Proteomic responses to lead-induced oxidative stress in Talinum ...

Environ Sci Pollut Res (2014) 21:8750–8764 DOI 10.1007/s11356-014-2808-9

RESEARCH ARTICLE

Proteomic responses to lead-induced oxidative stress in Talinum triangulare Jacq. (Willd.) roots: identification of key biomarkers related to glutathione metabolisms Abhay Kumar & Narasimha Vara Prasad Majeti

Received: 16 December 2013 / Accepted: 17 March 2014 / Published online: 8 April 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract In this study, Talinum triangulare Jacq. (Willd.) treated with different lead (Pb) concentrations for 7 days has been investigated to understand the mechanisms of ascorbate– glutathione metabolisms in response to Pb-induced oxidative stress. Proteomic study was performed for control and 1.25 mM Pb-treated plants to examine the root protein dynamics in the presence of Pb. Results of our analysis showed that Pb treatment caused a decrease in non-protein thiols, reduced glutathione (GSH), total ascorbate, total glutathione, GSH/oxidized glutathione (GSSG) ratio, and activities of glutathione reductase and γ-glutamylcysteine synthetase. Conversely, cysteine and GSSG contents and glutathione-Stransferase activity was increased after Pb treatment. Fourier transform infrared spectroscopy confirmed our metabolic and proteomic studies and showed that amino, phenolic, and carboxylic acids as well as alcoholic, amide, and ester-containing biomolecules had key roles in detoxification of Pb/Pb-induced toxic metabolites. Proteomic analysis revealed an increase in relative abundance of 20 major proteins and 3 new proteins (appeared only in 1.25 mM Pb). Abundant proteins during 1.25 mM Pb stress conditions have given a very clear indication about their involvement in root architecture, energy metabolism, reactive oxygen species (ROS) detoxification, cell signaling, primary and secondary metabolisms, and molecular transport systems. Relative accumulation patterns of both common and newly identified proteins are highly correlated Responsible editor: Elena Maestri A. Kumar : N. V. P. Majeti (*) Department of Plant Sciences, University of Hyderabad, Hyderabad 500046, India e-mail: [email protected] N. V. P. Majeti e-mail: [email protected] A. Kumar e-mail: [email protected]

with our other morphological, physiological, and biochemical parameters. Keywords Ascorbate–glutathione cycle . FTIR . Heavy metal stress . Mass spectrometry . Oxidative stress . Proteomics . Two-dimensional electrophoresis

Introduction Lead (Pb) is a potentially toxic metal, ranks second in toxicity among all hazardous material, and contaminates the environment and poses threat to all forms of life. Pb is extremely persistent in the soil, air, water, and food stuff through various natural, technogenic, and geogenic activities with no metabolic significance in biological system (Anonymous 2007, 2011). Pb can easily be accumulated in plant tissue and can result into phytotoxic manifestations, which includes generation of reactive oxygen species (ROS), inhibitions of antioxidative enzymes, cellular redox and ionic transport imbalance, and oxidative damage (Potters et al. 2010; Kumar et al. 2012). Despite the toxicity, several plants growing in metalpolluted soil are able to exclude, accumulate or hyperaccumulate heavy metals (HMs), and acquire a wide range of adaptive strategies (Sharma and Dietz 2006; Anjum et al. 2012). Pb exposure can modify physiological and biochemical state of plants that trigger several signaling pathways for adaptation to unfavorable conditions (Sharma and Dietz 2006; Kumar et al. 2013). Plant adaptive strategies include the activation of enzymatic and non-enzymatic antioxidant defense system against ROS (Sytar et al. 2013). Nonenzymatic antioxidants mostly involved glutathione biosynthetic metabolites and intermediates of the ascorbate–glutathione cycle (AGC) such as thiols, cysteine, reduced glutathione (GSH), oxidized glutathione (GSSG), ascorbate (AsA), dehydroascorbate, and monodehydroascorbate (MDA), where

Environ Sci Pollut Res (2014) 21:8750–8764

they play a major role in hydrogen peroxide (H2O2)-scavenging pathway in living cells (Potters et al. 2010; Anjum et al. 2012). Operations of the GSH biosynthesis and AGC not only maintain the reduced active forms of AsA and GSH at an appropriate level, thereby adjusting the cellular redox potential, but also participate in ROS detoxification (Potters et al. 2010). Glutathione (γ-Glu-Cys-Gly) is a widely distributed redox-active molecule, which is also involved in the biosynthesis of phytochelatins (PCs), a metal-binding bioligand (Sharma and Dietz 2006; Estrella-Gómez et al. 2009). Excessive amounts of HMs trigger a wide range of cellular responses including changes in gene expression, which facilitate synthesis of diverse metabolites that include specific amines, amino acids, and peptides (Sharma and Dietz 2006; Estrella-Gómez et al. 2009). Responses of plant proteins against HM stress include changes in expression and posttranslational modification of proteins to activate their defense mechanism (Visioli and Marmiroli 2013). Comparative quantitative analysis of protein is able to provide accurate information about major players involved in molecular responses of stress adaptive mechanisms. Proteomics is based on highthroughput biotechnological approaches, with its high resolution for separation and identification of (differentially) abundant proteins involved in cellular function of plant during various abiotic stress conditions (Sengupta et al. 2011; Kosová et al. 2011). Thus, use of proteomics-based approaches in various areas of plant biology research came up with a stunning rapidity over the past decade. Research on plant proteomes has provided advantageous information for a comprehensive understanding of the protein networks in plants in response to external HM stress (Visioli and Marmiroli 2013). The roots being the belowground or submerged (in the case of aquatic plants) portion of plants is the major access point to the metal, and as such, they play a vital role in metal accumulation and translocation of the aerial part of the plant. The regulation of proteins may act to re-establish root system homeostasis under HM stress condition. These proteins can also be considered as molecular markers for bioengineering plants for phytoremediation (Visioli and Marmiroli 2013). The protein expression profile of terrestrial and crop plants in response to various HMs have been described by various authors (Ahsan et al. 2007; Wang et al. 2011; Luque-Garcia et al. 2011; Visioli and Marmiroli 2013). Since proteins are directly involved in stress response, analysis of root protein expression during Pb stress can significantly unravel the possible relationships between protein abundance and plant stress acclimation. Talinum triangulare (Jacq.) Willd. (Ceylon spinach) is a common leafy vegetable among the other classes of vegetables that is grown in Nigeria, India, and other parts of the world. Though T. triangulare is known as a remarkable accumulator of HMs (Yusuf et al. 2003; Kumar et al. 2012), no report has been made on key aspects of proteomic and

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ascorbate–glutathione metabolisms for their significance in tolerance and detoxification of Pb-induced stress. Thus, the aim of the present study is to reveal and understand the (i) role of non-enzymatic antioxidants, ascorbate–glutathione related metabolites, and enzymes and (ii) dynamic role of root proteome in Pb stress alleviation.

Materials and methods Plant description and treatment in hydroponic experiment T. triangulare required for experiments was collected from a field bank maintained uniformly as a stock plant at the University of Hyderabad, India. Plants were propagated through stem cuttings (5.0–7.0-mm diameter and 17–22-cm height) in conical flask containing 100 mL of modified Hoagland’s nutrient media (Hoagland and Arnon 1950) in plant growth chamber at 16/8 h (day/night) at 28±2 °C for 3 weeks. The nutrient media was replaced every 3–4 days. After 3 weeks of acclimatization, uniform stem cuttings with adventitious roots and leaves were selected and treated with Pb(NO3)2 at different Pb concentrations of 0 (control), 0.25, 0.50, 0.75, 1.00, and 1.25 mM for 7 days under above conditions. Roots excised after 7 days of treatment were processed for analysis of various endpoints described as follows. Proteomic analysis was performed with plant roots treated with 0 (control) and 1.25 mM Pb(NO3)2. Non-protein thiols (NPSH) NPSH content in fresh root (0.5 g) was measured at 412 nm (extinction coefficient (ε)=13,600 M−1 cm−1) according to Sedlak and Lindsay (1968). Molecular cysteine Cysteine content in fresh root (0.5 g) was measured at 560 nm and was calculated by using the standard curve prepared from cysteine and expressed as nanomoles per gram fresh weight (fw) (Gaitonde 1967). Glutathione estimation Fresh root (0.5 g) was grinded in 4 mL of 0.1 M sodium phosphate-EDTA buffer (pH 8.0) containing 25w/v H3PO3. The homogenate was centrifuged at 15,000g for 20 min at 4 °C. In supernatant, glutathione (GSH and GSSG) content was determined fluorometrically after 15-min incubation with o-phthaldialdehyde (OPT) (Hissin and Hilf 1976). Fluorescence intensity was measured at 420 nm after excitation at 350 nm on a FluoroMax 3 fluorescence spectrophotometer.

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GSH and GSSG contents were calculated from the respective standard (Sigma-Aldrich) curves. Estimation of enzymes activities Fresh root (1.0 g) was homogenized in 50 mM sodium phosphate buffer (pH 7.0) containing 2 w/v polyvinyl polypyrrolidone and centrifuged at 13,000g for 20 min at 4 °C. Supernatants were collected, and the protein content was determined according to the method of Lowry et al. (1951) using a bovine serum albumin as a standard. γ-Glutamylcysteine synthetase (γ-ECS, E.C. 6.3.2.2) assay was performed with the estimation of phosphate content by phosphomolybdate estimation at 660 nm following the methodology of Reugsegger et al. (1992). Glutathione-S-transferase (GST, E.C. 2.5.1.18) activity was performed according to the method of Habig and Jakoby (1981). GST activity was measured at 340 nm for 1 min (ε=9.6 mM−1 cm−1), and the values represented as unit of micromoles per milligram of protein per minute. Glutathione reductase (GR, E.C. 1.6.4.2) activity was analyzed according to Jiang and Zhang (2001). NADPH oxidation was recorded as the changes in absorbance at 340 nm for 1 min and was expressed as unit of micromoles of oxidized NADPH per minute per milligram of protein (ε= 6.2 mM−1 cm−1). Cellular AsA content AsA content in fresh root (1.0 g) was recorded at 265 nm (ε= 14 mM−1 cm−1) and was calculated following the method of Logan et al. (1998) with minor modifications. Functional group analysis by FTIR spectroscopy Identification of functional groups was done by Fourier transform infrared (FTIR) spectroscopy (JASCO FTIR-5300, Easton, USA) as described by Wei et al. (2009) with modifications. Control and 1.25 mM Pb-treated roots (1.0 g) were washed with milliQ water followed by homogenization with milliQ. After that, aqueous phase was evaporated, and samples were obtained as a fine powder. The powdered sample was then mixed with potassium bromide (KBr) to make KBr tablets, which were directly used for FTIR analysis.


nitrogen and suspended in 5 mL of the extraction buffer containing 0.5 M Tris–HCl (pH 7.5), 0.7 M sucrose, 0.1 M potassium chloride, 50 mM EDTA, 2 β-mercaptoethanol, and 1 mM phenylmethanesulfonylfluoride. After thorough mixing, an equal volume of commercially available Trissaturated phenol (pH 7.5) was added to the extract suspension and mixed well, followed by centrifugation at 8,000g for 30 min at 4 °C. The upper phenolic phase was collected in centrifuge tubes, and an equal volume of extraction buffer was added to it. The above step was repeated, and the upper phenolic phase was re-extracted. To the final collected phenolic phase, four volumes of 0.1 M ammonium acetate in methanol were added and incubated overnight at −20 C for protein precipitation. The samples were then centrifuged at 10,000g at 4 °C for 30 min, and the precipitate was washed three times in ice-cold methanol, two times in ice-cold acetone, and at the end, air-dried for a few minutes. The final pellet was solubilized in 200 μL of the rehydration solution containing 8 M urea, 2 M thiourea, 4 % 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS), 30 mM 1,4-dithiothreitol (DTT), and 1.5 % immobilized pH gradient (IPG) buffer pH range 4–7 (GE Healthcare, Germany). The protein concentration was determined according to Bradford protein assay (Bradford 1976) using BSA as standard. After protein estimation, 0.004 % bromophenol blue was added to a final volume of 320 μL aliquots containing 800 μg proteins. Active rehydration of mixed protein was done on IPG strips (18 cm, 4–7 pH linear gradient; Amersham, GE Healthcare) for 12 h. Rehydration and isoelectric focusing (IEF) were carried out in Ettan IPGphor II (GE Healthcare, Germany) at 20 °C, using the following program: 30 min at 500 V, 3 h to increase from 500 to 10,000 V, and 6 h at 10,000 V (a total of 60,000 V h) (Sengupta et al. 2011). After IEF, the strips were equilibrated with two different equilibration buffers for 30 min each with a gentle rocking at 25±2 °C. The first equilibration was performed in a solution containing 50 mM Tris-HCl buffer (pH 8.8), 6 M urea, 2 % (w/v) sodium dodecyl sulfate (SDS), 30 % (w/v) glycerol, and 2 % DTT, and the second equilibration was performed by using 2.5 % (w/v) iodoacetamide by replacing DTT. The proteins were separated in the second dimension by 12 % SDS-PAGE at 10 mA gel−1 for 1 h and then 38 mA gel−1 for 6 h. Gel documentation and analysis

Protein extraction and two-dimensional electrophoresis (2-DE) Fresh roots from both control and 1.25 mM Pb-treated plants were washed thoroughly with milliQ and immediately frozen in liquid nitrogen and stored at −80 °C. Total root proteins were extracted according to Sarvanan and Rose (2004) with minor modifications according to Sengupta et al. (2011). Frozen root tissues were grinded to fine powder in liquid

The gels were stained with modified colloidal Coomassie staining (Wang et al. 2007; Sengupta et al. 2011). Protein spots in the gels were recorded using a calibrated densitometric scanner (GE Healthcare) and analyzed (normalization, spot matching, expression analyses, and statistics) using Image Master 2-D Platinum version 6 image analysis software (GE Healthcare, Germany). Only those spots which were reproducible and significantly (P≤0.05) exhibited at least >1.8-fold


relative abundances in 1.25 mM Pb-treated gel were considered for matrix-assisted laser desorption/ionization time of flight mass spectrometric (MALDI-TOF MS) analysis. In-gel trypsin digestion and MALDI-TOF MS analysis After protein spot analysis, in-gel digestion and MALDI-TOF MS analysis was conducted with a MALDI-TOF/TOF mass spectrometer (Bruker Autoflex III smartbeam, Bruker Daltonics, Germany) following the method explained by Shevchenko et al. (1996) with slight modifications (Sengupta et al. 2011). Stained protein spots were manually excised from each gel. Only those spots which were common in every replicate gel were picked. The picked gel pieces were first washed with 200 μL of 25 mM ammonium bicarbonate (NH4HCO3) solution to remove ammonium acetate followed by destaining with 100 μL of 50 % acetonitrile (ACN) in 25 mM NH4HCO3 for four times. Further, gel pieces were treated with 10 mM DTT in 25 mM NH4HCO3 and incubated at 55 °C for 1 h followed by treatment with 55 mM iodoacetamide prepared in 25 mM NH4HCO3 for 1 h at room temperature. Gel pieces were washed with 25 mM NH4HCO3 and ACN, dried in speed vac, and rehydrated in 20 μL of 25 mM NH4HCO3 solution containing 12.5 ng μL−1 trypsin (sequencing grade, Promega). Mixture was incubated on ice (4 °C) for 10 min and kept overnight for digestion at 37 °C. After digestion, a short spin for 10 min was given, and the supernatant was collected in a fresh centrifuge tube. The gel pieces were re-extracted with 50 μL of trifluoroacetic acid (TFA)-ACN solution, containing 0.1 % TFA and ACN in ratio of 1:1, for 15 min with frequent vortexing. The supernatants were pooled together and dried using speed vac and were reconstituted in 5 μL of TFA-ACN solution. An aliquot (2 μL) of the above sample was mixed with 2 μL of freshly prepared α-cyano-4-hydroxy-cinnamic acid (CHCA) matrix in 50 % ACN and 1 % TFA (1:1), and 1 μL was spotted on target plates for MALDI analysis. Peptide mass fingerprinting and MS/MS analysis Protein sample identification was performed by database search on the basis of peptide mass fingerprinting (PMF) and (MS/MS) using MASCOT program (http://www. matrixscience.com) employing biotools software (Bruker Daltonics, Germany). The similarity search for mass values was done with existing digests and sequence information from NCBInr and SwissProt database. The taxonomic category was set to Viridiplantae (green plants), and other search parameters were fixed with modification of carbamidomethyl (C), enzyme trypsin, peptide charge of 1+, and monoisotropic. According to the MASCOT probability analysis, only significant (P≤0.05) hits were accepted for protein identification.

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Statistical analysis The data presented correspond to the mean±standard error (SE) values of three replicates. One-way analysis of variance (ANOVA) and Duncan’s multiple range test by using SigmaStat software (version 11.0) were performed to confirm the variability of results and for the determination of significant (P≤0.05) difference between treatment group, respectively, which are denoted by different letters. Proteomic analysis was done with three biological replicates for both control and 1.25 mM Pb-treated root proteins. Each replication was composed of approximately 15 pooled plants. The normalized volume (%vol) of each spot was automatically calculated by the software as a ratio of the volume of a particular spot to the total volume of all spots present on the gel.

Results Pb-induced changes in NPSH, cysteine, and glutathione contents NPSH content was slightly increased at low Pb concentration and thereafter declined (Fig. 1a) and showed a negative linear graph (R2 =0.924). Pb induced a significant (P≤0.05) reduction in NPSH content by 35.0, 40.4, and 64.9 % during 0.75, 1.0, and 1.25 mM Pb in comparison with the control, respectively. The accumulation of cysteine contents showed dosedependent responses in Pb-treated T. triangulare roots (R2 = 0.912; Fig. 1b) over control root. Cysteine accumulation increased in stress responses and attained maximum significant value at Pb concentration of 1.25 mM. The amount of cysteine significantly (P≤0.05) increased to approximately 21–46 % during 0.25–1.25 mM Pb, respectively. GSH content in T. triangulare root was first increased followed by declining in comparison with the control, respectively, which showed a negative correlation (R2 =0.934) with an increasing Pb concentration (Fig. 1c). The maximum significant (P≤0.05) increase and decline in the GSH contents were observed at 0.25 and 1.25 mM Pb treatment, which accounted for 7.7 and 68.8 % in comparison with the control, respectively. On the other hand, GSSG level was found to gradually increase (R2 =0.956; Fig. 1d), and significant (P≤ 0.05) accumulations were observed at concentrations 0.25– 1.25 mM Pb (83.0–236.5 %) in comparison with the control, respectively. Total glutathione contents were calculated after adding both GSH and GSSG values (Fig. 1e), where it followed the same pattern as GSH (R2 =0.860). Furthermore, GSH/ GSSG ratio was significantly declined and was inversely related to increasing Pb treatments (Fig. 1f). The correlation analysis of GSH and GSSG showed a negative values of R2 =0.771.

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b

0.08 c

c 0.06

bc

b 0.04

ab a

0.02

15

Cysteine (µ mol g -1 fw)

a NPSH (m mole g–1 fw)

Fig. 1 Changes in (a) nonprotein thiols (NPSH) and (b) amino acid cysteine, (c) GSH, (d) GSSG, (e) total GSH contents, and (f) GSH/GSSG ratio in T. triangulare roots after 7 days of Pb treatment. Mean values (±SE) denoted by different letters are significantly different (P≤0.05) between each other


e

d

6

c b

4

a 2

GSSG (µ mol g –1 fw)

GSH (µ mol g –1 fw)

f

6 3

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1

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10 e 8

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GSH/GSSG ratio

Total GSH (µ mol g –1 fw)

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Pb concentration in nutrient media (mM)

Pb-induced changes in metabolic enzyme activities

Pb-induced changes in AsA content

A linear decline in γ-ECS activity was observed with increasing Pb concentration in T. triangulare roots (R2 =0.874; Fig. 2a). Activity of γ-ECS significantly (P≤0.05) declined during 0.75 (32.7 %), 1.0 (42.7 %), and 1.25 (45.5 %) in comparison with the control, respectively. However, GST activity was increased as the Pb treatment increased, which showed a strong positive correlation (R2 =0.947) with increasing Pb treatments (Fig. 2b). Pb treatments at 0.25 and 0.50 mM induced very slightly the GST activities, which were not significantly different from control, while maximum significant (P≤0.05) increase in GST activities were observed during 0.75 (71.4 %), 1.0 (89.1 %), and 1.25 (91.3 %) mM Pb treatments in comparison with the control, respectively. Our results also showed that GR activity was not much affected by Pb treatments in T triangulare roots (Fig. 2c). Interestingly, Pb treatments at 0.25 and 0.50 mM Pb cause a slight insignificant increase in GR activities, which were insignificantly declined at further treatment concentrations.

Spectrophotometric analysis of root extracts showed that Pb treatments caused dose-dependent reduction in AsA contents in comparison with the control (R2 =0.965; Fig. 2d). The significant decline in the AsA contents was observed in 0.25–1.25 mM Pb-treated T. triangulare roots and was accounted for reduction of 23–53 % over control, respectively. The tendencies of AsA contents were almost similar to that of the total glutathione contents, and both were shown a good positive correlation (R2 =0.810) with each other. Functional group analysis by FTIR spectroscopy FTIR spectral analysis is important to identify the stretching, bending vibration, and reduction in transmittance of characteristic peaks representing the characteristic functional group. These characteristic functional groups are from active compounds and metabolites and possibly involved in detoxification of Pb and Pb-induced toxic radicals. After Pb treatment,


a

b 150 d

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GR (µ mol NADPH oxidized min–1 mg–1 protein)

24

GST (µ mol mg–1 protein min–1)

-ECS (µg Pi mg–1 protein min–1)

Fig. 2 Changes in (a) γglutamylcysteine synthetase (γ-ECS), (b) glutathione-Stransferase (GST), (c) glutathione reductase (GR) activities, and (d) total ascorbate (AsA) contents in T. triangulare roots after 7 days of Pb treatment. Mean values (±SE) denoted by different letters are significantly different (P≤0.05) between each other

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a

2.7 1.8 0.9 0

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e d c

500

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250

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Pb concentration in nutrient media (mM)

very less difference in stretching and bending vibration of peaks was observed when compared with the control peaks. FTIR spectra of Pb-treated sample revealed that noticeable reduction in %transmittance of characteristic peaks have been observed when compared with the control spectra (Fig. 3, Table 1). The major reductions in peak transmittance values were observed approximately at 3,391, 2,922, 1,643, 1,381, and 1,055 cm−1. The value below 1,000 cm−1 wavenumber are considered as fingerprint zone and cannot be assigned to any particular functional group due to the complex interacting vibration system.

61 spots were significantly (P≤0.05) accumulated by >1.8fold. The criteria used to identify and explain the Pbresponsive proteins among the number of protein spots was that the protein spot in 1.25 mM Pb-treated gel should have at least 1.8-fold (P≤0.05) higher protein relative abundance than protein spot observed at the same location in the control gel. A total of 33 major spots, 27 upregulated spots and 6 new spots, which were well distinct, separated, and of considerable intensity, were selected for protein identification by MALDITOF analysis. Out of 33 selected spots, 23 proteins were successfully identified, which include 20 differently abundant under Pb stress in comparison with the control and 3 new

2-DE and protein accumulation profiling 57 B

B

%Transmittance

Our previous (Kumar et al. 2012, 2013) and present studies related to antioxidative enzymes and metabolites showed that maximum significant changes in analyzed parameters were observed at higher Pb treatment concentration, i.e., 1.25 mM. Based on these observations, we were further motivated to investigate the changes in protein pattern only at higher Pb treatment, i.e., 1.25 mM concentration, in comparison with the control sample through 2-DE for better understanding of dynamics of root proteins’ relative accumulation or abundance. The relative accumulation patterns of total root protein in T. triangulare exposed to 1.25 mM Pb concentration against respective control showed that 2-DE gels were reproducible and clearly revealed hundreds of well-resolved protein spots (Fig. 4). Overall analysis showed that out of the number of matched spots between control and Pb stress, approximately

A 769

A

661 603

A B

1738

1516 1249 1321 1381

2852

CH 3391

2922

NH OH

1643

1055

CO

CO

28

Wavenumber (cm−1) Fig. 3 Stretching, bending, and changes in %transmittance of functional groups involved in binding and detoxification of Pb and Pb-induced free radicals by Fourier transform infrared (FTIR) spectroscopy in roots: control sample (A) and 1.25 mM Pb-treated (B) sample spectra

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Table 1 Changes in %transmittance of peaks representing the characteristic functional group involved in detoxification of Pb and Pb-induced toxic radicals or metabolites Wavenumber

%Transmittance Control

16

43

27.8 38.6 42.1 43.6 32.6 44.7

8.9 2.7 1.5 1.7 8.0 2.5

1,381.16 1,321.36 1,249.99 1,055.16 769.67 661.64 603.77

43.2 44.4 45.8 40.4 53.3 50.0 49.4

40.4 41.7 42.9 32.9 52.0 48.5 48.6

2.8 2.7 2.9 7.5 1.3 1.5 0.8

15

11

18

13 12

14

N3

17

29 10

N1

20.1

N2

6

9

8

7

5

1

14.3

b

spots visible only in Pb-stressed protein gel. Positions of the 23 identified proteins were numbered in numerical values for 20 abundant spots and newly detected spots were marked as N1, N2, and N3, depicted accordingly in the master gel (Fig. 5a). Few of the identified spots are enlarged in Fig. 5b to visualize their relative abundant patterns during 1.25 mM

pH 7.0

Control

2

Pb

3

4

Control

Pb

Control

2

11

N1

8

12

N2

9

14

N3

10

18

New

Fig. 5 Two-dimensional electrophoresis master gel from the 1.25 mM Pb-treated root sample illustrating 23 identified proteins (a). The spot numbering in the master gel shown corresponds to the spot numbers given in Table 2. Enlarged view of the accumulation patterns of a few spots during control and 1.25 mM Pb stress conditions (b)

Pb stress. Relative spot volumes for each spot were expressed as %spot vol indicating the normalized values of the ratio of the individual spot to the total volume of all the spots in the gel (Fig. 6a, b).

20.1

Identification of differentially abundant proteins during Pb stress

14.3

Control

b

20.1 14.3

pH 7 20 19

63

1.25 mM Pb

36.7 41.3 43.6 45.3 40.6 47.2

97.4 63 43 29

pH 4 kDa 97.4

Difference in transmittance

3,391.16 2,922.42 2,852.98 1,738.02 1,643.50 1,516.19

4.0 kDa a 97.4 63 43 29

a

1.25 mM Pb

Fig. 4 Two-dimensional electrophoresis protein profile of T. triangulare roots grown under control (a), and 1.25 mM Pb (b)

Identified 23 proteins were grouped as differentially abundant and newly detected during 1.25 mM Pb treatment. Each of the 23 spots contained only one protein. The relative spot intensities during control and 1.25 mM Pb stress, accession number, source organism, sequence coverage, experimental and theoretical molecular weight and isoelectric point (pI), MS/ MS score, matched peptide sequence, fold changes, and respective functions of each individual protein are shown in Table 2. In some cases, more than one spot was identified as the same protein, for example, ATP synthase subunit β (spot 10 and 13) and chalcone–flavanone isomerase (spot 3 and N1). The identified proteins were categorized into five major groups based on their biological functions (Fig. 6c), i.e., (1)


b

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1

20 Control

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1.25 mM Pb

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16 b

12 b

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4 0

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Spot volume (%)

Spot volume (%)

Fig. 6 (a), (b) Percentage volumes of the spots on the gel for understanding the relative abundance pattern. Data are presented in mean±SE value (n=3) denoted by different letters are significantly different (P≤0.05) between each other. c Functional categorization and percentage contribution of identified proteins in their respective groups

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a

a

a

1

2

3

4

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6

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0.4

b b

a a

a

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0 7

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Spot number

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0.2

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11 12 13 14 15 16 17 18 19 20

Spot number

Hypothetical protein 13.04 Transporter 4.347

ROS-detoxification/ defense 21.739

Root morphology 8.69

Cell signaling/ Metabolisms 30.434

ROS detoxification and defense, (2) protein synthesis/energy metabolism, (3) cell signaling/cell metabolism, (4) root morphology, and finally, (5) transport proteins. Auxin transport, chalcone–flavanone isomerase and DNA-directed RNApolymerase subunit β were identified as newly appeared proteins only in Pb stress gel. The relative accumulation patterns of identified proteins showed that the spot 1 was highly abundant by a value of 23.3-fold, while spot 12 was least abundant which accounted for 1.8-fold (P≤0.05) in comparison with the control (Fig. 6a).

Discussion T. triangulare is an efficient Pb accumulator, where accumulated Pb caused Pb-induced oxidative stress through ROS production. T. triangulare has been reported to be well equipped with ROS scavengers and antioxidative systems (Kumar et al. 2012, 2013). In our present study, plants treated with Pb showed a variety of responses viz, alteration in AGC intermediates, related metabolites, and enzymes. Decrease in NPSH and increase in cysteine contents were observed in response to Pb-induced oxidative stress in T. triangulare roots. The decline in NPSH in our study indicates the possible

21.739 Protein synthesis/ Energy metabolism

incorporation of sulfur molecule into sulfur containing organic complex such as cysteine and thio-containing peptides, which is related to the level of plant tolerance mechanisms (Mishra et al. 2006; Singh et al. 2010; Anjum et al. 2012). Similar responses of significant reduction in NPSH content and induction in cysteine content have been observed in Najas indica after 7 days of Pb treatments (Singh et al. 2010). Treatment of Pb to Salvinia minima resulted into both formations of thiol-enriched Pb complexion peptides and synthesis of low-molecular weight metal chelators (Estrella-Gómez et al. 2009). Glutathione is one of the most abundant intracellular nonprotein thiols, which maintains the cellular redox status and acts as a chelating bioligand responsible for detoxification of HMs (Singh et al. 2010; Anjum et al. 2012). Our results of GSH and GSSG estimations showed that Pb treatments declined GSH and stimulated GSSG and total glutathione contents. Furthermore, Pb treatment increased γ-ECS activity, which catalyzes the first step of GSH biosynthesis, i.e., γ-EC from L-glutamate and cysteine. In our analysis, NPSH (R2 = 0.903) and γ-ECS (R2 =0.903) followed a similar trend and showed a good correlation with GSH, which indicated the key role of NPSH and γ-ECS in regulation of GSH biosynthesis in T. triangulare (Mishra et al. 2006; Anjum et al. 2012; Sytar

Superoxide dismutase (Cu–Zn)

Peroxidase (fragments)

Monodehydroascorbate reductase

5

6

12

ATPB_CUSGR gi|226493589

ATP synthase subunit β

ATP synthase β-chain

13

15

ATP synthase subunit α, mitochondrial Cell signaling/metabolisms

Triosephosphate isomerase

Probable receptor-like protein kinase Adenosylhomocysteinase

2,3-Bisphospho-glycerateindependent phosphoglycerate mutase 1 Chalcone–flavonone isomerase

8

16

20

N2

Auxin transport

DNA-directed RNA polymerase subunit β Root morphology 14 Actin-1

N3

N1

17

Chalcone–flavonone isomerase

3

18

Arabidopsis thaliana

Daucus carota

gi|113217 BIG_ARATH

Cryptomeria japonica

Pueraria montana

Mesembryanthemum crystallinum Arabidopsis thaliana


Secale cereale

Lotus japonicus

Nicotiana plumbaginifolia

Zea mays

Cuscuta gronovii

Chlamydomonasreinhardtii

Chlamydomonasreinhardtii

Vitis vinifera

Ginkgo biloba

Mesembryanthemum crystallinum

Arabidopsis lyrata


Reference organism

RPOC2_CRYJA

CFI_PUEML

PMG1_ARATH

SAHH_MESCR

Y5613_ARATH

TPIS_SECCE

CFI2_LOTJA

ATPAM_NICPL

HSP70_CHLRE

11

ATP synthase subunit β, mitochondrial Heat shock 70 kDa protein gi|5852203

gi|146432261

PER_GINBI

SODC1_MESCR

gi|297824877

10

Protein synthesis/energy metabolism

Glutathione-S-transferase

AKR6_ARATH

ROS detoxification and defense 1 Probable aldo-keto reductase

2

Accession no.

Protein identified

Spot no.

574.5/5.6

42.2/5.6

139.6/9.47

23.84/5.3

60.77/5.3

53.77/5.7

93.03/5.9

27.13/5.2

23.99/5.7

55.47/5.8

59.05/5.9

54.13/5.2

71.29/5.2

61.95/4.9

47.47/5.9

9.4/4.7

15.27/5.4

24.32/6.3

36.7/5.4

T

Mr (kDa)/pI

21/5.3

49/5.4

42/5.2

22/4.6

80/5.5

67/5.8

70/5.3

25/6.3

17/5.1

63/5.9

63/5.6

50/5.7

67/6.6

28/5.1

51/5.9

25/5.4

18/5.7

17/5.0

17/4.8

O

38/0

277/15

53/2

30/19

70/2

32/4

31/5

49/11

48/25

104/6

61/7

49/12

25/4

87/5

125/6

30/19

30/23

57/11

49/5

MS–MS score/SC (%)

Table 2 Major differentially accumulated and newly identified root proteins of T. triangulare during 1.25 mM Pb stress

–

4.93

–

–

3.03

1.82

5.66

3.54

3.86

3.56

4.12

2.86

5.39

3.39

1.8

2.92

5.12

6.03

23.39

FC

AVFPSIVGRPRNYELPDGQVITIGAER TTGIVLDSGDGVSHTVPIYEGYA LPHAILR EQTMGKSAPAVQEKLLGVVKVTSIL SSR

VMENCVAHMKSVGTYGDAEAAAIE KFAEAFKNVNFQPGATVFYR ENIASSPFLFHNKSISNNDSNNFQLAK

FMQVTMILPLTGQQYSEKVSE-NCVA IWKHLGIYTDEEGKDQTFPPGSS ILFTVLPK EAGSTMEVVAEQTKAIADKQAQEV HANLR INIGGDLISPKIDPLSRDAPMDEGHV STAVKGSFGYLDPEYFR DQADYISVPVEGPYKPAHYRL SKDQADYISVPVEGPYKPAHYR ALEYEDFDKFDRALEYEDFDKF DRVR

VVDLLAPYQRIPSAVGYQPTLATD LGGLQER ATAGDTHLGGEDFDERARFEEL CMDLFR GYIVQIIGPVLDVAFSPGMMPSIYNA LVVQGRHNQEPNVTCEVQQLL GNNRTVLIMELINNIAK VVDLLAPYQRAHGGFSVFAGV GERYDEGLPPILTALEVLDNDIR EAFPGDVFYLHSRATSESETLYCV YVAIGQKR

LTPEEMVELEAIAQPDFVKMVELE AIAQPDFVK MATIKVHGVPMSTATMRATIKVHG VPMSTATMRVHGVPMSTATM RVLAALYEK HAGDLGNITVGDDGTATFTII-DSQIP LTGPNSIVGRVGDDGTATFTIIDSQ IPLTGPNSIVGR LSPTFYATSXPNVXXTRPTFYATSX PNVXXTR LPGFHVCVGSGGERSVEEYDYLP YFYSR

Peptide sequences matched

Transport phytohormone auxin

Cellular morphology

Protein biosynthesis

Flavonoid biosynthesis

Carbohydrate metabolism

Amino acid metabolisms

Signal transductions

Carbohydrate metabolism

Flavonoid biosynthesis

Bioenergetic metabolism



Molecular chaperone


ROS detoxification and stress responses Glutathione metabolism

ROS detoxification and stress responses

Defense and stress responses

ROS detoxification

Function

8758 Environ Sci Pollut Res (2014) 21:8750–8764

Function unknown

Function unknown

Function unknown

8.54 35/5

Mr relative molecular weight, T theoretical, O observed, SC sequence coverage, FC fold change

70/5.6 68.4/6.03 Selaginella moellendorffii Hypothetical protein 19

gi|302811229

18.59 58/18 24/5.9 23.9/5.3 Selaginella moellendorffii Hypothetical protein 7

gi|242050434 Hypothetical protein SORBIDRAFT 4

Uncharacterized proteins

gi|302770993

Sorghum bicolor

Zea mays NIP11_MAIZE Aquaporin NIP1-(homologue) 9

Transport

8759

EEIVYNYFKGILGTPFQRGLRQGD PISPMLFVIVMESLNSLFKEAD REVVLIDSQADR MDVENVKVDDNDVKDLHFDLLTL HFIELVRLQQEQGGK SEVHVDENVAAPSPRTCMFTLVFQ DAAESSQR 17.69 43.34/10.0

17/5.4

26/6.4 29.65/8.5

O T

Mr (kDa)/pI Reference organism Accession no. Protein identified Spot no.

Table 2 (continued)

98/15

30/15

MS–MS score/SC (%)

FC

3.42

Peptide sequences matched

EEFADQGCAAMVVSVPFIQKIIAEIF GTYFLMFAGCGAVTINASK

Function

Transport water or neutral solutes


et al. 2013). It can also suggest that Pb-induced depletion of GSH facilitates PC syntheses, which further bind to Pb and carry them to the vacuole for detoxification and ultimately suppress the activation of stress-related responses in plant (Mishra et al. 2006; Estrella-Gómez et al. 2009). Pb-induced changes in GSH have been reported in Vicia faba and Phaseolus vulgaris (Piechalak et al. 2002). In our experimental condition, Pb treatments caused significant (P≤0.05) induction in GSSG content (Fig. 1d). GSSG may be increased as a result of oxidation of glutathione due to oxidative stress (Mishra et al. 2006; Anjum et al. 2012). The GSSG is reduced into GSH through NADPH-dependent reaction catalyzed by enzyme GR. Pb treatments caused an insignificant decline in GR activities when compared with the control (Fig. 2c), suggesting that GR does not have an important role in GSH regeneration from GSSG under Pb-induced stress conditions. GSTs are GSH-dependent ROS-detoxifying enzymes involved in GSH metabolism and play important roles in the cellular antioxidant defense mechanisms (Mishra et al. 2006; Anjum et al. 2012). GSTs catalyze the conjugation of GSH to a wide range of toxic electrophilic metabolites, generated through ROS action, converted them into inactive or nontoxic form and transported to vacuoles for sequestration (Coleman et al. 1997; Singh et al. 2010). In our result, the increase in GST might be attributed toward the conjugation of GSH with Pb or other toxic electrophiles, which further detoxify them and strengthen the plant cell against Pb-induced oxidative stress (Singh et al. 2010). On the other hand, we observed a decrease in AsA content representing a clear indication about oxidation of AsA to MDA. This oxidation takes place by the activity of ascorbate peroxidase (APX) with concomitant detoxification of H2O2 into a water molecule (Sytar et al. 2013). AsA is a strong ROS scavenger, because it has ability to donate electrons in a number of enzymatic and non-enzymatic reactions (Mishra et al. 2006). In our previous study, we observed almost similar responses of APX activity at higher Pb concentrations (Kumar et al. 2013) that might corroborate with the regulation of AsA content. FTIR spectral analysis of 1.25 mM Pb-treated sample showed noticeable reductions in %transmittance of few characteristic peaks when compared with the control spectra. At 3,400–3,200 cm−1, reduction in 8.9 %transmittance was observed, representing the –OH (alcoholic or hydroxyl) and N– H (amide) functional groups (Yalçın et al. 2012). Alcoholic or hydroxyl group might be donated from phenolic, alcoholic, carboxylic, and amino acids, whereas amide was from amino acids. Additionally, at 1,643 cm−1, reduction of 8.0 %transmittance was noted. This peak represents C=O (carboxylic) functional group from amide I or amino acid or peptides (Nahar and Tajmir-Riahiha 1996). Furthermore, reduction in 7.5 %transmittance was observed at 1,055 cm−1, representing the C–O (ester or amide) functional group. This might be

8760


donated by amino acid or peptide or lipid (Marmiroli et al. 2005). Along with these peaks, minor reductions in few more peaks were observed (Table 1), which clearly indicated the non-involvement of respective functional group in tolerance and detoxification process. Comparative proteomic analysis revealed the dynamic role of root proteins involved in different cellular functions. Our proteomic study showed that various defense mechanisms were activated in T. triangulare roots during Pb treatment with concomitant changes in many physiological and biochemical mechanisms (Fig. 7). Total 23 protein spots were identified and categorized in various groups based on their functions (Fig. 6c, Table 2), which will be discussed in the subsequent categories. ROS detoxification and defense This group constituted 21.739 % of the total identified proteins which encircle five spots, i.e., 1, 2, 5, 6, and 12 representing aldo-keto reductase, GSTs, Cu-Zn superoxide dismutase (SOD), peroxidase, and monodehydroascorbate reductase (MDAR) proteins, respectively. Aldo-keto reductase, a group of enzymes related to oxidoreductases, facilitates the interconversion of hydrogen molecules between aldehydes or ketones with the reduction of nicotinamide adenine dinucleotide (NAD+ to NADH) and is known to detoxify the highly cytotoxic ROS molecules (Sengupta et al. 2012). A similar response was also observed in the Cannabis sativa roots under copper (Cu) stress condition (Elisa et al. 2007). In addition to this, Cu-Zn SOD (spot 5) has also been identified as differentially accumulated during Pb stress. The increased abundance of Cu-Zn SOD was also observed in Vigna radiata roots under water deficit

condition (Sengupta et al. 2011) and in Arabidopsis seeds to cope against Cu stress (Gill et al. 2012). Further, enzyme peroxidase (fragment) was also differentially accumulated (spot 6) during 1.25 mM Pb treatment. Peroxidases are well known for detoxification of H2O2 into H2O as well as also involved in H2O2 signaling in plant cell (Potters et al. 2010). Thus, the significant high induction of probable aldo-keto reductase, CuZn SOD, and peroxidase in T. triangulare under Pb stress indicate that detoxification pathways are highly crucial to ameliorate Pb-induced oxidative stress. Spots 2 and 12 were identified as GST and MDAR enzymes, where they were differentially accumulated by 6.0and 1.8-fold during 1.25 mM Pb treatment, respectively. Almost all of the cytosolic GSTs have been known to occur as homo- or hetero-dimers of subunits with molecular weights of 23–29 kDa, which is quite similar to the 17 kDa protein as identified in our experiment. The differential abundance of GSTs has been identified in various plant root proteomic studies under several metal stress conditions such as in soybean under aluminium (Al) stress (Duressa et al. 2011), in rice under Cu stress (Ahsan et al. 2007), and in tomato roots under cadmium (Cd) stress (Rodríguez-Celma et al. 2010). On the other hand, MDAR is an important enzyme of AGC, which has a major role in H2O2-scavenging pathway, maintaining the AsA level in the cell and adjusting the cellular redox potential (Singh et al. 2010; Potters et al. 2010). Increased relative abundances of GST and MDAR during Pb stress condition are strongly corroborated by our spectrophotometric analysis of AGC metabolites and enzyme activities and suggest that glutathione metabolism has a vital role in detoxification of Pbinduced oxidative stress.

Lead (Pb) Ascorbate-glutathione metabolisms and Pb-detoxification mechanisms

Redox imbalance

Glu + Cys

γ-Glu–Cys

γ-ECS Gly

ROS production

Activation of signaling pathways

Vacuole PC–Pb

GS GSH

Pb PCs

Induction of stress responsive genes Hypothetical protein Transporter

ROS-detoxification/ defense

Rootmorphology Cell signaling/ Metabolisms

Protein synthesis/ Energy metabolism

Fig. 7 Schematic representation of the networking mechanisms for the identified groups of proteins and overall ascorbate–glutathione metabolisms in response to Pb-induced oxidative stress in T. triangulare. APX ascorbate reductase, Cys cysteine, DHA dehydroascorbate, DHAR dehydroascorbate reductase, γ-ECS γ-glutamylcysteine synthetase, Glu glutamine, Gly glycine, GR glutathione reductase, GS glutathione

(detoxified)

ROS

GR PCS

GSSG GSH

GSH-Pb

Pb

GST DHAR Lipid hydroxyperoxide DHA GST–Lipid hydroxyperoxide H2O2 H 2O (detoxified) APX Ascorbate MDA MDAR NAD(P)+ NAD(P)H

synthetase, GSH glutathione (reduced), GSSG glutathione (oxidized), GST glutathione-S-transferase, H 2 O 2 hydrogen peroxide, MDA monodehydroascorbate, MDAR monodehydroascorbate reductase, NAD(P)+ nicotinamide adenine dinucleotide phosphate, NAD(P)H NAD(P) reduced, PCs phytochelatins, PCS phytochelatin synthase, ROS reactive oxygen species


Protein synthesis/energy metabolism The peptides identified in spot 11 matched to HSP70 of Chlamydomonas reinhardtii with two matched peptides. The heat-shock proteins are commonly induced under stressful conditions and play a crucial role protecting against stress by re-establishing cellular homeostasis (Wang et al. 2004; Rodríguez-Celma et al. 2010). Members of HSP70 family were reported to be differentially accumulated in response to various abiotic stresses (Wang et al. 2004; Kosová et al. 2011; Hossain et al. 2012). HSP70 acts as a molecular chaperone and has a wide range of functions including protein folding, assembly, translocation, degradation, transport of various proteins across membranes, and preventing proteins from arbitrary aggregation under stress conditions (Wang et al. 2004, 2011). It has been suggested that the increased relative abundance of HSP70 under Pb or various HMs in different plants such as rice (Ahsan et al. 2007), wheat roots (Wang et al. 2011), and soybean roots (Duressa et al. 2011) was probably related to cellular protection against HM-induced damage. Spots 10 and 13 were identified as ATP synthase subunit β, spot 15 representing ATP synthase β chain, and spot 18 was confirmed as ATPase synthase subunit α, which were differentially abundant by 3.3-, 2.8-, 4.1-, and 3.6-folds, respectively, during 1.25 mM Pb stress when compared with the control. These proteins belong to bioenergetic metabolism and were associated with the energy production in the form of adenosine triphosphate (ATP) as well as transport ions and protons across the membrane electrochemical gradient. The increased abundance of four different subunits of ATP synthase revealed that high energies are required to cope against Pb stress in T. triangulare roots (Rodríguez-Celma et al. 2010; Wang et al. 2011). The increased energy production might be related to an enhanced utilization of energy in the synthesis of defenserelated metabolites required for combating against Pb-induced toxicity. Our results are in a good accordance with roots of Cdtreated wheat (Wang et al. 2011) and tomato (RodríguezCelma et al. 2010), where increased abundance of ATP synthase were discussed for an increase in energy production required during Cd stress. Cell signaling/cell metabolism In response to Pb stress, an increased relative abundance of two proteins for carbohydrate metabolisms has been observed in T. triangulare root. Spots 8 and 20 identified as triosephosphate isomerase (EC 5.3.1.1; 3.5-fold) and 2,3bisphosphoglycerate-independent phosphoglycerate mutase 1 (3.1-fold), which catalyzes the fifth and eight steps of glycolysis, respectively. The differential relative abundance of these enzymes suggests that glycolysis might have a key role to adjust and accomplish the energy required for synthesis of various metabolites useful for stress acclimatization in root

8761

cells under Pb stress condition (Wang et al. 2004). Changes in relative abundance of several glycolysis metabolic enzymes such as glucose phosphate isomerase, triosephosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase have been observed during various abiotic stress conditions (Ahsan et al. 2007; Duressa et al. 2011; Sengupta et al. 2011; Hossain et al. 2012). Spot 17 was relatively abundant by 3.8-fold during 1.25 mM Pb stress and identified as S-adenosylhomocysteinase. SAdenosylhomocysteinase catalyzes the lysis of Sadenosylhomocysteine to form homocysteine with the elimination of adenosine. The intermediate homocysteine can either synthesize methionine or form cysteine through respective metabolic pathways (Ravanel et al. 2004; Sharma and Dietz 2006). Methionine is a sulfur-containing amino acid which serves as a component of the universal activated methyl donor S-adenosylmethionine and the building block in protein synthesis (Sengupta et al. 2011; Hossain et al. 2012). In addition, cysteine is required for the synthesis of methionine and GSH/PCs (Sharma and Dietz 2006; Singh et al. 2010). It might be possible that an increased accumulation of amino acids during HM stress conditions indicate a significant protective role as well as regulatory function (Sharma and Dietz 2006), as we observed in our spectrophotometric analysis of cysteine content. The adenosine produced as the action of Sadenosylhomocysteinase may also act as the precursor for purine nucleotide biosynthesis, which serves multiple roles in the plant cells (Stasolla et al. 2003). Thus, it can be suggested that the S-adenosylhomocysteinase might be a key regulator of various plant metabolic pathways which ensure an optimum plant performance during Pb-induced oxidative stress. Sensitivity of cells to sense the stress signal and subsequent changes in gene expression and cell metabolism were the key events in plant adaptation response to a stress factor (Sharma and Dietz 2006; Estrella-Gómez et al. 2009). In our experiment, spot 16 is identified as probable receptor-like protein kinase (RLPK) of Arabidopsis thaliana. RLPK membrane proteins belong to serine/threonine protein kinase superfamily and contain one protein kinase domain. This superfamily belongs to the family of transferases, and it is recognized as the largest class of transmembrane sensors in A. thaliana (Wigniewska et al. 2003). The increase in relative abundance of RLPK might have relevance in Pb-induced oxidative stress signal. It is possible that RLPK play crucial roles in communication between cells or organs that control the defense mechanisms in response to Pb stress in T. triangulare. Flavonoids are considered as a large family of plant secondary metabolites, and are well known antioxidants. Through MALDI-TOF, spot 3 was identified as a chalcone– flavanone isomerase (CFI) and was differentially accumulated by 3.8-fold, which belongs to the family of isomerases. This enzyme catalyzes the isomerization of chalcones to their

8762

corresponding flavanones in flavonoid biosynthetic pathway. Flavanones are a type of flavonoids belonging to plant polyphenolic secondary metabolites and possessing strong ROS scavenger groups, which are known for capturing free radicals ions by donation of its phenolic hydrogen atoms (Ravanel et al. 2004; Hossain et al. 2012; Sytar et al. 2013). These results are in strong accordance with our FTIR result explaining that the phenolic functional group may be involved in tolerance mechanisms. Thus, the increased relative accumulation of CFI demonstrates that a rapid isomerization of chalcones to flavanones is an important strategy of T. triangulare against high Pb concentrations.


hand, the active efflux of As(III) from roots through aquaporin showed a potential role in decreased As accumulation and increased As detoxification strategy in plants (Duan et al. 2012). Furthermore, the transgenic study of A. thaliana mutants with independent mutations in AtNIP1;1 showed a decrease in As contents in comparison to wild type confirmed that NIP1;1 is the key determinant of As(III) uptake and transport (Duan et al. 2012). It can be suggested that an increased relative abundance of NIP1;1 is possibly responsible for metal transport and maintaining the water status during Pb stress condition. Newly identified proteins

Root morphology/cell structure Spot 14 was identified as actin 1 (cytoskeleton protein) and was relatively accumulated by approximately 5-fold during Pb stress when compared with the control. Apart from being the essential housekeeping gene, actin also performs certain special cellular functions including cell division, cytokinesis, cell signaling, vesicle, organelle movement, and maintenance of cell junctions and cell shape. Actin is also known to have a dynamic plasticity with respect to its structure under different abiotic stress conditions (Sengupta et al. 2011; Duressa et al. 2011). It has also been reported about increased relative abundance of actin in Cu-treated Cannabis sativa roots (Elisa et al. 2007), in Cd-treated wheat roots (Wang et al. 2011), and Altreated soybean roots (Duressa et al. 2011). The response of actin protein to Pb stress may be related to cellular adaptive strategies against structural modification involving cell rupture and formation of pores, as we observed in our previous electron microscopic study of T. triangulare roots under different Pb treatment (Kumar et al. 2013). Transporters Aquaporins are channel proteins that facilitate the transport of water and small neutral molecules across cell membranes (Dynowski et al. 2008). Spot 9 was identified as aquaporin nodulin-26-like intrinsic protein 1;1 (NIP1;1) protein and was relatively accumulated by 3.5-fold during 1.25 mM Pb stress. Plant aquaporins can be divided into four subfamilies; among them, NIPs are unique to plants (Kamiya et al. 2009; MitaniUeno et al. 2011). NIPs are involved in the transport of a wide variety of solutes, but the mechanisms controlling the selectivity of transport substrates are poorly understood (MitaniUeno et al. 2011). Pb treatment has been reported to downregulate the aquaporin plasma membrane intrinsic protein (PIP1-6) in maize roots (Visioli and Marmiroli 2013), which was suggested to be responsible for an inhibition in plant water transport. Aquaporin homologues facilitate the uptake and rapid transport of arsenic (As) in Pteris vittata and other plants (Bienert et al. 2008; Mathews et al. 2011). On the other

In our experimental conditions, we have identified only three new spots as they marked by N1, N2, and N3 in 2-DE gel (Fig. 5a), which represents CFI, auxin transport, and DNAdirected RNA polymerase subunit β proteins, respectively. As CFI already identified as spot 3 with different Mr/pI (23.84/ 5.3), here, an increased relative accumulation as a new spot confirmed the presence of different isoforms with significant importance in Pb-induced stress acclimatization. Usually, the phenomenon results from due to posttranslational modification or protein degradation. In addition, the phytohormone auxin has well-established functional roles in embryogenesis, cell division and elongation, lateral root formation, and other physiological processes of plants (Friml et al. 2003). Auxin is synthesized in the leaves and then transported to the roots via vascular tissues, and this is mediated through various auxintransport proteins along with the diffusive flux of auxin across apoplast (Titapiwatanakun and Murphy 2009). Modulations in the expression of auxin transport proteins were highly crucial as the concentration gradient created by directional auxin movement could be strongly correlated to the plant morphological adaptations to the environment along with the establishment of plant axial polarity and organ patterning (De Smet and Jurgens 2007). In the present study, an auxin transport protein (spot N2) was found to be newly accumulated in T. triangulare roots subjected to 1.25-mM Pb concentrations. It is known that in hydroponic culture, high metal concentrations result in osmotic stress to plant due to a reduced uptake of water through the roots and under such conditions, plants try to modulate their root morphology for maximizing water conductivity. Thus, based on our results, we can hypothesize that Pb-induced auxin transport protein is for altering its root morphology through lateral root formation in order to tolerate Pb-induced osmotic stress. The osmotic stress may alleviate by aquaporin (spot 9) channel, which may enhance water conductivity during Pb-induced water limitations. Furthermore, DNA-directed RNA polymerase subunit β is known to be involved in the transcription of DNA to mRNA that further translated into peptide with the help of various translation factors. The two isoforms of DNA-directed RNA


polymerase subunit β were expressed in cobalt-treated Pseudomonas putida (Ray et al. 2013). Certainly, the increased accumulation of transcription factor, RNA polymerase subunit β, during Pb stress in our experiment indicates that T. triangulare roots possibly promote protein synthesis during Pb stress, which may reveal key responses of its tolerance mechanisms against Pb stress. Hypothetical proteins Spots 4, 7, and 19 were identified as hypothetical proteins, and hence, no defined role could be assigned to these spots. Since these proteins exhibited an increased relative abundance during 1.25 mM Pb when compared with the control, the possibility existed that these hypothetical proteins might be involved in tolerance to Pb-induced oxidative stress.

Conclusion Our results concluded that NPSH, cysteine, GSH, GSSG, and GST played vital roles in T. triangulare during Pb treatment. The decline in NPSH and GSH clearly indicates their major involvement in defense reaction through synthesis of sulfurrich bioligands. From the results, it can be suggested that ascorbate–glutathione and other related metabolites and enzymes cumulatively form an efficient defense system against ROS in addition to their significance for the detoxification and compartmentalization of Pb in the plant tissues (Fig. 7). Functional group identification gave confirmatory evidence for our spectrophotometric and proteomic results which showed amino, phenolic, carboxylic acids and other alcoholic, amide, and ester-containing biomolecules have a key role in detoxification of Pb or Pb-induced toxic metabolites. Significant differential abundance of identified proteins involved in ROS detoxification and defense, protein synthesis/energy metabolism, cell signaling/metabolism, root morphology, and transporters provides an interesting insight into the root system dynamics against Pb-induced toxicity (Fig. 7). Under high Pb concentrations, T. triangulare primarily regulates its water conductivity through induction of water conducting aquaporins and root morphology-related proteins including actins and auxin transporters. Identification of new protein spots possibly revealed the importance of those proteins in secondary metabolisms, root morphology, and tolerance mechanisms against Pb stress. Acknowledgments A.K. gratefully acknowledged the University of Hyderabad Research Scholarship through the University Grant Commission, New Delhi. The authors express their gratitude to Ms. Monika and Ms. Kalpana, Proteomics facility, UoH and Dean, School of Chemistry, UoH, for FTIR analysis.

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