REGULAR PAPER

Salt and UV-B induced changes in Anabaena PCC 7120:physiological, proteomic and bioinformatic perspectives

Snigdha Rai • Shilpi Singh • Alok Kumar Shrivastava •

L. C. Rai

Received: 10 April 2013 / Accepted: 23 September 2013 / Published online: 11 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract This study examines response of Anabaena sp.

PCC 7120 to salt and UV-B stress by combining physio-

logical, biochemical, proteomics and bioinformatics

approaches. Sixty five significantly altered protein spots

corresponding to 51 protein genes identified using MALDI-

TOF MS/MS were divided into nine functional categories.

Based on relative abundance, these proteins were grouped

into four major sets. Of these, 27 and 5 proteins were up- and

downregulated, respectively, both under salt and UV-B

while 8 and 11 proteins showed accumulation in salt and

UV-B applied singly. Some responses common to salt and

UV-B included (i) enhanced expression of FeSOD, alr3090

and accumulation of MDA indicating oxidative stress, (ii)

accumulation of PDH, G6P isomerase, FBPaldolase, TK,

GAPDH and PGK suggesting enhanced glycolysis, (iii)

upregulation of 6-PGD, 6PGL and NADPH levels signify-

ing operation of pentose phosphate pathway, (iv) upregu-

lation of Dps, NDK and alr3199 indicating DNA damage,

and (v) accumulation of proteins of ribosome assembly,

transcriptional and translational processing. In contrast,

enhanced expression of RUBISCO, increased glycolate

oxidase activity and ammonium content under salt signify

the difference. Salt was found to be more damaging than

UV-B probably due to a cumulative effect of ionic, osmotic

and oxidative damage. A group of proteins having common

expression represent decreased toxicity of salt and UV-B

when applied in combination.

Keywords Anabaena PCC 7120 � Salt and UV-B

stress � Comparative proteomics � 2-DE � MALDI-

TOF MS/MS � Bioinformatics

Abbreviations

2DE Two-dimensional gel electrophoresis

ACN Acetonitrile

CHAPS 3-[(3-cholamidopropyl)dimethy

lammonio]-1-propanesulfonate

CHCA Cyano-4-hydroxycinnamic acid

DTT Dithiothreitol

MALDI-TOF-MS Matrix-assisted laser desorption

ionization–time of flight–mass

spectrometry

PMSF Phenyl methyl sulfonyl fluoride

SDS Sodium dodecyl sulfate

PAGE Polyacrylamide gel electrophoresis

ROS Reactive oxygen species

TBA 2-Thiobarbituric acid

MDA Malonildialdehyde

SOD Superoxide dismutase

CAT Catalase

APX Ascorbate peroxidase

GR Glutathione reductase

NBT Nitro blue tetrazolium

CAT Catalase

GR Glutathione reductase

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11120-013-9931-1) contains supplementarymaterial, which is available to authorized users.

S. Rai � A. K. Shrivastava � L. C. Rai (&)

Molecular Biology Section, Centre of Advanced Study in

Botany, Banaras Hindu University, Varanasi 221005, India

e-mail: lcrbhu15@gmail.com; lcrai@bhu.ac.in

S. Singh

Department of Bioinformatics, Shobhit University, Meerut, India

123

Photosynth Res (2013) 118:105–114

DOI 10.1007/s11120-013-9931-1

Introduction

Anabaena, a filamentous heterocystous cyanobacterium

inhabiting paddy fields (Singh 1961) fixes *20–30 kg

N/ha/season and supports crop productivity (Chaurasia and

Apte 2011). Occupying the top crust of water logged paddy

fields, it undergoes continuous exposure of environmental

vagaries as higher plants do. Cyanobacteria in general and

Anabaena in particular have been extensively studied

under nutrient limitation, heat, cold, salinity, UV-B, metals

and other abiotic stresses. Unfortunately most of the

stresses investigated were ‘‘single stress’’ type, hence they

fail to reflect the conditions prevalent in the paddy fields

(Latifi et al. 2005; Rajaram and Apte 2008; El-Fahmawi

and Owttrim 2007; Srivastava et al. 2011, Mishra et al.

2009; Bhargava et al. 2006; Pandey et al. 2012). The

combined effect of two abiotic stresses is unique and

cannot be extrapolated from the responses to each of them

when applied individually (Mittler 2006). It is logical to

assume that molecular signaling pathways and the complex

network of inter-related genes may synergize and/or

antagonize each other’s effects. Combination stress studies

in Synechocystis PCC 6803, Microcoleus vaginatous,

Anabaena doliolum (Hagemann et al. 1991; Srivastava

et al. 2006; Mishra et al. 2008; Lan et al. 2010) are among

the few reports in cyanobacteria. Anabaena PCC 7120, the

model organism with fully sequenced genome closely

resembles higher plants photosynthesis, survives under a

wide array of abiotic stresses and apt for genetic manipu-

lation (Chaurasia and Apte 2008), was chosen for exploring

the molecular basis of single and combined stress impacts.

Among the agriculturally important abiotic stresses,

salinity and UV-B are of particular interest. Salinity affects

19.5 % of irrigated and 2.1 % of dry agricultural land

worldwide (FAO 2000). It is known to upset osmotic and

ionic balance within cells (Fernandes et al. 1993) leading to

arrested growth, oxidative damage, arrested growth and

finally death (Foyer and Noctor 2003; Tijen and Ismail

2006). Likewise, increase in UV-B (280 and 320 nm) due to

ozone depletion has engrossed scientific attention in recent

years. High level of UV-B radiation may lead to an

increased production of ROS, oxidative stress and damage

to DNA, RNA and aromatic proteins (He et al. 2002).

Analysis of salt and UV-B stresses revealed some common

effects such as damage of photosynthetic pigments,

increased respiration, protein denaturation and altered car-

bohydrate metabolism (Srivastava et al. 2005; Bhargava

et al. 2007; Sudhir et al. 2005; Donkor and Hader 1996;

Chen et al. 2006; Gao et al. 2009). However, there is a

complete lack of data on the combined effect of salt and UV-

B in cyanobacteria in general and Anabaena in particular.

Anabaena exhibits wide distribution across a range of

salinity (Srivastava et al. 2009) and reported to enhance

osmolyte synthesis under high salt (Salerno et al. 2004;

Hagemann 2011). UV-B interrupts active DNA repair and

enhance production of compounds like MAA and scytone-

min (He et al. 2002). Taking recourse to the fact that (i) both

salt and UV-B trigger anti-oxidative defense system, (ii)

salt-induced osmoprotectants (Hagemann 2011) are chem-

ical chaperone (Diamant et al. 2001) function as molecular

chaperons, and (iii) accumulation of heat shock proteins and

transcription factors (hsp and hsf) are of common occur-

rence, it was exciting to find out (i) as how Anabaena

responds to combined effects of salt and UV-B, (ii) how

their combined effects differ from individual effects, and

(iii) which of the two stresses is more detrimental to Ana-

baena PCC 7120. In view of high salt stress causing ionic,

osmotic and oxidative imbalance in cells, it was envisioned

more detrimental than UV-B. This paper suggests that

combination stress studies may be more critical for a holistic

view of stress management in cyanobacteria.

Materials and methods

Organism culture conditions, experimental design

and stress application

Anabaena sp. PCC 7120 was cultured as per Pandey et al.

(2012). The single and combined salt and UV-B treatments

were given at their LC50 doses as determined by the plate

colony count method (Rai and Raizada 1985). A 5.0 M

stock solution of NaCl was used for salt treatment. UV-B

radiation intensity of 12.9 m Wm-2 nm-1 was given by

UV-B lamp for 30 min, which corresponded to

2.34 9 106lE cm-2 UV-B dose. For the salt?UV-B study,

the two doses were applied simultaneously. Biochemical

estimations and proteomics for control and all treatments

were performed simultaneously after 24 h of exposure. All

experiments were done in triplicate.

Biochemical assays

Lipid peroxidation was measured in terms of the total content

of 2-thiobarbituric acid (TBA) reactive substances and

expressed as equivalent of MDA (malonildialdehyde),

extinction coefficient 155 mM-1, using the method of

Cakmak and Horst (1991). The total peroxide was measured

according to Sagisaka (1976). Total thiol content was esti-

mated according to the method of Ellman (1959). Proline

was measured following Bates et al. (1973). For assay of

superoxide dismutase (SOD), catalase (CAT), ascorbate

peroxidase (APX) and glutathione reductase (GR) cell

extract was prepared in the cell lysis buffer containing 1 mM

EDTA and 1 % (w/v) poly vinyl pyrrolidone (PVP) with the

106 Photosynth Res (2013) 118:105–114

123

addition of 1 mM ascorbate in case of APX assay. SOD

activity was assayed by monitoring the inhibition in the

reduction of nitro blue tetrazolium (NBT) according to the

method of Gianopolitis and Ries (1977). CAT activity was

determined by measuring the consumption of H2O2

(extinction coefficient 39.4 mM-1 cm-1) according to Aebi

(1984). Activity of GR was determined as per the method of

Schaedle and Bassham (1977). APX activity was determined

at an absorbance of 290 nm (extinction coefficient

2.8 mM-1 cm-1) for 1 min in 1-ml reaction mixture con-

taining 50-mM potassium phosphate buffer (pH 7.0), 0.5-

mM ascorbic acid, 0.1-mM H2O2, and 200 ll of enzyme

extract (Nakano and Asada 1981). Total soluble protein was

measured by the method of Bradford (1976). Glycolate

oxidase was assayed as per the method of Baker and Tolbert

(1966) and the ammonium content according to Bergmeyer

(1965). Please refer to the supplementary file for the detailed

procedure of the biochemical assays.

Measurement of physiological parameters

The rate of respiration was determined by measuring the

total O2 consumed in the dark for a given time period

minus non-specific O2 uptake (Mallick and Rai 1993). The

size of the ATP pool was measured as per Larsson and

Olsson (1979). NADPH/NADH level of the cell extract in

Tris–Cl (pH 8.0) was measured by recording absorbance at

340 nm (Smyth and Dugger 1981). Electron transport

activities (photosystem I & II) were measured according to

Tripathy and Mohanty (1980).

Protein extraction, quantification, 2-DE separation

of protein and 2DE gel analysis

Protein extraction, quantification, 2-DE separation of pro-

tein was performed as per Pandey et al. (2012) 2-DE gel

image analysis software PDQuestTM basic version 8.0.1

(Bio-Rad) was used for gel analysis. Spot quantification in

the control and treated gels was done by measuring spot

volumes (intensity 9 mm2). Sixty five protein spots with

an abundance ratio of at least 1.4-fold were taken as dif-

ferentially expressed proteins, excised manually and

digested according to Shevchenko et al. (2006). The

digested protein spots were subjected to MALDI-TOF/

MS–MS analysis followed by homology search using

MASCOT employing NCBInr (The National Center for

Biotechnology Information non-redundant) as the protein

database. In brief, 1 ll of trypsinized peptide samples was

mixed with matrix, CHCA and spotted on ground steel

plate. The dried spots were subjected to Bruker Daltonics

auto flex speed MALDI-TOF/TOF for mass spectrometric

identification. Data acquisition and analysis were per-

formed using flex control and flex analysis/biotools version

3.2 software, respectively (Bruker, Daltonics). The peptide

MS/MS ion search was performed with the following

MASCOT settings: taxonomy as bacteria; peptide mass

tolerance of ±50 to ±100 ppm for peptide mass finger-

printing and ±1.2 Da for MS/MS, monoisotopic mass,

alkylation of cysteine by carbamidomethylation as a fixed

modification, and oxidation of methionine as a variable

modification.

Bioinformatics predictions

The identified protein sequences were retrieved from cyano-

base (http://genome.microbedb.jp/cyanobase/Anabaena). A

variety of In silico tools were used to predict physicochemical

and functional attributes of hypothetical proteins. Expasy

Protparam was used to decipher the physico-chemical prop-

erties and PSORTb for protein localization study. Motifscan

and CDD were used for predicting functional motifs and

conserved domain study. BlastP was used to infer functional

relationships and gene families by comparing with the

databases.

Statistical analysis

All experimental values are expressed as mean ± SD. The

results of the physiological data were statistically analysed

by one-way ANOVA and the Duncan’s new multiple range

test (DMRT) to determine the significant difference among

group means. A p value B 0.05 was considered statistically

significant (SPSS 16.0).

Results and discussion

Biochemical and physiological responses of Anabaena

PCC 7120 to salt and UV-B stresses

The salt and UV-B-induced oxidative damages were

measured in terms of lipid peroxidation, total peroxide

content, proline and thiol in the control and the treated cells

(Fig. 1a). An increase of 52, 61.29 and 57.14 % in total

peroxide and 24.39, 46.9, 14.84 % in TBARS content was

recorded for salt, UV-B and salt?UV-B, respectively.

Proline and thiol contents were observed to increase by

29.41, 45.45, 63.7 and 9.4, 31.8, 4.92 % for salt, UV-B and

salt?UV-B, respectively, as compared to control. Thiol

plays a prominent role in protecting membrane against free

radical damage (Rennenberg 1995; Nagalakshmi and Pra-

sad 2001). Activity assay for SOD, CAT, APX and GR

(Fig. 1b) followed this trend and further validated the role

of antioxidant defense system under above stresses

(Halliwell and Gutteridge 2006). However, it needs to be

mentioned that except SOD and proline which showed

Photosynth Res (2013) 118:105–114 107

123

additive response, salt and UV-B in combination produced

antagonistic effect. A much higher level of proline in UV-

B and salt?UV-B might be due to its dual role as an osmo-

protectant (Ashraf and Foolad 2007) and ROS detoxifying

antioxidant (Xie et al. 2009).

Physiological parameters (Table 1) indicated inhibited

photosynthesis, increased respiration, elevated ATP and

NADPH levels. Inhibited PSI and PSII can be explained in

light of the work of Mishra et al. (2008) and Thapar et al.

(2007) where (i) pigment biosyntheses are considered pri-

mary targets of salinity, and (ii) genes encoding PS poly-

peptides are repressed under UV-B. This inhibited

photosystem activity also finds support from Giardi et al.

(1997) and Sudhir et al. (2005). An appreciably lowered

PSII activity may be correlated with the susceptibility of

D1 and D2 proteins hindering electron transport between

QA and QB (Perez-Perez et al. 2012; Srivastava et al.

2008; Allakhverdiev et al. 2000). The recorded increase in

respiration under all treatments may be a requirement for

cellular repair and maintenance under stress (Srivastava

et al. 2007; Bhargava et al. 2008). Increased expression of

ATPsyn., ATP content and NADPH under salt and UV-B

(Table 1) might be a requirement (i) to recover and revi-

talize cellular processes after stress, (ii) to support ATP

dependant activity of Clp A and Clp B proteases. In

addition, increase of ATP under salt stress might also be

related with active efflux of sodium to normalize the ionic

state within cells.

Glycolate oxidase assay and ammonium content mea-

surement suggested the operation of photorespiration in

salt-treated Anabaena cells (Fig. 1c). While glycolate

content was increased by 4.96- and 2.34-fold in salt and

salt?UV-B treated cells, respectively, no worthwhile

change was noticed in UV-B alone. The observed increase

in the enzyme finds support from the report of Srivastava

et al. (2011). Ammonium content was estimated with a

view that a considerable quantity is released in photores-

piration during conversion of glycine to serine (Srivastava

et al. 2008).

Proteomic response of Anabaena PCC 7120 to salt

and UV-B stresses

A total of 453, 386, 273 and 233 protein spots were ana-

lysed by PDQuest software in 2-DE gels of control, salt,

UV-B and salt?UV-B treatements, respectively (Fig. 2).

65 protein spots corresponding to 51 protein genes were

divided into four major groups on the basis of their

GO ammonium

Enz

yme

activ

ity/c

onte

nt

0

2

4

6

8

10

12

14

16

SOD CAT APX GR

Enz

yme

activ

ity

0.0

0.1

0.2

0.3

0.4

0.5control salt UV salt+UV

LPO TPRX Proline Thiol

Con

tent

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8control salt UV salt+UV

control salt UV salt+UV

Fig. 1 a Lipid peroxidation

(LPO) and total peroxide

(TPRX) content in mM/mg

protein, Proline and thiol in nM/

mg protein, b SOD (U enzyme/

mg protein), CAT (lM/min/mg

protein), APX (nM/min/mg

protein) and GR (mM/min/mg

protein) and c Glycolate oxidase

(GO) as U enzyme/mg protein

and ammonium content in lM/

cell 9 10-5

108 Photosynth Res (2013) 118:105–114

123

expression pattern. While the first group comprising of 27

proteins depicted common upregulation, the second group

with 5 proteins was downregulated under salt and UV-B

(Table 2). Interestingly however, eight proteins (RBPE,

6-PGL, AKR, RUBISCO, UROD, alr0803, all4050 and

alr3199) were upregulated in salt, but downregulated in

UV-B. Likewise 11 proteins were upregulated in UV-B and

downregulated under salt. The above results represent a

significant difference in the response of Anabaena to two

stresses and call for further investigation. A detailed

information about the identified proteins spots is provided

in Table (Supplementary data) and their relative abundance

is given in Fig. 3.

A common accumulation of Prx and alr3090 (similar to

catalase) proteins substantiates the biochemical findings

and points towards oxidative stress. However, thiol-specific

Table 1 Physiological parameters of Anabaena sp. PCC 7120 under control and salt, UV-B, salt?UV-B treatment

Physiological parameters Control Salt UV-B Salt?UV-B

PS-I activity (lmol O2 consumed lg-1 protein h-1) 9.39 ± 0.05a 8.90 ± 0.03b (-1.06) 7.45 ± 0.05d (-1.26) 7.89 ± 0.07c (-1.19)

PS-II activity (lmol O2 evolved lg protein-1 h-1) 8.80 ± 0.02a 6.47 ± 0.23c (-1.36) 7.32 ± 0.02b (-1.20) 7.30 ± 0.5lb (-1.21)

ng ATP mg protein-1 6.45 ± 0.09c 6.79 ± 0.03b (?1.05) 6.48 ± 0.04c (?1.005) 6.88 ± 0.15a (?1.06)

mMNADPH mg protein-1 0.61 ± 0.04c 0.89 ± 0.21a (?1.46) 0.67 ± 0.14b (?1.10) 0.69 ± 0.32b (?1.13)

Respiration (lM O2 consumed mg protein-1 min-1) 6.27 ± 0.23d 10.06 ± 0.9a (?1.60) 8.20 ± 0.52c (?1.31) 9.07 ± 0.36b (?1.45)

All values are mean ± SD. Values within (? and - signs) parenthesis indicate fold increase or decrease, respectively, as compared to control.

Superscript letters are the result of one way analysis of variance (ANOVA) and denote the significant differences within the dataset

Fig. 2 The 2-DE images of total cytosolic protein extract from Anabaena (a) control, b 100 mM salt (NaCl), c 30 min of UV-B exposure, and

d Salt?UV-B. The protein (500 lg) was applied to pH 4–7 IPG dry strips with 12 % linear vertical SDS-PAGE as the second dimension

Photosynth Res (2013) 118:105–114 109

123

antioxidant protein AhpC (Mishra et al. 2011) declined

under salt and UV-B. Similar observation was reported in

arsenic where high intracellular H2O2 inhibited AhpC and

the detoxification was carried out by upregulated CAT and

OR proteins (Pandey et al. 2012).The increased expression

of OR protein suggested a notable alteration in redox status

under the two stresses. Of the 14 proteins belonging to

energy metabolism, three were found associated with phy-

cobilisome complex formation and pigment biosynthesis.

Among these, PRCLP was found to accumulate under both

salt and UV-B stresses (1.4- to 1.67-fold), while RUBISCO

and UROD depicted increased expression under salt and

decreased expression under UV-B. Five proteins G-6PI,

FBPald., GAPDH, TK and PGK functioning in glycolysis

presented a common upregulation under the two stresses

indicating enhanced glycolysis, hence, respiration which

was attested physiologically also. However, PDH (inter-

links glycolysis to the TCA cycle) was found to decrease in

salt and increase in UV-B and salt?UV-B. A common

accumulation of GAPDH and TK in stressed Anabaena

cells along with enhanced NADPH content indicated

operation of pentose phosphate pathway under short-term

salt and UV-B stresses. The increased level of 6PGL pro-

teins involved pentose phosphate pathway further attests the

above proposition. An increase in the above and conse-

quently the ribose content may be viewed as requirement of

nucleotide synthesis for DNA damage repair. An increase in

ATPsyn. and ATP pool may be viewed to (i) efflux the

accumulated intracellular sodium, (ii) maintain energy

reservoir for chaperone activity (Hoffmann et al. 2010) and

repair cellular metabolism. Thus salt and UV-B stresses

produced oxidative damage, inhibited photosynthesis and

increased respiration in the cells.

The identified AKR and G proteins are novel entries to

salt and UV-B stressed Anabaena proteome. Enzyme AKR

Table 2 List of the identified proteins categorized into four major

groups on the basis of their performance under salt and UV-B stresses

Proteins upregulated

by salt and UV-B

Acronym Spot no.

ATP synthase ATPsyn. 32

Probable GTP-binding protein G protein 62

Glyceraldehyde-3-p-dehydrogenase GAPDH 10, 37

30S ribosomal protein S6 30sRPs6 51

50S ribosomal protein L4 50sRPL4 23

Phycobilisome rod core linker protein PRCLP 21, 22

RNA binding protein D RBPD 53

Chain A Fd-NADP reductase FNR 38, 60

Glutamine synthetase GS 6, 18, 24

Nucleoside diphosphate kinase NDK 50

Fe-super oxide dismutase Fe-SOD 29, 31, 52

ACP phosphodiesterase ACPphos. 35, 42

Peroxiredoxin Prx. 13, 27

ATP-dependant clp protease ClpA 64

Endopeptidase ClpB ClpB 49

Lysyl tRNA synthetase LysRS 28

Response regulator RR 30

NusG 63

NADH dehydrogenase NDH 61

Transketolase TK 16, 34

Glutamate-1semialdehyde

aminotransferase

GSAT 43

Phycobilisome rod core linker protein PRCLP 21, 22

all5218 44

alr0882 20

all3014 58

alr3090 15

alr3904 66

Proteins downregulated

by salt and UV-B

Acronym Spot no.

GroEL 2,3

Heat shock protein 1 HSP1 57

Alkyl hydro peroxide reductase AhpC 36

all1124 46

30S ribosomal protein S1 30sRPs1 11

Proteins upregulated

by salt and downregulated by UV-B

Acronym Spot no.

RNA binding protein E RBPE 46

6-phosphogluconolactonase 6-PGL 65

Uroporphyrinogen decarboxylase UROD 9

Aldo-keto reductase AKR 56

RUBISCO 5, 8, 19

alr3199 12

all4050 47

alr0803 7

Table 2 continued

Proteins downregulated

by salt and upregulated by UV-B

Acronym Spot no.

Phosphoglycerate kinase PGK 4, 45

Nutrient stress-induced DNA binding protein Dps 48

Fructose 1,6 bis phosphate aldolase FBPald. 25

Elongation factor Ts EFTs 39

Oxidoreductase OR 55

Pyruvate dehydrogenase PDH 53

Glucose 6-P isomerase G6PI 33

DnaK 1

all3325 40

Polynucleotide phosphorylase/polyadenylase PNPP/PA 17

6-Phosphogluconate dehydrogenase 6-PGD 26

Protein spots and acronyms for the corresponding proteins have been

provided for reference to the text

110 Photosynth Res (2013) 118:105–114

123

showing upregulation under salt, catalyses reduction of

aldehydes and ketones and is known to efficiently reduce

methyl glyoxal using NADPH (Xu et al. 2006). In addition,

accumulation of Alr3904 and All3014 (predicted glyoxa-

lase proteins) as discussed below might be due to operation

of glyoxalase pathway for methyl glyoxal detoxification

but requires further investigation. G protein acts as

molecular switch, involved in transmitting signals inside

cell, was enhanced under both the stresses. Besides, the

protein ACPphos., commonly upregulated under the two

stresses participates in pantothenate and CoA biosynthesis.

A common decline in DnaK, GroEL and HSP1 may be

due to their utilization in active refolding of proteins. The

ClpA and B proteins (Clp protease family) regulate protein

turnover and remove denatured polypeptides (Clarke

1999). LysRS catalyses the formation of lysyl transfer

RNA which inserts lysine into proteins (Freist and Gauss

1995) was found to be significantly induced.

Of the seven proteins involved in protein synthesis-

50sRPL4, 30sRPs1 and 30sRPs6 constitute the ribosome

assembly, EF-TS assists elongation of peptide and PNPP/

PA, RBPD and RBPE help in post-transcriptinal processing

and post-translational modification. While 30sRPs6 and

50sRPL4 were found to increase under the two stresses,

though to different degrees, 30sRPs1 showed a common

decrease under stress. PNPP/PA showed accumulation

under UV-B and decline under salt and salt?UV-B treat-

ments. Protein NusG showed appreciable accumulation in

salt and UV-B stresses. NusG like NusB (Kumari et al.

2009) influences transcription termination and anti-termi-

nation under stress. In the light of the limited number of

proteins investigated, it is difficult to propose existence of

broad difference in the two stresses with respect to tran-

scription, translational and protein folding in the

cyanobacterium.

Four proteins—NDK (enzyme mediating synthesis of

nucleotide triphosphates from nucleotide diphosphates

using ATP), Dps (DNA binding protein protecting DNA

from oxidative damage; Narayan et al. 2010), PBP

(involved in purine biosynthesis) and alr3199 (having a

possible role in stress-induced DNA damage and repair in

Anabaena; Padmaja et al. (2011) have role in nucleotide

synthesis and damage repair. Of these, while Dps registered

a decline in salt, a remarkable upregulation of others under

DnaK GroEL HSP1 ATPsyn. G Protein PGK G6PI 6PGD GAPDH FBP ald. NDH TK EfTs 30sRPs1 RBPD RBPE AKR

Rel

ativ

e ab

unda

nce

0

50

100

150

200

250

300

ControlSaltUV-BSalt+UV-B

Prx FeSOD OR AhpC GS NDK PBP 6PGL PRCLP UROD GSAT PDH 30sRPs6 50sRPL4 PNPP DPS ACP Phos.

Rel

ativ

e ab

unda

nce

0

50

100

150

200

250

300

350

FNR ClpA ClpB NusG RUBISCO RR LysRS alr0803 alr0882 all5218 all3014 alr3904 alr3199 all4050 alr3090 all1124 all3325

Rel

ativ

e ab

unda

nce

0

50

100

150

200

250

Fig. 3 Changes in expression (relative abundance) of the identified proteins in Anabaena sp. PCC 7120 under control, salt, UV-B and salt?UV-

B measured as spot volumes (mean ± SD) using PDQuest

Photosynth Res (2013) 118:105–114 111

123

single and combined treatments was observed. It was

exciting, therefore, to note that salt stress like UV-B

appears to be linked to DNA-associated processes and

warrants further investigation.

In silico analysis of salt and UV-B induced hypothetical

proteins

Of the ten proteins in this category alr0882, all5218, all3014,

alr3904 and alr3090 showed a common upregulation under

the two stresses. Among these, alr0882 is a universal stress

protein (uspA). Heterologous expression of uspA in E.coli

DuspA offered tolerance to multiple abiotic stresses

including salinity where it registered 41.3 % increase in

specific growth rate over the mutant (Shrivastava et al.

2012). Upregulation of Alr3904 and All3014 (predicted

glyoxalase II) along with AKR might be due to operation of

methyl glyoxal detoxification pathway under salt stress.

Alr3090 was predicted to be catalase, and all5218 as putative

modulator of DNA gyrase. Hypothetical proteins Alr0803,

Alr3199 and Alr4050 showed significant accumulation

under salt, but a decrease in UV-B. Alr3199 has been char-

acterized as hemerythrin protein having a possible role in

stress-induced DNA damage and repair in Anabaena (Pad-

maja et al. 2011). Appreciable accumulation of Alr3199

under salt and negligible effect under UV-B entails its stress

specific response. Similarly, All4050 was predicted to be

PRC-barrel domain-containing protein while All1124,

showing a common decline under salt and UV-B was

homologous to glutathione S transferase.

Interestingly, while most biochemical and physiological

parameters indicated a more damaging effect for salt than

UV-B, their combination mitigated the salt toxicity. A

similar situation was observed in protein expression study.

Proteins corresponding to (i) antioxidants Alr3090, Prx and

FeSOD, (ii) peptide translation 30sRPs1, 50sRPL4 and

PNPP/PA, (iii) photosynthetic pigment proteins PRCLP

and UROD, (iv) hypothetical all5218 and all3014 showed

antagonism in salt?UV-B. Likewise, NusG, ACP Phospo.

and RR showed a reduced expression in salt and UV-B

combination than their individual treatments. Thus, the

above results indicated antagonistic effect of salt and UV-B

when used in combination and supports the view that

stresses in combination may produce impacts which cannot

be extrapolated from single stress studies.

Appendices

Detailed information of the identified protein spots (pro-

vided as Supplementary Table) and the particulars of in

silico tools for hypothetical protein function prediction are

provided as supplementary data.

Acknowledgments L. C. Rai is thankful to SERB for the project

and J. C. Bose National Fellowship. Snigdha Rai and Alok Kumar

Shrivastava thank the University Grants Commission and the Council

of Scientific and Industrial Research, New Delhi for SRF. We thank

the Head and the Programme Coordinator CAS in Botany for facili-

ties and ISLS Banaras Hindu University, Varanasi, India for MALDI-

TOF/MS analysis.

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