 times for 30 min with wash solution (50% acetonitrile and 50 mM ammonium bi carbonate), until the coomassie dye was completely removed. The destained gel pieces were dehydrated in 100% acetonitrile for 5 min and rehydrated in reduction solution (10 mM DTT/ 100 mM ammonium bicarbonate, 50:50 v/v) for 30 min at 50 °C. Reduction solution was removed and samples were then alkylated using 50 mM iodoactamide/100 mM ammonium bicarbonate (50:50 v/v) for 30 min in dark at RT. The gels were incubated with 100% acetonitrile, air dried and covered with digestion solution (25 μg of lyophilized modified sequencing grade trypsin (Promega) per 1 ml of 25 mM ammonium bicarbonate). Following overnight digestion at 37 °C, the tryptic peptides were extracted twice with extraction solution containing 50% ACN and 1% TFA (in water) with continuous agitation for 10 min. Extracts were pooled, transferred to a fresh micro centrifuge tube and vacuum concentrated in a SpeedVac. 1 μl of trypsinized peptide samples were mixed with 1 μl of α-Cyano-4-hydroxycinnamic acid (CHCA) matrix, (2.5 mg/ml in 50% ACN) on the target plate (MTP 384 ground steel, Bruker Daltonics, Germany) and subjected to Auto flex speed MALDI– TOF/TOF instrument (Bruker Daltonics, Germany) having Nd: YAG smart laser beam of 335 nm wavelength for mass spectrometric identification. External calibration was done with peptide calibration standard supplied by Bruker, with masses ranging from 1046 to 3147 Da. Data acquisition and analysis (peak list) was performed using flex analysis and biotools version 3.3 and 3.2 respectively (Bruker, Daltonics). All spectra were smoothened and internally calibrated. The human keratin and trypsin autodigest peptide ions were excluded. The number of miss cleavage permitted was 1. The measured tryptic peptide masses were transferred through MS BioTool program as inputs to search against the taxonomy of other bacteria in the databases using MASCOT algorithm version 2.4.1 (Matrix Science Ltd., UK). The protein database employed was NCBInr (The National Center for Biotechnology Information non-redundant). A combined peptide mass fingerprinting (PMF) and tandem MS/MS was performed with the following MASCOT settings: taxonomy as other bacteria; peptide mass tolerance of ±100 ppm for peptide mass fingerprinting and 200 ppm (for precursor ions) and ±1.2 Da (for fragment ions) for MS/MS, peptide charge setting as +1, alkylation of cysteine by carbamidomethylation as a fixed modification and oxidation of methionine as a variable modification. Monoisotopic masses were obtained using a SNAP averagine algorithm and a signal-to-noise (S/N) threshold of 3. The protein score automatically calculated by the software was −10 × Log (P) where P is the probability that the observed match is a random event. Proteins with MASCOT score > 75 indicated

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identity or significant homology (P < 0.05) to a database entry. Protein scores were derived from ions scores as a nonprobabilistic basis for ranking protein hits. False discovery rate (FDR) estimations were calculated using a reverse target decoy to endure an FDR of no more than 25%. The protein identity was assigned as the protein that produced the highest score, had the best coverage of its peptide sequence with at least two matched peptides and good agreement between experimental and theoretical Mr and pI. To avoid false positives, the peptide sequences with highest scores of each spot were validated by BLAST P analysis.

2.8.

3.

Results

3.1. Growth behavior of three Anabaena spp. under butachlor stress

Statistical analysis

The statistically significant differences between groups were analyzed using Student's t test. A P value of less than 0.05 was considered significant. All values shown in the figures and tables are means ± SD. Principal component analysis (PCA) was performed to examine the relationship between protein abundance and the different time points selected for the study (protein abundance was taken as variables and time points as observations). PCA was performed by open source R-programming language using function prcomp. A biplot was constructed by using the singular value decomposition (SVD).

2.9.

com/web). For docking procedure PDB file of both ligand and protein were uploaded in the docking server and the docking was done for all the proteins separately. The docked ligand protein interaction was visualized in discovery studio visualizer and the interacting residues between the ligand and the protein were traced out.

Butachlor toxicity on three Anabaena species was investigated by measuring their growth and percent survival. The LC50 concentration of butachlor for three species varied e.g., 32, 25, and 36 μM for Anabaena 7120, A. doliolum, and Anabaena L31, respectively (Fig. 1). Based on the LC50 values Anabaena L31

In silico analysis and molecular docking of proteins

The identified proteins were divided into thirteen functional categories as per Cyanobase (http://genomekazusa.or.jp/ Cyanobase/anabaena). PSORT-B version 3.0.2 was used to categorize the proteins as cytoplasmic, cytoplasmic membrane, and periplasmic taking into account the N terminus signal sequence and the hydrophobic regions in the peptide. ExPASy ProtParam to compute the physico-chemical properties like molecular weight and theoretical pI of the butachlor stress responsive proteins. The five most abundant hypothetical and unknown proteins were selected for molecular docking study with butachlor. Their protein sequences were retrieved from cyanobase (http:// genome.microbedb.jp/cyanobase/Anabaena). These sequences were used to search template structure using PDB database (http://www.pdb.org/pdb/home/home.do). Templates with >40% similarity were used for homology modeling of selected proteins using three different homology tools namely Easy Pred 3D [56] (http://www.unamur.be/sciences/biologie/urbm/ bioinfo/esypred/), Swiss-Model [57] (http://swissmodel.expasy. org/workspace/index.php?func=modelling_simple1) and Discovery studio 3.0 module DS MODELLER [58]. The validation and quality assessment of the structures so generated were done using RAMPAGE [59] (http://mordred.bioc.cam.ac.uk/~rapper/ rampage.php). The structures with most suitable scores within the favored, allowed and outlier regions were selected for further study. Active site analysis was done by using Q-site finder [60] (http://www.modelling.leeds.ac.uk/qsitefinder/). Structure of the ligand butachlor was retrieved in the SDF format from pubchem site (http://www.ncbi.nlm.nih.gov/ pccompound) and converted to PDB by using discovery studio visualizer and saved for further analysis. The docking of ligand molecule with the selected proteins was performed using online docking server [61] (http://www.dockingserver.

Fig. 1 – Growth curve of (A) Anabaena sp. PCC 7120 (B) A. doliolum and (C) Anabaena L31 in terms of absorbance at 750 nm; broken lines with solid circles, untreated control cells; and solid lines with hollow circles, butachlor treated cells. Bars indicate ± SD.

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emerged as the most tolerant and A. doliolum as the most sensitive among the three species employed in this study.

3.2.

Physiological and biochemical parameters

Mechanism of butachlor toxicity in Anabaena spp. was examined through measurement of selected physiological and biochemical variables such as activity of photosynthetic electron transport through PSI, PSII and whole chain, rate of respiration, and NADPH and ATP contents. After 1, 3, 5 and 7 day of butachlor treatment all physiological activities except respiration were significantly inhibited. PSI, PSII and whole chain activity in Anabaena 7120 registered significant decline on day 1 (40.6%, 42.72% and 33.91% respectively) which gradually recovered on subsequent days while in A. doliolum and Anabaena L31 all above activities showed continuous decrease over time being maximum in A. doliolum, and minimum in Anabaena L31 (Table 1). A gradual decrease in ATP content was also observed for A. doliolum and Anabaena L31 during entire time course whereas, Anabaena 7120 with a marked recovery in ATP pool after 5 day treatment constituted an exception. Furthermore, the respiration was maximum in A. doliolum followed by Anabaena 7120 and Anabaena L31 (Table 1). A decrease in NADPH level was observed in

Anabaena L31 (58.53%) and A. doliolum (45%). Anabaena 7120, in contrast registered a mild increase (33.89%) following butachlor treatment for 7 days (Fig. 2A). Cellular redox and detoxification status of the test organisms under butachlor stress was assessed in terms of the activity of glutathione S-transferasae (GST), glutathione reductase (GR) and the total thiol content. A. doliolum possessed a higher level of total thiol than Anabaena 7120 and Anabaena L31 (Fig. 2B) under control condition. It is notable that while butachlor treatment produced a mild increase in the total thiol level of A. doliolum (1.22 fold), the induction was better in case of Anabaena 7120 (1.94 fold) and Anabaena L31 (2.37 fold). The GST activity was appreciably increased in all the strains (Fig. 2C); this being maximum in Anabaena L31 (2.49 fold), followed by Anabaena 7120 (2.1 fold) and A. doliolum (1.92 fold) on day 7. Furthermore, GR required to maintain a high GSH/GSSG ratio also registered a slightly higher increase in Anabaena L31 (2.7 fold) than in Anabaena 7120 (2.5 fold) and A. doliolum (2.48 fold) (Fig. 2D).

3.3.

2DE analysis of total soluble proteins

A comparative proteomics analysis was conducted to study the butachlor induced temporal changes in protein profile

Table 1 – Effect of butachlor on physiological parameters of Anabaena sp. PCC7120, A. doliolum and A. L31. Physiological parameter Anabaena PCC7120 PS-I activity (μmol O2 consumed μg protein−1 h−1) PS-II activity (μmol O2 evolved μg protein−1 h−1) Whole chain activity (μmol O2 evolved μg protein−1 h−1) ATP content (ng ATP mg protein−1) Respiration (μM O2 consumed mg protein−1 min−1) Anabaena doliolum PS-I activity (μmol O2 consumed μg protein−1 h−1) PS-II activity (μmol O2 evolved μg protein−1 h−1) Whole chain activity (μmol O2 evolved μg protein−1 h−1) ATP content (ng ATP mg protein−1) Respiration (μM O2 consumed mg protein−1 min−1) Anabaena L31 PS-I activity (μmol O2 consumed μg protein−1 h−1) PS-II activity (μmol O2 evolved μg protein−1 h−1) Whole chain activity (μmol O2 evolved μg protein−1 h−1) ATP content (ng ATP mg protein−1) Respiration (μM O2 consumed mg protein−1 min−1)

Control

1 day

3 day

5 day

7 day

17.46 ± 0.11

10.37 ± 0.02 ⁎ (−40.6)

12.84 ± 0.10 ⁎ (−26.46)

14.64 ± 0.04 ⁎ (−16.15)

19.9 ± 0.05 ⁎ (+13.97)

19.85 ± 0.10

11.37 ± 0.11 ⁎ (−42.72)

13.99 ± 0.08 ⁎ (−29.52)

16.75 ± 0.02 ⁎ (−15.61)

21.21 ± 0.01 ⁎(+6.85)

13.65 ± 0.01

9.02 ± 0.19 ⁎ (−33.91)

9.65 ± 0.09 ⁎ (−29.3)

11.06 ± 0.13 ⁎(−18.97)

13.97 ± 0.02 ⁎ (+2.34)

27.62 ± 0.02 2.65 ± 0.04

16.01 ± 0.21 ⁎ (−42.03) 3.0 ± 0.17 ⁎ (+ 13.2)

21.99 ± 0.18 ⁎ (−20.38) 3.39 ± 0.12 ⁎ (+28.46)

33.59 ± 0.11 ⁎ (+ 21.61) 4.2 ± 0.26 ⁎ (+ 58.48)

42.67 ± 0.08 ⁎ (+54.49) 4.5 ± 0.51 ⁎ (+69.8)

19.39 ± 0.01

14.49 ± 0.21 ⁎ (−25.27)

13.57 ± 0.11 ⁎ (−30.01)

8.98 ± 0.15 ⁎ (−53.77)

7.13 ± 0.02 ⁎ (−63.22)

17.09 ± 0.04

11.59 ± 0.03 ⁎ (−32.18)

8.90 ± 0.03 ⁎ (−47.92)

7.40 ± 0.22 ⁎ (−56.7)

4.14 ± 0.17 ⁎ (−75.77)

17.66 ± 1.01

10.74 ± 0.12 ⁎ (−39.18)

10.72 ± 0.05 ⁎ (−39.29)

7.89 ± 1.05 ⁎ (−55.32)

4.57 ± 0.13 ⁎ (−74.12)

37.58 ± 0.09 2.5 ± 0.61

29.65 ± 0.04 ⁎ (−21.10) 2.75 ± 0.21 ⁎ (+ 10.0)

21.82 ± 0.01 ⁎ (−41.93) 3.1 ± 0.14 ⁎ (+24.0)

12.83 ± 0.11 ⁎ (−65.85) 3.2 ± 0.28 ⁎ (+ 28.0)

12.1 ± 0.19 ⁎ (−67.61) 4.9 ± 0.31 ⁎ (+96.0)

13.13 ± 0.10

13.75 ± 0.13 ⁎ (+ 4.72)

11.68 ± 0.07 ⁎ (−11.04)

10.01 ± 0.08 ⁎ (−23.76)

7.11 ± 0.02 ⁎ (−45.84)

16.75 ± 0.11

16.23 ± 0.09 ⁎ (−3.1)

13.23 ± 0.06 ⁎ (−21.01)

11.47 ± 0.12 ⁎ (−31.52)

7.77 ± 0.19 ⁎ (−53.61)

16.25 ± 0.03

14.92 ± 0.05 ⁎ (−8.18)

11.85 ± 0.02 ⁎ (−27.07)

10.74 ± 1.01 ⁎ (−33.90)

8.37 ± 0.11 ⁎ (−48.49)

44.36 ± 0.09 3.8 ± 0.09

37.62 ± 0.02 ⁎ (−15.19) 4.09 ± 0.41 ⁎ (+ 7.63)

32.24 ± 0.26 ⁎ (−27.32) 4.5 ± 0.35 ⁎ (+18.42)

30.32 ± 0.12 ⁎ (−31.65) 5.7 ± 0.06 ⁎ (+ 50.0)

25.43 ± 0.03 ⁎ (−42.67) 6.2 ± 0.02 ⁎ (+63.15)

All values are mean ± SD. Values in parenthesis indicate percent inhibition (−) or stimulation (+). ⁎ Indicates significantly different values (P < 0.05).

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Fig. 2 – Effect of butachlor on (A) NADPH, (B) thiol contents, (C) GST, and (D) GR activity of butachlor treated Anabaena spp. at different time points. Bars indicate ±SD.

of Anabaena 7120, A. doliolum and Anabaena L31 at four time points i.e. 1d, 3d, 5d and 7d. Three set of experiments presenting 2DE protein profile under control, 1, 3, 5 and 7 day of butachlor treatment for each of (1) Anabaena 7120, (2) A. doliolum, and (3) Anabaena L31 were conducted. For each sample at least triplicate gels (a total of 45 gels) giving high reproducibility were performed. The average number of protein spots in control and treated gels were between 788– 846, 200–684 and 848–921 in the three species respectively. The 2D gel images revealed both qualitative and quantitative changes at different time points with respect to control (Table 2). The gels with most significant differences over time (control, 1 day and 7 day) have been displayed as Fig. 3A, B and C in the text while gels of 3 and 5 day treatment are shown in Figs. S2, S3, S5 of the supplementary data. 75 differentially expressed proteins having greater than 1.5 fold changes in spot volume were selected for further study. While A. doliolum possessed the maximum number (44–45) of down-accumulated proteins as compared to Anabaena 7120 and Anabaena L31 (Fig. 4), the Anabaena L31 possessed a large number of up-accumulated proteins on day1 and 3 as compared to Anabaena 7120. Contrary to this the long term exposure (5 and 7 day) produced a higher number of differentially expressed and up-accumulated proteins in Anabaena 7120 than Anabaena L31 (Fig. 4).

3.4. Identification and functional classification of differentially expressed proteins A total of 225 protein spots (75 from each set) were identified by MALDI–TOF MS/MS analysis (Table S1, S2 and S3; supplementary information). These proteins were further classified into 13 functional categories as per cyanobase (http://genome. microbedb.jp/cyanobase/Anabaena): (1) photosynthesis and respiration; (2) energy metabolism; (3) central intermediary metabolism; (4) transcription; (5) translation; (6) cellular process; (7) regulatory functions; (8) amino acid metabolism; (9) purines, pyrimidines, nucleosides and nucleotides; (10) biosynthesis of cofactors, prosthetic groups and carriers; (11) transport and binding proteins; (12) hypothetical and unknown proteins and (13) others (Fig. 5). The largest proportion was constituted by hypothetical and unknown proteins (29%, 31%, and 38% in Anabaena 7120, A. doliolum and Anabaena L31 respectively). From the list of identified hypothetical and unknown proteins, a total of 11 were common across the three Anabaena spp. and represented by 16, 17 and 20 spots in Anabaena 7120, A. doliolum and Anabaena L31 respectively. However, 6 of these proteins, e.g. Alr0803 (a signal transduction histidine kinase homologue), Alr0806 (a high light inducible protein homologue), Alr3090 (Mn catalase homologue), Alr3199 (HHE/DNA nickase homologue), Alr4050 and Alr4051 (PRCH domain

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Table 2 – Comparative analysis of differentially expressed protein spots in three Anabaena spp. at different time points. Sample gel

Anabaena PCC 7120 Control 1 day 3 day 5 day 7 day Anabaena doliolum Control 1 day 3 day 5 day 7 day Anabaena L31 Control 1 day 3 day 5 day 7 day

No. of spots ⁎

No. of variable spots ⁎⁎

Quantitative changes Up-accumulated

Down-accumulated

Qualitative changes (unique expressed)

788.5 818.0 822.5 805.0 846.5

± ± ± ± ±

16.26 18.38 38.89 49.49 31.81

0 105 108 109 92

0 57 67 73 56

0 42 35 29 28

0 6 6 7 8

684.5 550.0 264.0 200.0 364.5

± ± ± ± ±

4.94 79.19 29.69 21.21 20.50

0 96 101 88 87

0 50 46 29 20

0 42 50 54 64

0 4 5 5 3

885.5 921.0 848.0 893.0 878.5

± ± ± ± ±

28.08 7.07 14.14 4.24 25.66

0 124 119 104 78

0 95 78 56 42

0 21 32 42 31

0 8 9 6 5

⁎ Total number of spots detected (mean + SD). ⁎⁎ Total number of variable spots, taking control gel of each respective species as reference.

containing protein) registered increased accumulation in all the three species demonstrating their stress acclimation potential under butachlor stress. Furthermore, the functions deduced from sequence homology are compiled in Table 3. Relative spot abundance is expressed as the ratio of spot volumes of high abundant (plus value) or low abundant (minus value) proteins to the control (Figs. S5, S6, S7; supplementary information). Of the total identified proteins, 7 (linker protein cpcC, glutathione S transferase, biotin carboxy carrier protein, putative heterocyst to vegetative cell connection protein, hypothetical proteins Alr0267, Alr0882, and All2110), 9 (phycocyanin alpha chain, ribulose-phosphate 3-epimerase, dihydroxy-acid dehydratase, L-sorbosone dehydrogenase, bifunctional purine biosynthesis protein, hypothetical proteins Alr1063, Alr1850, Alr0617, and Alr3277), and 7 (isocitrate dehydrogenase, nusG, D-3-phosphoglycerate dehydrogenase, carbohydrate selective porin, Alr0765, All5091 and All3398) proteins were detected specifically in Anabaena 7120, A. doliolum and Anabaena L31 respectively. Many proteins were represented by multiple spots on 2D gels. In Anabaena 7120 for instance, 34 protein spots were represented by 16 proteins of which 14 proteins corresponding to 30 spots may be regarded as isoforms having same accumulation pattern, whereas the cpcB and All2110 represented by two spots each on the same gel showed opposite expression from each other (Fig. S5). Likewise, similar pattern was also observed with A. doliolum and Anabaena L31. Notably many isoforms exhibiting opposite expression pattern had different molecular weight and pI, such proteins may be the result of posttranslational modification.

3.5. PCA analysis and comparison of butachlor induced Anabaena proteomes 75 proteins each from three Anabaena spp. on subjecting to principal component analysis demonstrated segregation of

data of significant variability into two principle components (PC1 and PC2) (see table S4; supplementary information). Plotting of these proteins against PC1 and PC2 provided the following information (see Fig. 6 A, B and C): (i) Distribution of proteins of Anabaena L31 into three major clusters, where cluster I, the largest one, with 31 proteins depicting maximum accumulation on day 1. The cluster II (28 proteins) included proteins maximally accumulated on day 3 and cluster III (12 proteins) was comprised of down-accumulated proteins on all time points (Figs. 6A, 7A). (ii) 75 butachlor responsive proteins of A. doliolum were distributed into three major clusters of which cluster I was represented by a group of 36 down-accumulated proteins on all time points. However, Cluster II (23 proteins) and cluster III (16 proteins) contained proteins having maximum accumulation on day 1 and 3 respectively (Figs. 6B, 7B). (iii) Anabaena 7120 possessed four major clusters, where cluster I contained 9 proteins depicting down-accumulation beginning with day 1 to 7 and cluster II (12 proteins) harbored proteins showing maximum accumulation only on day 3. Cluster III (36 proteins) and IV (12 proteins) constituted a large chunk of proteins depicting maximum accumulation on day 5 and 7 respectively (Figs. 6C, 7C). The PCA analysis, thus clearly revealed a time and species specific protein dynamics in three Anabaena species.

3.6. Molecular docking of some high abundant hypothetical proteins The five most over accumulated hypothetical proteins (Alr3090, Alr0803, Alr3199, All4050 and All4051) from all the three Anabaena spp. used to visualize their interaction with the ligand butachlor, gave seven possible structures for each protein (except for Alr0803) following homology modeling using Easypred, Swiss-model and DS-MODELLER. No template

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Fig. 3 – (A). 2DE protein profile of cytosolic proteins of Anabaena PCC7120 from control (C) and cells treated with 32 μM butachlor for 1 day (1), and 7 day (7). Images for 3 and 5 day are given as Supplementary information (Fig. S1). (B). 2DE protein profile of cytosolic proteins of Anabaena doliolum from control (C) and cells treated with 25 μM butachlor for 1 day (1), and 7 day (7). Images for 3 and 5 day are given as Supplementary information (Fig. S2). (C). 2DE protein profile of cytosolic proteins of Anabaena L31 from control (C) and cells treated with 36 μM butachlor for 1 day (1), and 7 day (7). Images for 3 and 5 day are given as Supplementary information (Fig. S3).

Fig. 4 – Number of differentially expressed proteins (P < 0.05 and greater than 1.5 fold difference) in the proteome of Anabaena 7120, A.doliolum and Anabaena L31 in response to butachlor stress for 1, 3, 5 and 7 day (number following P, D and L).

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Fig. 5 – Pie diagram showing functional classification and distribution of all 75 identified proteins in (A) Anabaena sp. PCC7120, (B) A. doliolum and (C) Anabaena L31.

Table 3 – Corresponding homologues of the hypothetical/unknown proteins. Spot no.

Protein ID a

P67 D58, L58 L54

Alr0267 Alr0617 Alr0765

P1, P51, D1, D51, L1, L51 P16, D15, D16,L15, L16 P64 P69, D69, L62, L63 D62, D63 P24, P25

Alr0803

Protein ID b

Protein name

Identity Positive (%) (%)

Anabaena variabilis ATCC 29413 Synechococcus sp. JA-3-3Ab Anabaena variabilis ATCC 29413

97.3 58.9 94

99 71.7 97.5

Anabaena variabilis ATCC 29413

96.3

97.9

Alr0806

Integrins alpha chain CpeS-like protein CBS domain containing membrane protein Ava_0595 Signal Transduction Histidine Kinase (STHK), LytS SynRCC307_0520 High light inducible protein

Synechococcus sp. RCC307

57.9

63.2

Alr0882 Alr0893 Alr1850 All2110

Ava_4486 Ava_4496 Ava_4264 SYNW2427

Anabaena variabilis ATCC 29413 Anabaena variabilis ATCC 29413 Anabaena variabilis ATCC 29413 Synechococcus sp. WH8102

96.8 96.4 94.8 55.6

100 98.4 97.3 66.7

P53, D44, L53 P17, D41, L41 P48, L48

All2893 Alr3090 Alr3091

Ava_1009 Ava_3821 NATL1_17551

96.3 99.1 46.7

98.1 99.6 80

P32, P70, D7, D8, L7, L59 D72

Alr3199

Ava_3897

Alr3277

P9215_19591

Hemerythrin HHE cation binding region Arginase family protein

Anabaena variabilis ATCC 29413 Anabaena variabilis ATCC 29413 Prochlorococcus marinus str. NATL1A Anabaena variabilis ATCC 29413

47.6

66.7

P23, D23, L23

All3324

RPA4231

putative oxidoreductase

55.6

66.7

D20, D21, L20, L21 P22, P75, D65, L65, L68, L69 P8, P54, P58, D6, D48, L6, L8, L57, L70 D17, L17

All3586 All4050

Syncc9902_1685 Npun_F5452

62.5 71.4

68.8 84.9

All4051

Npun_F5451

Nostoc punctiformeATCC 29133

73.6

83.6

All4782

PMM1657

Prochlorococcus marinus MED4

48.6

65.7

P72, D24, L24 L64 L74

All4894 All5091 All3398

Ava_2167 PMT0115 P9303_25371

Thermostablecarboxypeptidase 1 PRC-barrel domain-containing protein AvaK PRC-barrel domain-containing protein Clp protease ATP-binding subunit, ClpX Beta-Ig-H3/fasciclin dTDP-glucose-4,6-dehydratase short chain dehydrogenase

Prochlorococcus marinus str. MIT 9215 Rhodopseudomonas palustris CGA009 Synechococcus sp. CC9902 Nostoc punctiformeATCC 29133

Anabaena variabilis ATCC 29413 Prochlorococcus marinus MIT9313 Prochlorococcus marinus MIT 9303

97.9 53.3 80.0

99.3 66.7 80.0

a b

Ava_2778 CYA_2807 Ava_4660

Organism

Protein ID of Unknown/Hypothetical proteins. Protein ID of the homolouges.

Universal stress protein A Peptidase C56, PfpI Transketolase, central region Possible type II alternative RNA polymerase sigma factor TPR repeat-containing protein Mn containing catalase orotidine 5′-phosphate decarboxylase

98.6

100

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Fig. 6 – PCA of proteins from (A) Anabaena L31 (B) A. doliolum (C) Anabaena 7120. Percentage of total protein variability explained in PC1 and PC2 is given in parenthesis. Protein in red color represent variable whereas text in blue color indicates time points as observation. Ellipse with yellow dashed line indicates clusters.

protein with significant homology was found for Alr0803, therefore, its de novo structure was generated using I-TASSER program (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). The overall stereochemical quality and accuracy of the modeled proteins were evaluated using RAMPAGE program (http:// mordred.bioc.cam.ac.uk/~rapper/rampage.php). The best structures showing maximum homology and most suitable score on rampage were taken for further study. Table 4 compiles a list of these proteins along with their templates used for structure prediction and calculation of percentage of residues falling in favored, allowed and outlier regions of the modeled protein. The selected models (Fig. 8) were further used as a receptor for docking calculations with butachlor ligand. The interaction of the ligand molecule with the protein molecule i.e. docking was analyzed using discovery studio. All the five proteins,

Alr0803, Alr3090, Alr3199, All4050 and All4051 showed docking with butachlor with an estimated free energy of binding as − 2.69, −4.11, −4.56, −5.32, and −3.25 kcal/mol and an interacting surface of 530.38, 538.474, 530.53, 727.404 and 494.196 respectively (Table S5; supplementary information). Since low binding energy indicates low entropy and more stable interaction, the All4050, Alr3199 and Alr3090 depicted better interaction as compared to the other two. The Q-site results demonstrated the presence of ten binding sites within all these proteins (Fig. S8; supplementary information). The active site of Alr3090 where ligand docks (site 7) comprised of ALA32,38,131,132,136, THR33, GLY34,135, SER37, GLN41, TYR115, ASN117 (Fig. 9B) while in case of all other proteins butachlor failed to dock successfully with any of the predicted active sites. Therefore, putative domain prediction for all the proteins was carried out using

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conserved domain database search (CDD search). The Alr3090 possessed a conserved manganese catalase, a ferritin like domain and all the five highly conserved residues of a dimanganese cluster such as Glu35, 65, 133 and His68, 166 thereby suggesting it to function as Mn catalase. Analysis of All4050 and All4051 revealed the presence of a PRCH (photosynthetic reaction center subunit H) domain between amino acid residue 1–145 and 1–132 respectively. This domain is a key regulator of electron transfer between quinones in photosynthetic reaction centers of purple bacteria [62]. Nine (SER45, LYS48, ILE49, VAL57, 79, ASP58, HIS62, GLY76, and LUE80) of the 12 interacting residues of All4050

(Fig. 9D) and 5 (ASP21,59, LEU25, TRY28, and THR56) of the 6 interacting residues of All4051 (Fig. 9E) with butachlor fall within PRCH conserved domain thereby indicating functional impairment of these proteins. Further, Alr3199 contains two hemerythrin domains in the C-terminal region where the first 197 amino acid (N-terminal non-hemerythrin region) encompasses DNase/nickase activity [63]. Butachlor docking depicted interaction with nine residues e.g. SER113, GLY114,169, LEU116, VAL117, ASP165, ALA166, ILE170 and ARG173 of Alr3199 (Fig. 9C) which fall in the N-terminal non-hemerythrin region thus indicating its dysfunction. Contrary to the above, no conserved domain was

Fig. 7 – Functional clustering of butachlor responsive proteins in (A) Anabaena L31, (B) A. doliolum and (C) Anabaena 7120. Green color denotes maximally up accumulated and red color down accumulated proteins.

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Fig. 7 (continued).

found for Alr0803 of which 9 residues e.g. ILE152,237, VAL153, ASP154, GLY155, ARG205, SER235, LEU236, and LYS239 (Fig. 9A) appeared to dock with butachlor, thus difficult to predicting function.

unknown proteins revealed the existence of species specific protein dynamics in three cyanobacteria employed in this study.

4.1.

4.

Discussion

Diazotrophic cyanobacteria being an integral part of rice fields in tropical countries have attracted attention of scientist over the world which resulted into the study of their interaction with agriculturally relevant herbicides. Despite this, impact of butachlor on cyanobacteria has not yet been addressed satisfactorily. Butachlor is known to inhibit growth of plants and microorganisms by negatively affecting photosynthesis, RNA, protein and lipid biosynthesis etc. [13,24,27–29]. Of the three closely related species of Anabaena, Anabaena L31 appeared more tolerant followed by Anabaena 7120 and A. doliolum as adjudged by their LC50 values for butachlor. Two dimensional gel electrophoresis (2-DE) coupled with MALDI– TOF MS/MS and molecular docking of some hypothetical and

Altered photosynthesis under butachlor stress

Photosynthesis measured by polarographic oxygen assay not only was significantly inhibited by butachlor treatment but also was in good agreement with the accumulation profile of photosynthesis related proteins as also supported by earlier observation in Aulosira fertilissima [46]. Among the photosynthesis related proteins, a large part (~ 54%) was constituted by the antenna proteins acclaimed as most abundant constituent (~ 50%) of the total soluble proteins [64]. Decreased accumulation of phycobiliproteins in Anabaena 7120, despite up-accumulated linker peptides (cpcC, cpcG1) crucial for stability and assembly of phycobiliproteins, indicates sensitivity of antenna complex to butachlor. However, Anabaena L31 having decreased accumulation of phycoerythrin (pecA) and increased phycocyanin (cpcB) and allophycocyanin (apcA, apcB) indicates rapid turnover of these proteins under stress.

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Fig. 7 (continued).

Low abundance of ferredoxin NADP + oxidoreductase (petH) catalyzing the electron flow from ferredoxin to NADP+ both in A. doliolum and Anabaena L31 results in a decreased NADPH/NADP + level thus supporting our physiological data.

A significant decrease in rpaB, an ompR type DNA binding response regulator was also observed in the above two species under butachlor stress. A low copy number of rpaB has been reported to boost electron transfer from phycobilisomes to PSI

Table 4 – Ramachandran plot values obtained from RAMPAGE program. Protein name

Template used

Source

Identity

Favored region

Allowed region

Outlier region

All4051

1EYS DS 1EYS DS 1JKU DS – 3R9V

Photosynthetic reaction center from a thermophilic bacterium, Thermochromatium tepidum Photosynthetic reaction center from a thermophilic bacterium, Thermochromatium tepidum Manganese Catalase from Lactobacillus plantarum

57%

93.8%

5.1%

1.1%

57%

93.9%

4.4%

1.7%

31%

96.0%

4.0%

0.0%

– 23%

86.1% 93.0%

5.6% 4.3%

8.3% 2.7%

All4050 Alr3090 Alr0803 Alr3199

Proteolytically truncated form of IpaD from Shigella flexneri bound to deoxycholate

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Fig. 8 – Schematic representation of secondary structure of (A) Alr0803, (B) Alr3090, (C) Alr3199, (D) All4050, and (E) All4051. The α-helix is represented by red, β-sheet by cyan blue and loops by green colors.

in Synechocystis 6803 [65]. Since increased PSI activity is often associated with augmented cyclic electron transport, the decreased accumulation of rpaB may accelerate cyclic electron flow to produce more ATP required for an efficient carbon fixation and protection from photo damage under butachlor stress. Photosynthesis relies on the cooperation of light (photosynthetic electron transport) and dark reactions (CO2 fixation). Rubisco large subunit (rbcL) the very first Calvin cycle enzyme and transketolase (TK) known for marked role in carbon fixation [66] were down accumulated in all the three species rendering carbon fixation as one of the most pretentious pathways under butachlor stress in cyanobacteria. Furthermore, low abundance of phosphoribulokinase (prk) required for RuBP regeneration during Calvin cycle under butachlor stress only in A. doliolum indicates that photosynthesis was most severely affected in this organism. However, a high abundance of phycobiliproteins in A. doliolum need further investigation.

4.2. Increased carbon metabolism at early stage of butachlor stress ensures better growth Carbon metabolism includes various energy generating pathways like glycolysis, TCA cycle and pentose phosphate pathway that fuel all other cellular metabolism by providing metabolic precursors such as carbon skeleton, ATP and reducing equivalents thereby affecting growth and survival of the organism. The time course accumulation pattern of proteins related to

glycolysis namely phosphoglyceratekinase (pgk), fructose-1, 6 bisphosphatase (fbp), fructose bisphosphate aldolase (fba) and TCA cycle e.g. pyruvate dehydrogenase E1 beta subunit (pdhE1B) was quite species specific in the three studied organisms. Anabaena L31 showed early accumulation of these proteins reflecting an early shock response during which organism tries to synthesize enough metabolic initials to sustain cellular metabolism. While in Anabaena 7120 their late accumulation (on day 5 and 7) could be an acclimation strategy to replenish the damage manifested during initial stage of butachlor stress. Contrary to this, decreased abundance of these metabolic proteins in A. doliolum indicates escalated butachlor toxicity in this cyanobacterium. Subunits of ATP synthase (atpA and atpB) also followed a similar pattern of accumulation across the three species. An increased accumulation of adenylate kinase (ADK) was observed at two different time points in all Anabaena species. High abundance of ATP synthase and adenylate kinase required for ATP synthesis from ADP, suggests a positive impact of butachlor on energy metabolism. In contrast to the above a decrease in cellular ATP over time (Table 1) may be due to its requirement to repair the metabolic variables impaired by butachlor stress.

4.3.

Other cellular metabolism under butachlor stress

Since nitrogen assimilation and carbon metabolism are tightly coordinated in cyanobacteria effect on C metabolism would affect nitrogen metabolism as well. In A. doliolum, this

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Fig. 9 – Hydrophobic interactions between butachlor and protein: (A) Alr0803, (B) Alr3090, (C) Alr3199, (D) All4050, and (E) All4051(amino acid residues in blue color, green hatched lines represent hydrophobic interactions).

proposition was corroborated by a pronounced decrease in gutamine synthetase (GlnA), a key enzyme for ammonia assimilation in heterocyst [67]. In contrast, Anabaena 7120 exhibited large increase in abundance of glnA whereas Anabaena L31 showed a transient increase on day 1. Increase of glnA is often correlated with nitrogen deprivation [68]. Decrease in phycobiliproteins and heterocyst to vegetative cell connection proteins (fraH) in Anabaena 7120 and increased abundance of cyanophycinase (cphB2) in A. doliolum and Anabaena L31 indicate the onset of N-starvation as a result of repressed growth. In addition to above, proteins such as 3-phosphogluconate dehydrogenase (PGDH), argininosuccinate synthetase (argG)

and dihydroxy acid dehydratase (DHAD) catalyzing biosynthesis of serine, arginine and branched chain amino acid respectively and nucleic acid biosynthesis proteins (e.g. purine biosynthesis protein purH and dihydroorotase) were largely down accumulated reflecting a butachlor mediated compromised amino acid and nucleic acid metabolism across the three Anabaena species.

4.4.

Butachlor stress negatively affects protein biosynthesis

Different stresses are generally responded by an efficient regulation of gene expression that includes mRNA transcription, processing, translation and protein folding. A number of

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butachlor responsive proteins related to transcription and translation were observed in the proteome of Anabaena. The polynucleotide phosphorylase (PNPase) functions in RNA processing [69–71] and its presence appears indispensible under low temperature [72] and phosphorus deprivation [73]. In coherence with the above, the observed increase of PNPase in Anabaena 7120 might offer protection during stress while the reverse appears true for A. doliolum. Proteins involved in translation process such as EF-Tu, EF-Ts, EF-G, peptidyl prolyl cis/trans isomerase (PPIase) and 30S ribosomal subunit S2 (rpsB) showed dramatic decrease in A. doliolum while late accumulation in Anabaena 7120 and only transient increase in Anabaena L31. Since, translation is a highly energy demanding process and consequently one of the main targets to be inhibited under abiotic stresses including butachlor. Molecular chaperones are responsible for protein folding, assembly, translocation, degradation and also assist in the refolding of damaged proteins under stress. Their overaccumulation correlates positively with increased tolerance under stress. Results indicate early accumulation of groEL in A. doliolum (after 1 day) and Anabaena L31 (after 3 day) while on 7th day in Anabaena 7120. The other two chaperones e.g. the trigger factor (Tig) and Dnak perform cooperative folding of newly synthesized proteins [74] and deletion of both causes synthetic lethality in Escherichia coli [74–76]. In view of the above, inhibition of Tig has to be compensated by DnaK accumulation in Anabaena 7120. However, one of the reasons for an increased sensitivity of A. doliolum to butachlor could be the down accumulation of both DnaK and Tig.

4.5.

Induction of oxidative stress under butachlor stress

The high abundance of superoxide dismutase (sodB) on day1 in A. doliolum and day 3 and 5 in Anabaena L31 indicates butachlorinduced oxidative stress in the test organisms. Furthermore, a significant down accumulation of 1-cys peroxiredoxin (AhpC) and up accumulation of All3090 (homologue of catalase) as observed in all three Anabaena species appear logical for the decomposition of H2O2. Catalase works more efficiently under lofty H2O2 concentration than peroxiredoxin [77], thus augmented accumulation of catalase mirrors increased accumulation of H2O2 under butachlor stress. An appreciable increase of stress responsive oxidoreductase (OR) in all three species as also reported for arsenic [45] and desiccation stress [78] suggests operation of defense mechanism in the cell to fight against butachlor. Earlier studies point toward participation of aldo/ keto reductase (AKR) in oxidative defense and transcriptional regulation [79] in plants [80–82] and cyanobacteria [83]. An increased accumulation of AKR in Anabaena L31 and Anabaena 7120 but appreciable decrease in A. doliolum was observed. The low abundance of All1124 encoding probable glutathione S-transferase in Anabaena 7120 is at variance with our biochemical results presenting enhanced GST activity upon butachlor treatment. This may be due to the presence of seven GST isoforms in Anabaena 7120 genome. Mounting evidence suggest that GST catalyzed glutathione conjugation is the primary mode of butachlor detoxification [84,85] hence increased GST confers tolerance [25,85,86]. In view of the relationship between GST, GR and thiol, the activities of GST, GR and total thiol

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content were estimated in all the three selected species. A maximum increase in GST, GR and thiol in Anabaena L31 followed by Anabaena 7120 and A. doliolum was observed. These results adequately support the increased tolerance of Anabaena L31 to butachlor over the other two species.

4.6.

Effect on regulatory and other proteins

Cyanoabcteria like other prokaryotes harbor two component signal transduction system e.g. histidine kinase and a response regulator for stimulus perception and gene expression regulation under environment stress. The response regulator orrA identified in butachlor stressed proteome, showed a significant increase in both Anabaena L31 and Anabaena 7120. orrA is known to regulate the expression of alpha-glucanotransferase (AGTase) [57] involved in carbohydrate metabolism and synthesis of cryoprotectants [87]. An increased accumulation of AGTase observed across the three species under butachlor stress appears relevant because it is known to protect cells from stresses like low temperature [57,87–89], salt, osmotic stress [57] and desiccation [90]. Increased expression of NusG (2.6 fold) known to increase transcription elongation rates and anti termination by decreasing pausing [91] in Anabaena L31 may be regarded as an adaptive strategy for maintaining RNA transcript level during stress; increased transcript level of NusG is already known for low temperature stress in Synechocystis PCC6803 [92]. Biotin carboxyl carrier protein (accB) significantly up accumulated in Anabaena 7120, constitutes one of the four components of acetyl CoA carboxylase that catalyzes the first committed step of fatty acid biosynthesis. Considering that very long chain fatty acids (VLCFAs) are the primary target of chloroacteanilides, the high abundance of accB could be to ameliorate butachlor toxicity.

4.7. Molecular docking of butachlor with hypothetical and unknown protein Based on the presence of conserved domains and information available in literature [63,93], the high abundance hypothetical proteins such as putative catalase (Alr3090), PRC domain containing proteins (All4050 and All4051), and Alr3199 are envisioned to support the antioxidative defense system, photosynthesis and DNA damage repair respectively. Molecular docking revealed that butachlor binds with conserved domains or N-terminal region (in case of Alr3199) which are primarily responsible for performing such functions. Thus, in the event of binding of butachlor with the hypothetical proteins Alr3090, All4050, All4051 and Alr3199, the cyanobacteria would be required to produce them in high abundance (see Figs. S5, S6, S7, 6) to counteract butachlor stress. Furthermore, Alr0803 having 97% similarity with the signal transduction histidine kinase of Anabaena variabilis showed a considerable docking with butachlor. It requires mentioning that up accumulation of Alr0803 has been reported under a variety of stresses like copper [36], arsenic [45], low temperature [87], desiccation [90], and salt [94] which are known to cause oxidative stress hence its up accumulation under butachlor is quite likely. Since no conserved domain could be identified in this hypothetical protein, it is hard to predict its function under butachlor stress.

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Conclusions

Comparative proteomics of three closely related diazotrophic Anabaena spp. under butachlor stress provided the following information: (1) existence of species specific protein dynamics in the test organisms, (2) significant inhibition of proteins related to photosynthesis, amino acid, nucleic acid and protein biosynthesis across the three species suggesting them to be marker of sensitivity to butachlor, (3) high accumulation of some known, hypothetical and unknown proteins suggests their role in stress tolerance in the three species studied. Taking recourse to the above it was tempting to recommend application of a mixed population of Anabaena L31 and Anabaena PCC7120 as an efficient biofertilizer in paddy fields receiving butachlor treatment. Molecular docking of butachlor with a group of up accumulated proteins requires further investigation of their potential in stress management and transgenic development.

Acknowledgments L.C. Rai is grateful to the Science and Engineering Research Board, DST, New Delhi for the project, and to J. C. Bose National Fellowship. Chhavi Agrawal thanks the Department of Biotechnology, New Delhi, for senior research fellowship (SRF), Sonia Sen, Snigdha Rai, and Prashant Kumar Singh thank the CSIR and UGC New Delhi for JRF and SRF. We thank Mr. Anubhav Shukla for helping in the statistical analysis. We thank the Head and the Programme Coordinator CAS in Botany and ISLS Banaras Hindu University, Varanasi, India for MALDI–TOF/MS analysis.

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2013.11.015.

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