Halimione portulacoides (L.) physiological/biochemical characterizationfor its adaptive responses to environmental mercury exposure

Naser A. Anjum a, Mohd Israr b, Armando C. Duarte a, Maria E. Pereira a, Iqbal Ahmad a,b,c,n

a CESAM-Centre for Environmental and Marine Studies, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugalb Department of Microbiology and Immunology, University of North Carolina, School of Medicine, Chapel Hill, NC 27599, USAc CESAM-Centre for Environmental and Marine Studies, Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal

a r t i c l e i n f o

Article history:Received 5 October 2013Received in revised form6 January 2014Accepted 20 February 2014

Keywords:Salt marshMercuryMacrophyteHalimione portulacoidesAntioxidant defense systemPolypeptide pattern

a b s t r a c t

This study investigates largely unexplored physiological/biochemical strategies adopted by salt marshmacrophyte Halimione portulacoides (L.) Aellen for its adaptation/tolerance to environmental mercury(Hg)-exposure in a coastal lagoon prototype. To this end, a battery of damage (hydrogen peroxide, H2O2;thiobarbituric acid reactive substances, TBARS; electrolyte leakage, EL; reactive carbonyls; osmolyte,proline) and defense [ascorbate peroxidase, APX; catalase, CAT; glutathione peroxidase, GPX; glutathionesulfo-transferase, GST; glutathione reductase, GR; reduced and oxidized glutathione (GSH and GSSG,respectively), and GSH/GSSG ratio] biomarkers, and polypeptide patterns were assessed in H. portula-coides roots and leaves at reference (R) and the sites with highest (L1), moderate (L2) and the lowest (L3)Hg-contamination gradients. Corresponding to the Hg-burdens, roots and leaves exhibited a differentialmodulation of damage- and defense-endpoints and polypeptide-patterns. Roots exhibiting the highestHg-burden (at L3) failed to maintain a coordination among enzymatic-defense endpoint responses whichresulted into increased oxidation of reduced glutathione (GSH) pool, lowest GSH/GSSG (oxidized) ratioand partial H2O2-metabolism. In contrast, the highest Hg-burden exhibiting leaves (at L1) successfullymaintained a coordination among enzymatic-defense endpoints responses which resulted intodecreased GSH-oxidation, enhanced reduced GSH pool and GSH/GSSG ratio and lower extent of damage.Additionally, increased leaf-carotenoids content with increasing Hg-burden implies its protectivefunction. H. portulacoides leaf-polypeptides did not respond as per its Hg-burden but the roots did.Overall, the physiological/biochemical characterization of below (roots)- and above (leaves)-groundorgans (studied in terms of damage and defense endpoints, and polypeptides modulation) revealed theadaptive responses of H. portulacoides to environmental Hg at whole plant level which cumulativelyhelped this plant to sustain and execute its Hg-remediation potential.

& 2014 Elsevier Inc. All rights reserved.

1. Introduction

The physiological/biochemical or chemical characterization ofplant responses to metal/metalloid exposure has been accepted asa representative basic step towards revealing plant's tolerance/adaptive potential to metal/metalloid contaminated conditions(Maestri et al., 2010; Anjum et al., 2013a). Owing to its persistence,bio-accumulation, bio-magnification nature and toxicity to biota,mercury (Hg) is severely deteriorating varied environmentalcompartments including marine systems (Pereira et al., 2009;Ahmad et al., 2012). Salt marshes represent marine ecosystem

where salt marsh plants (SMPs) have been widely reported toperform essential ecological functions mainly by minimizing Hg-availability to other biota (Pereira et al., 2009; Anjum et al., 2011,2012a, 2013a,b). Thus far, extensive reports available on differentSMPs-tolerance/adaptation or survival strategies under Hg-contamination scenario have considered mainly the chemicalcharacterization of either sites, plants and/or their organs(Pereira et al., 2009; Anjum et al., 2011, 2012a, 2013a,b). Moreover,though the studies on the exploration of SMP's physiological/biochemical mechanisms underlying their tolerance/adaptation orsurvival under adverse conditions have been considered aprerequisite to affirm plant's: (a) inherent adaptive capabilityunder contaminated salt marshes, (b) cleanup-suitability forcontaminated salt marshes, as well as (c) significance for theelaboration of innovative plant based-remediation techno-logies for Hg-contaminated salt marshes (Anjum et al., 2013a),

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Environmental Research

http://dx.doi.org/10.1016/j.envres.2014.02.0080013-9351 & 2014 Elsevier Inc. All rights reserved.

n Corresponding author at: CESAM-Centre for Environmental and Marine Studies,Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal.Fax: þ351 234 37 0084.

E-mail address: ahmadr@ua.pt (I. Ahmad).

Environmental Research 131 (2014) 39–49

a complete lacuna is perceptible on the physiology/biochemistry ofbasic strategies for SMP's tolerance/adaptation to environmentalHg-exposure.

Plant's capacity to tolerate/adapt contaminated scenarios hasbeen earlier judged by their inherent potential to maintain abalance between the environmental insults (including metal/metalloid)-accrued damages and the counter action of defensivemachinery (Gill and Tuteja, 2010; Anjum et al., 2012b). Addition-ally, the modulation of polypeptide patterns has been widelyreported as a basic but important marker to mirror the extentof damage and the defense system strength which can beeasily visualized using sodium dodecyl sulphate-polyacrylamide-gel electrophoresis (SDS-PAGE) (Sobkowiak and Deckert, 2006;Ahsan et al., 2007). Nevertheless, plant protein-SDS-PAGE hasbeen considered as a fundamental step toward proteomic studies(Sobkowiak and Deckert, 2006; Ahsan et al., 2007). On theperspective of plant's damage responses to Hg stress, photosyn-thetic pigments (chlorophyll; carotenoids) and the membrane-damage traits (such as cellular membrane integrity and lipidperoxidation) have been regarded as sensitive indicators of Hg-modulated events at the organism level (Patra and Sharma, 2000;Zhou et al., 2008). Hg-accrued enhanced generation of reactiveoxygen species (ROS; such as O2

d� , OHd, and H2O2) and subsequentROS-mediated oxidative damages to vital bio-molecules includingnucleic acids, proteins and membrane lipids are clearly perceptiblein literature (Patra and Sharma, 2000; Gill and Tuteja, 2010;Anjum et al., 2012b, 2013c). Nonetheless, in plants, efficient andsynchronous action of various enzymatic (such as catalase, CAT;ascorbate peroxidase, APX; glutathione reductase, GR; glutathioneperoxidase, GPX; glutathione sulfo-transferase, GST) and non-enzymatic (such as tripeptide glutathione, GSH; ascorbate, AsA;carotenoids and tocopherols) components of antioxidant defensesystem directly or indirectly metabolize varied ROS and theirreaction products. Moreover, proteinogenic amino acid andosmolyte-proline has been reported to significantly help plantsadapt to varied stress conditions through osmotic adjustment,stabilization of sub-cellular structures, conferring rigidity to thepeptide chain, scavenging of free radicals and retaining of energy(Sharma and Dietz, 2006). Together, enzymatic and non-enzymaticcomponents of antioxidant defense system and the osmolyte helpplants to maintain cellular metabolism, growth and productivityunder unfavorable conditions (Gill and Tuteja, 2010; Anjum et al.,2012a,b, 2013c). However, exhaustive literature search depicted

absence of reports highlighting a complete physiological/biochem-ical characterization of strategies adopted by salt marsh macro-phyte Halimione portulacoides (L.) Aellen for its adaptation/tolerance to environmental Hg-exposure.

Given the above description, the current study aimed to testthe hypothesis: can the salt marsh macrophyte's physiological/biochemical traits and the polypeptides be modulated byHg-contamination under realistic condition? If yes, to what extentthe coordination is maintained among the physiological/biochem-ical defense and damage mechanisms as well as the polypeptidesin below (roots)- and above (leaves)-ground organs of a sameplant species in order to tolerate and execute its ecologicalfunctions? To test the hypothesis in H. portulacoides, this studyaimed to: (i) determine organ specific Hg-burdens and analyzedamage (H2O2; TBARS; EL; reactive carbonyls; proline) endpointresponses, (ii) investigate the modulation of defense endpoints(APX; CAT; GPX; GST, GR, and (iii) establish the potential relation-ship of previous damage and defense endpoint responses with themodulation of organ-specific polypeptide patterns. Efforts havebeen made to critically cross-talk the aforesaid damage anddefense endpoint responses and polypeptide patterns modulationwith currently and previously determined H. portulacoides organ-specific Hg-burdens (Anjum et al., 2011).

2. Materials and methods

2.1. Study area

The Ria de Aveiro (Portugal) has been used as a coastal lagoon prototype incontext with the current study (Fig. 1). It is a shallow coastal lagoon (45 km length;10 km wide; located along the Atlantic Ocean on the northwest coast of Portugal;400380N, 80440W) with several channels and extensive inter-tidal areas andrepresents a southwest European mesotidal system. Between 1950 and 1994, achlor-alkali industry discharged Hg-rich effluents into one of the remotest branchesthat end in an inner basin (Laranjo basin) (Pereira et al., 1998). Though the Hg-discharge diminished considerably in the last two decades, Hg-concentration in thesurface sediments of Laranjo basin is still much higher than pre-industrial levels(Pereira, 1997). Since no other important sources of contaminants other thanmercury have been reported in this area, Laranjo basin has been regarded as “fieldlaboratory” for assessing mercury toxicity under realistic environmental conditions(Pereira et al., 2009). Additionally, these conditions may also provide an in situmodel in context with the current study for the assessment of physiological/biochemical strategies adopted by H. portulacoides for its tolerance to andHg-remediation service execution under highly Hg-polluted Ria de Aveiro coastallagoon. Along transects defined by the distance from the main Hg-source, three

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sampling sites depicting the highest (L1), moderate (L2) and the lowest (L3)Hg-contamination were selected. Additionally, samples were also collected fromthe Hg-free site (R) (Fig. 1).

2.2. Plant material

The salt marsh macrophyte choosen for the current investigation-Halimioneportulacoides (L.) Aellen. is a dicot, C3, perennial, creeping sub-shrub that attainsheight between 30 and 80-cm, and belongs to family Chenopodiaceae. The plant isfound in acid, neutral and basic soils and can grow in very alkaline and saline soils.It can grow in semi-shade (light woodland) or no shade conditions, and occurs inaerated spots in salt marshes and estuaries along the European Mediterranean andAtlantic coasts. H. portulacoides is one of the most extensively studied salt marshmacrophyte species with respect to its role in metal/metalloid speciation, mobilityand remediation processes due to its worldwide distribution (Anjum et al., 2013a).

2.3. Samplings

H. portulacoides plant samples were collected in five replicates during low tideat three sampling sites depicting the highest (L1), moderate (L2) and the lowest(L3) Hg-contamination along transects defined by the distance from the main Hg-source. Additionally, samples were also collected from the Hg-free site (R). Plantswere dug out carefully with a shovel and later, rinsed well with lagoon water toremove adhered dust particles. All samples were placed into plastic bags andtransported to the laboratory under refrigerated conditions. Once in the laboratory,plants were separated into shoots and roots/rhizomes, and were dipped into liquidnitrogen, powdered and were either used immediately or stored at �80 1C. Thepowdered plant tissues were used for various physiological/biochemical (damageand defense) traits set out in the current study. A composite sample was used fordetermination of Hg concentration.

2.4. Chemical analyses

2.4.1. Plant biomass-mercury determinationMercury concentrations in H. portulacoides roots and leaves were determined

as per the method described previously (Anjum et al., 2011) by atomic absorptionspectrophotometer with thermal decomposition through an advanced Hg analyzer(LECO 254, USA).

2.5. Biochemical analyses

A brief description of the methodologies adopted for various biochemicalanalyses are presented hereunder. However, detailed methodologies adopted fordifferent bio-assays can be found in the ‘Supplementary materials'.

2.5.1. Damage endpoint estimationsMercury-mediated oxidative stress was tested in H. portulacoides by analyzing

tissue (root and leaf)-specific H2O2 level and damages to cell membranes indicatedby the leakage of electrolytes and the peroxidation of membrane lipids andoxidation of proteins. Proline, a proteinogenic amino acid was determined as arepresentative major osmolyte in H. portulacoides tissues.

Root and leaf H2O2 content was determined following the method of Loretoand Velikova, 2001 as adopted and described by Dipierro et al. (2005). Electrolyteleakage (EL) assessment was considered as a measure of cell membranepermeability and cell viability, and was determined following the method asdescribed by Anjum et al. (2013c). The content of thiobarbituric acid reactivesubstances (TBARS) was determined as a status of membrane lipid peroxidationin fresh roots and leaves as per the method adopted and described by Anjumet al. (2013c). For the estimation of organ-specific (leaves–roots)-protein oxida-tion, carbonyls reaction with 2,4-dinitrophenylhydrazine (DNPH)-based metho-dology of Levine et al. (1994) was followed. Root and leaf proline content wasestimated by the method of Bates et al. (1973) and expressed as mmol g�1 freshweight; whereas, the contents of total chlorophyll and carotenoids in leaves wereestimated following the method of Alan (1994) as adopted and described byAnjum et al. (2013c).

2.5.2. Defense endpoint estimationsBoth enzymatic (APX, CAT, GPX, GST, GR) and non-enzymatic (GSH, GSSG,

GSH/GSSG) antioxidant defense components were considered under defenseendpoints. Samples for the enzymatic and non-enzymatic antioxidants protectionassays were prepared following the previously described method (Anjum et al.,2013c). Enzymatic and non-enzymatic antioxidants assays: the activity of APXwas determined by monitoring the AsA decomposition per minute at 25 1C andwas calculated using an extinction coefficient of 2.8 mM�1 cm�1 (Nakano andAsada, 1981) whereas, the activity of CAT was determined by monitoring thedisappearance of H2O2 at 240 nm (Aebi, 1984). The activity was calculated byusing extinction coefficient 0.036 mM�1 cm�1 and expressed as μmol of

H2O2 g�1 fresh weight/min. The activity of GSH-regenerating enzyme-GR wasdetermined by monitoring the GSH-dependent oxidation of NADPH (Foyer andHalliwell, 1976). The activity of GSH-metabolizing enzymes such as GPX (bymeasuring the oxidation of NADPH at 340 nm for 3 min using H2O2 as substrate)and GST (by measuring increase in absorbance at 340 nm for 3 min due to theformation of the conjugate 1-chloro-2,4-dinitrobenzene (CDNB) (substratum)/GSH, catalyzed by GST) was assayed following the methods of Mohandas et al.(1984) and Drotar et al. (1985), as adopted and described by Anjum et al. (2013c).Root and leaf contents of non-enzymatic antioxidant glutathione (GSH; reduced,oxidized, and total glutathione) were determined following the method ofAnderson (1985).

2.6. Protein extraction, quantification and characterization

Root and leaf tissues (0.25 g) were homogenized with phosphate buffer(25 mM, pH 7.0) using a chilled mortar and pestle. The homogenate wascentrifuged at 15,000g for 20 min at 4 1C. Protein concentration was quantifiedby the Coomassie Brilliant Blue G-250 assay (Bradford, 1976). Subsequently,equal amounts (15 mg) of proteins were denatured, loaded onto each lane andseparated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis(SDS-PAGE) (5% and 12.5% acrylamide for stacking and resolving gels, respec-tively) at a constant current of 20 mA using a vertical slab gel (BioRad MiniProtean II, Richmond, CA, USA) (Laemmli, 1970). Separated polypeptides on thegel were visualized by staining with Coomassie Brilliant Blue R-250 solution andsubsequently detained with glacial acetic acid–methanol solution (1:4, v/v). Theproteins fractionated into bands were scanned using the Molecular Imager GelDoc XRþSystem (Bio-Rad).

2.7. Data analysis

SPSS (PASW statistics 18) for Windows was used for statistical analysis. One-way analysis of variance (ANOVA) followed by pairwise multiple comparisonsemploying the Tukey test was executed in order to detect significant data amongtreatments. The data are expressed as mean values7SD of three independentexperiments with at least five replicates for each. The significance level was set at Po0.05.

3. Results

Results depiction has been performed taking into account thedescription of H. portulacoides root and leaf damage and defenseendpoint responses individually. On each individual organ, resultshave been presented describing the significant changes in damageand defense indices and the modulation of polypeptide patternstaking into account both inter-site variations within same organ,and inter-organ comparisons at reference and contaminated sites.In order to highlight significant differences among the testedendpoints, the organ-specific responses have been detailed inthe ‘Supplementary materials'.

3.1. H. portulacoides root damage endpoint responses

3.1.1. Oxidative stress indices and proline levelIndices of oxidative stress such as the contents of H2O2 and

TBARS, percent EL, and also the contents of reactive carbonylsand proline displayed significant increases maximally at L3followed by L1 and L2 (vs. R). Contaminated site comparisonsrevealed significantly lower extents of these traits at L2 (vs. L1and L3) (Fig. 2a–e).

3.2. H. portulacoides root defense endpoint responses

3.2.1. Enzymatic and non-enzymatic antioxidants protectionThe activities of root-enzymatic antioxidants such as APX, CAT,

GPX, GST and GR significantly increased maximally at L3 followedby L1 and L2 (vs. R); whereas, comparisons among contaminatedsites revealed significantly lower activities of APX, CAT, GPX, GSTand GR at L2 (vs. L1 and L3) (Fig. 3a–e). On the perspective of thechanges in non-enzymatic antioxidants, the reduced GSH contentand GSH/GSSG ratio significantly decreased maximally at L3

N.A. Anjum et al. / Environmental Research 131 (2014) 39–49 41

followed by L1 and L2 (vs. R); whereas, significant increase in theoxidized GSH (GSSG) was perceptible maximally at L3 followed byL1 and L2 (vs. R). Comparisons among contaminated sites revealedsignificantly higher content of reduced GSH at L2 (vs. L1 and L3).Taking into account the oxidized GSH level and GSH/GSSG ratiovariations among contaminated sites, significantly lower oxidizedGSH content and GSH/GSSG ratio were displayed at L2 (vs. L1 andL3) (Fig. 4a–c).

3.3. H. portulacoides root-polypeptide patterns

The visualization of SDS-PAGE intensity of H. portulacoides root-protein bands of different kDa revealed differential responses. Newpolypeptides of approx. 225, 100, 90, 60, 45 and 35 kDa wereappeared at L1 (vs. R). At L2, polypeptide of approx. 90, 45 and 35appeared but with reduced intensity; however, at the same site,new polypeptides of approx. 95, 65 and 25 kDa appeared. Poly-peptides of approx. 95, 65 and 45 (with good intensities), and 35and 25 kDa polypeptides (with less intensities)-appeared earlier atL2, were re-appeared at L3. Interestingly enough, polypeptides ofapprox. 90, 60 and 45 which were present at L1 again re-appearedat L3 (Fig. 5).

3.4. H. portulacoides leaf damage endpoint responses

3.4.1. Oxidative stress indices, proline and photosynthetic pigmentlevels

Leaf-EL percent and the contents of H2O2, TBARS and reactivecarbonyls displayed significant increases maximally at L1 followedby L2 and L3 (vs. R); whereas, significant maximum decrease inleaf-proline was depicted at L1 followed by L2 and L3 (vs. R).Contaminated sites comparisons revealed both significantly lower(vs. L1) and higher (vs. L3) EL percent and the contents of H2O2,TBARS and reactive carbonyls at L2; whereas, significantly lowerproline content was perceptible at L2 (vs. L3) (Fig. 6a–e). Photo-synthetic pigments namely chlorophyll and carotenoids respondeddifferentially to Hg-contamination. Chlorophyll content signifi-cantly decreased maximally at L1 (depicting highest Hg-contam-ination) followed by L2 (depicting moderate Hg-contamination)and L3 (depicting lowest Hg-contamination) (vs. R); whereas,carotenoids content displayed significant increases maximally atL1 followed by L2 and L3 (vs. R). Comparisons among contami-nated sites revealed significantly higher chlorophyll content at L3followed by L2 (vs. L1); while L2 displayed both significantly lowerand higher carotenoids content when compared to L1 and L3,respectively (Table 1).

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Fig. 2. Contents of H2O2 (A) and thiobarbituric acid reactive substances (TBARS) (B), electrolyte leakage (C), reactive carbonyls (D) and proline (E) in Halimione portulacoidesroots at reference (R) and sites with highest (L1), moderate (L2) and lowest (L3) mercury contamination in Laranjo bay salt marsh (Ria de Aveiro), Portugal. Values are themeans of five replicates7standard deviation. Significant differences (between sites): avs. R, bvs. L1, cvs. L3. Fresh weight, f.w.

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3.5. H. portulacoides leaf defense endpoint responses

3.5.1. Enzymatic and non-enzymatic antioxidantsSignificantly increased activities of APX, CAT, GPX, GST and GR

were depicted maximum at L1 followed by L3 and L3 (vs. R).Considering contaminated sites comparisons, both significantlylower and higher activities of APX, CAT, GPX, GST and GR weredisplayed at L2 when compared to L1 and L3 (Fig. 7a–e).Concerning the changes in reduced (GSH) and oxidized (GSSG)glutathione pools and GSH/GSSG ratio, the reduced GSH contentsignificantly decreased maximally at L1 followed by L2 and L3 (vs.R); whereas, significant increases in oxidized GSH content andGSH/GSSG ratio were perceptible maximally at L1 followed by L2and L3 (vs. R). On the comparisons among contaminated sites,significantly higher content of reduced GSH was perceptible at L3followed by L2 (vs. L1); while L2, displayed both significantlyhigher (vs. L1) and lower (vs. L3) content of reduced GSH.Considering oxidized GSH level and GSH/GSSG ratio comparisonsamong contaminated sites, significantly lower oxidized GSH wasdisplayed at L3 followed by L2 (vs. L1); while L2 displayed bothsignificantly lower (vs. L1) and higher (L3) oxidized GSH contentand GSH/GSSG ratio (Fig. 8a–c).

3.6. H. portulacoides leaf-polypeptide patterns

The visualization of SDS-PAGE intensity of H. portulacoides leafprotein bands of different kDa revealed differential responses.The intensity of leaf-polypeptide of approx. 75 kDa graduallydecreased maximally at L3 followed L2 and L1 (vs. R). Contrarily,the intensity of a polypeptide of approx. 45 kDa was highermaximally at L2 followed by L3 and L1 (vs. R). Interestingly atL2, a new polypeptide of approx. 6–8 kDa appeared with strongintensity (Fig. 9).

4. Discussion

In a sequence of our previous study presenting a completeH. portulacoides root–leaf Hg-burden analysis and sediment-physico-chemical characterization (Anjum et al., 2011), thecurrent study constitutes a further step in order to achieve theunderstanding of the significance of largely unexplored organ-specific physiological/biochemical (damage and defense) end-point responses, and also of polypeptides modulation for H.portulacoides tolerance to environmental Hg-exposure. Since, the

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Fig. 3. Activities of ascorbate peroxidase (APX) (A), catalase (CAT) (B), glutathione peroxidase (GPX) (C), glutathione sulfo-transferase (GST) (D) and glutathione reductase (GR)(E) in Halimione portulacoides roots at reference (R) and sites with highest (L1), moderate (L2) and lowest (L3) mercury contamination in Laranjo bay salt marsh (Ria de Aveiro),Portugal. Values are the means of five replicates7standard deviation. Significant differences (between sites): avs. R, bvs. L1, cvs. L3. Fresh weight, f.w.

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current analysis of H. portulacoides organs for their Hg-burdens(Supplement Table S1) confirmed the same pattern as obtained inour earlier study during ‘Autumn’ (Anjum et al., 2011) where thediscussion pertaining to H. portulacoides organs-Hg-burdens wasalready done, the discussion on organs-Hg pattern is not consid-ered here in the current paper in order to avoid the repetition andalso deviation from the major objective of the current paper.However, hereunder, a critical discussion on the damage anddefense endpoints responses and polypeptides patterns is per-formed considering both root and leaf together. Additionally, adetailed discussion on the organ-specific responses to Hg-burdenshas been specified in the ‘Supplementary materials’.

4.1. H. portulacoides root and leaf damage endpoints

Corresponding to our earlier report on the root and leaf-Hg-burdens (Anjum et al., 2011) and also that of the new confirmatorydata (Supplement Table S1), EL percent and the contents of H2O2,TBARS and reactive carbonyls displayed significant increasing

trend: L34L14L2 (vs. R). Enhanced H2O2 accumulation and itsconsequence measured as EL and TBARS in roots and leaves seemobvious because of Hg-burdens in these organs (Anjum et al.,2011) (Supplement Table S1). Earlier, Hg-accrued induction ofoxidative stress in terms of ROS (including H2O2) generation andH2O2-mediated consequences (such as EL, and damages to mem-branes, proteins, lipids) were reported or reviewed as obviousresponses in plants as a result of transition property of mercuricions (Patra and Sharma, 2000; Zhou et al., 2008; Gill and Tuteja,2010; Anjum et al., 2013c). As reported also in H. portulacoidesroots and leaves, the cellular proteins were earlier reportedvulnerable to oxidative stress where, stress mediated enhancedlevel of reactive carbonyls in plant cells have been reported and/orreviewed (Davies, 2005; Shulaev and Oliver, 2006; Anjum et al.,2013c, 2014). The exhibition of differential levels of proline indifferential Hg-burden displaying roots and leaves is very impor-tant. Considering root-proline level, the current study revealed theexhibition of enhanced accumulation of osmolyte-proline inH. portulacoides root maximally at L3 followed by L1 and L2.Although, proline accumulation in plants has been considered as abiomarker (damage or defense) of metal/metalloid stress butconcrete physiological functions of metal/metalloid-accruedenhanced proline accumulation in stressed plants are still con-troversial (Sharma and Dietz, 2006; Wang et al., 2009). However,proline accumulation in H. portulacoides roots can be considered asa symptom of injury since its' accumulation is also accompaniedwith enhanced oxidative stress measured in terms of H2O2, TBARS,EL and protein oxidation. Contrarily in leaves, a decreased prolinelevel due to increasing Hg-level points towards the significance ofthis proteinogenic amino acid and osmolyte in radical scavenging,electron sinking, and the stabilization of macromolecules and cellwall components (Sharma and Dietz, 2006; Wang et al., 2009).Furthermore, decreased protein hydrolysis and/or increased pro-line utilization during the detoxification of leaf-Hg mediatedenhanced ROS (such as H2O2) can also be attributed to theexhibited lower proline level by H. portulacoides leaves.

On the perspective of leaf photosynthetic pigments, signifi-cantly decreased leaf chlorophyll content observed herein, hasbeen reported earlier as an obvious response in metal/metalloidincluding Hg-treated non-salt marsh plants (Patra and Sharma,2000; Anjum et al., 2013c). However, Hg-mediated increase in leafcarotenoids content as obtained in the current study, is interesting.

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Fig. 5. Representative gel depicting the polypeptide patterns modulation inHalimione portulacoides roots at reference (R) and sites with highest (L1), moderate(L2) and lowest (L3) mercury contamination in Laranjo bay salt marsh (Ria deAveiro), Portugal. Arrows in white color indicate significant changes and extrapolypeptides. The gel was stained with Coomassie Brilliant Blue (CBB)-R-250.

N.A. Anjum et al. / Environmental Research 131 (2014) 39–4944

Since, carotenoids were reported earlier as accessory light harvest-ing pigments and important quenchers of the singlet state ofchlorophyll and singlet oxygen, increases in carotenoids withincreasing Hg-contamination imply the protective function of thispigment against Hg-mediated potential anomalies in leaves(Saxena and Saiful-Arfeen, 2009; Anjum et al., 2013c).

4.2. H. portulacoides root and leaf defense endpoints

In the present investigation, contingent to H. portulacoides rootand leaf Hg burdens, enzymatic (GR, APX, CAT, GPX and GST) and

non-enzymatic (GSH and GSSG contents; GSH/GSSG ratio) anti-oxidants displayed differential responses. In order to cope up withHg-accrued damages in roots and leaves, the maintenance of adifferential tuning was revealed between H2O2-metabolizingenzymes (such as APX, CAT, GPX, and GST) and GSH its redoxcouple (GSSG/GSH) and regenerating enzyme (GR) in these organs.GR-mediated maintenance of the high levels of both reduced GSHand GSH/GSSG ratio but decreased GSSG level were consideredearlier necessary to maintain a reduced cellular environment andhence, for running optimum cellular metabolic functions in plantsunder metal/metalloid stress (Rausch and Wachter, 2005; Gill andTuteja, 2010; Anjum et al., 2012b, 2013c,d). Though the sensitivityof GR activity to Hg concentrations has been earlier evidenced inplants under a hydroponic culture study (Sobrino-Plata et al.,2009), the elevation in the activity of GR may be an adaptivestrategy in H. portulacoides roots exhibiting high Hg-burden underenvironmentally realistic conditions. A decreased level of reducedGSH pool in H. portulacoides roots is an indicative of insufficiencyof enhanced GR activity for the maintenance of, as expected,enhanced level of reduced GSH. The GR insufficiency is furtheraccompanied by significantly increased level of oxidized glu-tathione (GSSG) but decreased GSH/GSSG ratio. Earlier, the accu-mulation of GSSG in plant tissues under stress conditions wasassessed as a measure of induced oxidative stress (Gill and Tuteja,2010; Anjum et al., 2012b, 2013c,d). Hence, it is apparent thatenhanced GR activity was not competent to maintain increased

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Fig. 6. Contents of H2O2 (A) and thiobarbituric acid reactive substances (TBARS) (B), electrolyte leakage (C), reactive carobonyls (D) and proline (E) in Halimione portulacoidesleaves at reference (R) and sites with highest (L1), moderate (L2) and lowest (L3) mercury contamination in Laranjo bay salt marsh (Ria de Aveiro), Portugal. Values are themeans of five replicates7standard deviation. Significant differences (between sites within same organ): avs. R, bvs. L1, cvs. L3. Fresh weight, f.w.

Table 1Chlorophyll and carotenoids (mg g�1 fresh weight) contents in Halimione portula-coides leaves at reference (R) and sites with highest (L1), moderate (L2) and lowest(L3) mercury contamination in Laranjo bay salt marsh (Ria de Aveiro), Portugal.Values are the means of five replicates7standard deviation. Significant differencesare-(i) between sites (within same organ):

Sites Chlorophyll Carotenoids

R 9.470.6 5.070.3L1 4.270.3a 11.370.8a

L2 7.770.5ab 9.070.6abc

L3 8.570.6ab 7.370.5ab

a vs. Rb vs. L1c vs. L3.

N.A. Anjum et al. / Environmental Research 131 (2014) 39–49 45

pool of reduced GSH and GSH/GSSG ratio but decreased GSSG leveldespite its significantly enhanced activity in H. portulacoides roots.In this context, the role of some other factors cannot be ignoredthat might have consumed/used the enhanced GR activity-mediated increased pool of reduced GSH. To this end, in thecurrent study, the enhanced activities of H2O2-metabolizingenzymes APX and CAT cannot be blamed since both the enzymesdo not need reduced GSH pool as substrate; rather, the formeralthough, utilizes AsA as substrate and but the later does not torequire any substrate to metabolize H2O2 into H2O and O2. Similarresults were reported by Zhou et al. (2008) and Ortega-Villasanteet al. (2007) where APX and CAT-mediated removal of excessiveH2O2 was reported in Hg-treated plants. Hence in the currentcontext, elevated activities of the other two enzymes namely GPXand GST can be attributed for the decreased pool of reduced GSHand GSH/GSSG ratio but increased GSSG level despite elevated GRactivity; since, both GPX and GST employ reduced GSH pool as amajor substrate during H2O2 metabolism (Gill and Tuteja, 2010;Anjum et al. 2012a, 2013d). It is worthy to mention that though inH. portulacoides roots, a fine coordination among GSH regenerating(i.e., GR), GSH-utilizing (i.e., GPX, GST) and also H2O2-detoxifying(i.e., APX, CAT, GPX, GST) enzymes was absent but this may be thestrategy of H. portulacoides roots to tightly control Hg-accruedobvious anomalies in this organ.

Considering the appraisal of the defense trait-responses in H.portulacoides leaves, though, Hg-stress mediated elevations in

H2O2-scavenging enzymes evidenced to effectively degrade ROS(Ortega‐Villasante et al., 2007; Zhou et al., 2008; Sobrino-Plataet al., 2009) but the elevations in H2O2-metabolizing enzymessuch as APX, CAT, GPX, and GST in H. portulacoides leaves withincrease in leaf-Hg burden were not able to scavenge ROS whichimplies the incapacity of these enzymes to scavenge H. portula-coides leaf-ROS (such as H2O2) generated due to leaf Hg burdens.However, a balanced tuning between GSH-regenerating (GR) andH2O2-metabolizing (APX, CAT, GPX; GST) enzymes is perceptible inleaves which resulted into decreased GSH oxidation but increasedreduced GSH pool and GSH/GSSG ratio. Together, these responsesled to a complete H2O2-metabolism and control of H2O2-accruedanomalies (such as TBARS, EL and protein oxidation). Our observa-tions on enzymatic and non-enzymatic antioxidants modulation inH. portucaloides leaves differing in Hg-burdens coincide with thefindings of Srivastava et al. (2005) and Requejo and Tena (2006) innon-salt marsh plants where, the authors attributed increases inthe activity of antioxidant enzymes to the induced transcription oftheir genes which cumulatively contribute to plant tolerance tooxidative stress.

4.3. H. portulacoides root and leaf polypeptide patterns

Earlier, the polypeptide patterns in different plant species werereported to be modulated by varied biotic and abiotic stressfactors including metal/metalloid. Herein, H. portulacoides root

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Fig. 7. Activities of ascorbate peroxidase (APX) (A), catalase (CAT) (B), glutathione peroxidase (GPX) (C), glutathione sulfo-transferase (GST) (D) and glutathione reductase (GR)(E) in Halimione portulacoides leaves at reference (R) and sites with highest (L1), moderate (L2) and lowest (L3) mercury contamination in Laranjo bay salt marsh (Ria de Aveiro),Portugal. Values are the means of five replicates7standard deviation. Significant differences (between sites within same organ): avs. R, bvs. L1, cvs. L3. Fresh weight, f.w.

N.A. Anjum et al. / Environmental Research 131 (2014) 39–4946

polypeptides responded to its Hg-burden but no exhibition of anyextra polypeptide was perceptible in H. poutulacoides leavesexhibiting the highest Hg burden (at L1). Particularly in roots,the occurrence of new polypeptides (225, 100, 90, 60, 45 and35 kDa at L1; 95, 65 and 25 kDa at L2) could be stress polypeptidesproduced to overcome moderate-Hg-burden accrued potentialtoxicity. The significance of polypeptides of approx. 95, 65 and45 (with good intensities), and 35 and 25 kDa polypeptides (withless intensities) (those were appeared earlier at L2-site withmoderate Hg) for protein synthesis induction is advocated dueto their re-appearance at site with the highest root-Hg burden(L3). Additionally, expressed polypeptides-mediated enhancedmetabolic activities in H. portulacoides roots with the highestHg-burden are envisaged. Moreover, the involvement of newpolypeptides in roots at L3 in the Hg-accrued parallel enhance-ments in oxidative indices, enzymatic antioxidants, and increasedGSSG but decreased GSH levels and GSH/GSSG ratio cannot beignored. However, disappearance of some root-polypeptides eitherat L1, L2 or L3 sites could be due to lowered protein synthesis and/or the depletion of reserve proteins to overcome potential stresscaused by Hg-burden. Either up or down regulation expression ofpolypeptides in roots reflected adaptive strategy of this organunder environmental Hg concentrations This is in agreement withthe findings of Sobkowiak and Deckert (2006) and Ahsan et al.(2007) where, the authors concluded that expression of proteinsrepresents directly the most biological processes in a living cellunder varied stress conditions. In context with Hg-burden inducedmodulation of polypeptides in leaves, the intensity of polypeptideof approx. 70–75 kDa appeared at reference site, followed theincreasing trend: L34L24L1. This is in agreement with the reportof Garcia et al. (2006) where, the authors noted higher intensitiesof the bands in the control plant leaves when compared to Cd, Cu,Pb and Zn treated plant leaves. To the other, the reduction of thepolypeptide intensities is in concurrence with the excessHg-burden accrued significantly elevated oxidative stress indices.Moreover, the appearance of a polypeptide of approx. 45 kDa withgood intensity (exhibited the increasing trend: L24L34L1) whencompared to R and the appearance of a new polypeptide of approx.6–8 kDa appeared with strong intensity at site with moderateHg-contamination can be considered an indication of the inhibi-tory effects of Hg-stress on transcriptional process (Riccardi et al.,1998). Additionally, polypeptides of low molecular weight:45–75 kDa, as appeared in the H. portulacoides leaves at differentsites may be stress proteins (Przedpełska-Wąsowicz et al., 2012);which, can also be potentially involved in H. portulacoidesresponses to Hg stress resulting into less oxidative stress but toinduction of strong antioxidant defense system in leaves whencompared to that of roots (Fig. 10).

5. Conclusions

In order to tolerate and execute Hg-remediation under envir-onmental Hg exposure, H. portulacoides organs (roots and leaves)exhibited a differential physiological/biochemical trait modulation.Particularly in roots, though a fine coordination among GSHregenerating (i.e., GR), GSH-utilizing (i.e., GPX, GST) and alsoH2O2-detoxifying (i.e., APX, CAT, GPX, GST) enzymes was absentbut in concurrence with the polypeptide patterns, this organsuccessfully kept the level of high Hg-burden accrued obviousimpairments under control. On the other, H. portulacoides took theadvantage of the physiological/biochemical strategy adapted by itsleaves where, a finely tuned coordination among the responses ofenzymatic-defense endpoints resulted into decreased GSH-oxidation but enhanced pools of reduced GSH and GSH/GSSGratio, which subsequently lowered the extent of Hg-accrued

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Fig. 8. Reduced (GSH) (A) and oxidized (GSSG) (B) glutathione contents, and GSSG/GSH ratio (C) in Halimione portulacoides leaves at reference (R) and sites withhighest (L1), moderate (L2) and lowest (L3) mercury contamination in Laranjo baysalt marsh (Ria de Aveiro), Portugal. Values are the means of five replicates7-standard deviation. Significant differences (between sites within same organ): avs.R, bvs. L1, cvs. L3. Fresh weight, f.w.

R L1 L2 L3

225

100

75

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35

M

Fig. 9. Representative gel depicting the polypeptide patterns modulation inHalimione portulacoides leaves (B) at reference (R) and sites with highest (L1),moderate (L2) and lowest (L3) mercury contamination in Laranjo bay salt marsh(Ria de Aveiro), Portugal. Arrows in white color indicate significant changes andextra polypeptides. The gel was stained with Coomassie Brilliant Blue (CBB)-R-250.

N.A. Anjum et al. / Environmental Research 131 (2014) 39–49 47

damages. Overall, the physiological/biochemical characterizationof below (roots)- and above (leaves)-ground organs (studied interms of damage and defense endpoints, and polypeptides mod-ulation) revealed the adaptive responses of H. portulacoides toenvironmental Hg at whole plant level which cumulatively helpedthis plant to sustain and execute its Hg-remediation potential.

Acknowledgments

Authors gratefully acknowledge the financial supports receivedfrom both FCT (Government of Portugal) through contract (FRH/BPD/64690/2009; SFRH/BPD/84671/2012) and by the Aveiro Uni-versity Research Institute/CESAM.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.envres.2014.02.008.

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Root Leaf

Halimione portulacoides Adaptive Responses to Environmental Mercury

Parallel Enhancements in GSH-regenerating (GR) and H2O2-

metabolizing (APX, CAT, GPX; GST) enzymes

Increased: GSH oxidation

Decreased: reduced GSH pool and

GSH/GSSG ratio

Partial: H2O2-metabolism and control of H2O2-accrued anomalies i.e. TBARS, EL, Protein Oxidation,

Osmolyte

BALANCED tuning between GSH-regenerating (GR) and H2O2-

metabolizing (APX, CAT, GPX; GST) enzymes

Decreased: GSH oxidation

Increased: reduced GSH pool and

GSH/GSSG ratio

Complete: H2O2-metabolism and control of H2O2-accrued anomalies i.e. TBARS, EL, Protein Oxidation,

Osmolyte

Root and leaf differential polypeptide expression patterns

Substantiated by

Control of H. portulacoides Health and Ecological-Function Execution

Fig. 10. Schematic representation of the summarized basic mechanisms underlyingHalimione portulacoides (root-leaf) physiological/biochemical responses and theirpotential relationship with polypeptide patterns under environmental mercuryexposure. See discussion for details.

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