Chemosphere 120 (2015) 309–320

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Ultrastructural, metabolic and proteomic changes in leaves of upland cotton in response to cadmium stress M.K. Daud a,b, He Quiling a, Mei Lei a, Basharat Ali a, S.J. Zhu a,⇑ a b

Department of Agronomy, College of Agriculture and Biotechnology, Zijingang Campus, Zhejiang University, Hangzhou, PR China Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat 26000, Pakistan

h i g h l i g h t s  Cd deposited more in roots than leaves.  Leaf’s physiology and ultramorphology did not drastically alter.  ROS-scavenging enzymes were active in leaves.  ROS-combating and mitochondrial respiration related proteins were upregulated.  Methionine synthase, involved in lignification process, was also upregulated.

a r t i c l e

i n f o

Article history: Received 20 January 2014 Received in revised form 9 June 2014 Accepted 22 July 2014

Handling Editor: A. Gies Keywords: Antioxidants Cadmium Energy-dispersive X-ray analysis (EDX) Gossypium hirsutum L. Proteomic changes Ultrastructural modifications

a b s t r a c t Present study explores physiological, biochemical and proteomic changes in leaves of upland cotton (ZMS-49) using 500 lM cadmium (Cd) along with control. Leaves’ biomass and chlorophyll pigments decreased at 500 lM Cd. Cd contents in roots were higher than leaves. Levels of ROS (O2A and H2O2) both in vivo and in vitro and MDA contents were significantly increased. Chlorophyll parameters (F0, Fm, F 0m and Fv/Fm), total soluble protein contents and APX showed a decline at 500 lM Cd. SOD, CAT and POD and GR activities significantly enhanced. Less ultrastructural alterations in leaves under Cd stress could be observed. Scanning micrographs at 500 lM Cd possessed less number of stomata as well as near absence of closed stomata. Cd could be located in cell wall, vacuoles and intracellular spaces. Important upregulated proteins were methionine synthase, ribulose 1,5-bisphosphate carboxylase, apoplastic anionic guaiacol peroxidase, glyceraldehydes-3-phosphate dehydrogenase (chloroplastic isoform) and ATP synthase D chain, (mitochondrial). Important downregulated proteins were seed storage proteins (vicilin and legumin), molecular chaperones (hsp70, chaperonin-60 alpha subunit; putative protein disulfide isomerase), ATP-dependent Clp protease, ribulose-1,5-bisphophate carboxylase/oxygenase large subunit. Increase in the activities of ROS-scavenging enzymes, less ultrastructural modification, Cd-deposition in dead parts of cells as well as active regulation of different proteins showed Cd-resistant nature of ZMS-49. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Cadmium (Cd) is a toxic pollutant, which mainly comes from different sources such as pesticides, mining and chemical fertilizers (Daud et al., 2013a) into environment. In comparison with ani-

Abbreviations: APX, ascorbate peroxidase; CAT, catalase; (EDX), energy-dispersive X-ray analysis; H2O2, hydrogen peroxide; OH, hydroxyl radical; MDA, malondialdehyde; POD, guaiacol peroxidase; ROS, reactive oxygen species; SEM, scanning electron microscopy; SOD, superoxide dismutase; O2, superoxide radical; TEM, transmission electron microscopy. ⇑ Corresponding author. Tel.: +86 13067922851. E-mail address: [email protected] (S.J. Zhu). http://dx.doi.org/10.1016/j.chemosphere.2014.07.060 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

mals, plants are more vulnerable to Cd stress (Daud et al., 2013b). In plants, it influences several physiological events such as water uptake, nutrient assimilation, photosynthesis and respiration (Lage-Pinto et al., 2008). At ultramorphological level, a number of sub-cellular alterations in roots and leaves under Cd stress have also been documented by Daud et al. (2009a) and Daud et al. (2009b). Cd can be deposited in different subcellular organelles. Knowledge about its deposition and distribution in cellular compartments is gained through energy-dispersive X-ray analyses (EDX) and electron energy loss spectroscopy (EELS) attached with transmission and scanning electron microscopy. Cd also disturbs the biochemistry of plant cells by causing stressful conditions (Lannig et al., 2006). Resultantly, reactive

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oxygen species (ROS) of toxic nature are generated (Ahsan et al., 2009). To avoid or to reduce their production, various mechanisms regarding its absorption and uptake, sequestration and synthesizing antioxidant molecules (Jin et al., 2008) become active. Important one is the activation of antioxidant defense mechanism, which is mainly comprised of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) etc. Alternatively, Cd greatly disturbs the expression levels of various proteins. How various proteins are up and down-regulated can be better understood through proteomic studies. These studies are based on the systematic analysis and documentation of expressed proteins and their study at the functional level (Nwugo and Huerta, 2011). Present study was designed to explore physiological, biochemical and proteomic changes in leaves of upland cotton induced by Cd stress. This is first comprehensive study by employing physiological, ultramorphological, biochemical and proteomic approaches in order to have a deeper look about various mechanisms being active in cotton leaves under Cd stress. 2. Materials and methods 2.1. Plant materials and growth conditions An upland cotton cultivar (ZMS-49, kindly provided by Chinese Academy of Agricultural Sciences), was used in the present growth chamber experiment. Uniform grade matured seeds were surface sterilized by first immersing in 70% ethanol for 3 min and then in 0.1% HgCl2 for 8–10 min. They were washed with ddH2O for three times and soaked overnight in dH2O. Sterilized seeds were sown in a mixture of peat and vermiculite (7:3, v:v) and grown in a growth chamber for ten days. Complete dark conditions were provided for three days and for seven days 16 h photoperiod of 50 lmol m2 s1 under white fluorescent light was provided. At eleventh day, uniformly grown seedlings were washed thoroughly with care and were grown in modified Hoagland solution for four hours for acclimatization. Then seedlings were transferred to fresh medium having 500 lM Cd as CdCl22.5H2O. Medium without Cd salt acted as control. Seedlings were grown in the Cd stressful conditions for 24 h. At the end of stress period, seedlings were divided into roots, stems and leaves. Leaves were subjected to various physiological, ultrastructural, biochemical, and proteomic studies. For each study, separate independent experiments were run. There were kept three replications per each level. Throughout experiment 28 ± 2 °C culture temperature as well as 60% relative humidity were maintained. 2.2. Measurements of biomass, photosynthetic pigments and Cd contents After 24 h Cd stress, leaves were subjected to biomass, photosynthetic pigments and Cd analyses. For each measurement, there were kept three replications with different number of plants. Regarding fresh and dry biomasses, three plants per replication were taken. In order to quantify photosynthetic pigments, 0.1 g fresh leaves/replication was used to determine the chlorophyll pigment composition. 80% acetone was used to extract the pigments, which were determined spectrophotometerically using the method of Ahammed et al. (2012). For Cd contents analysis in both roots and leaves, the number of plants per replication was fifteen. Seedlings were washed with tap and distilled water thrice, respectively. Roots were immersed in 20 mM EDTA-Na2 for 15 min in order to remove adhering metals and were washed with dH2O for three- four times. After that, seedlings were blotted dry to remove the excessive water and were separated into roots and leaves, oven dried at 80 °C for 48 h, and

then ground into powder. 0.2 g of each root and shoot samples were digested with a mixture of 5 ml of HNO3 + 1 ml of HClO4. The resultant solutions were diluted to 25 ml using 2% HNO3 and then filtered. The concentrations of Cd in the filtrate were determined using inductively coupled plasma atomic emission spectroscope (ICP-AES, IRIS/AP optical emission spectrometer, Thermo Jarrel Ash, San Jose, CA) following standard procedures. 2.3. In situ tissue localization of reactive oxygen species In situ localization of ROS (i.e. O2A and H2O2) was done according to Bernstein et al. (2010) with some modifications. Briefly, leaves were detached from the stem and washed for three times in ddH2O and were subjected to staining for both superoxide and hydrogen peroxide. For superoxide localization, NBT was used. Leaves were gently vacuum-infiltrated (5 min) with 0.01% NBT solution and incubated in the dark in the same solution for 2 h at 30 °C under very slow shaking. To determine that this staining was due to the formation of O2A , 10 mM MnCl2 was added together with NBT as a negative control. After staining for 2 h, chlorophyll was removed from the tissue by boiling the segments in a 9:1 solution of 99% ethanol and glycerin for 10 min. Leaves were observed for the presence of blue spots, which show the presence of superoxide. For H2O2 detection in leaves, 3, 3-diaminobenzidine (DAB) was used as staining agent. Leaves were gently vacuum-infiltrated with 1 mg ml1 DAB solution for 10 min and incubated in the dark in the same solution for 4 h at 30 °C under very slow shaking. As a negative control, 200 lM diphenyleneiodonium (DPI) was added together with DAB. After staining, the chlorophyll was removed from the leaf tissue by boiling the segments in 95% ethanol for 10 min. Brown spots were searched, which are the characteristics of the presence of H2O2. Three replications per treatment were done for each type of localization studies and stained segments were photographed with digital camera to obtain images. 2.4. Quantification of MDA contents and ROS 0.5 g leaves materials of young seedlings of ZMS-49 were used for the determination of lipid peroxidation and reactive oxygen species. Lipid peroxidation was estimated in terms of malondialdehyde (MDA) contents and was determined as 2-thiobarbituric acid (TBA) reactive substances following the method of Zhou and Leul (1998). For determination of hydrogen peroxide (H2O2) content, 0.5 g leaves were crushed with 5.0 ml of TCA (0.1%, w/v) in an ice cold conditions, and the homogenate was centrifuged at 14,000 g for 20 min (Velikova et al., 2000). In total 4 ml reaction mixture, there was added 1 ml supernatant, 1 ml phosphate buffer (pH 7.8) and 2 ml potassium iodide (1 M) and the absorbance was read at 390 nm. H2O2 content was determined using an extinction coefficient of 0.28 lM cm1 and expressed as lmol g1 FW. Superoxide radical (O2A ) content was determined according to Jiang and Zhang (2002) method with some modifications. Leaves samples (0.5 g) were homogenized in 3 ml of 50 mM potassium phosphate buffer (pH 7.8) and then homogenate was centrifuged at 10 000 g for 10 min at 4 °C. 1 ml of supernatant was mixed with 0.9 ml of 50 mM potassium phosphate buffer (pH 7.8) and 0.1 ml of 10 mM hydroxylamine hydrochloride, and then incubated it at 25 °C for 24 h. After incubation 1 ml of 17 mM sulphanilamide and 1 ml of 7 mM a-naphthylamine was mixed in 1 ml solution for further 20 min at 25 °C. After incubation, n-butanol in the same volume was added and centrifuged at 1500 g for 5 min. The absorbance of supernatant was noted at 530 nm in spectrophotometer. Standard curve was used to calculate the production rate of O2A in the leaves.

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2.5. Measurements of chlorophyll florescence parameters Photochemical quenching parameters (F0, Fm, F 0m and Fv/Fm) were measured using an imaging pulse amplitude-modulated (PAM) fluorimeter (IMAG-MAXI; Heinz Walz, Effeltrich, Germany). F0 is minimal fluorescence yield when the PSII reaction center is open. Fm is maximal fluorescence yield at the time of closure of PSII reaction center. F 0m is the maximal fluorescence in the light-adapted state. Fv/Fm is the photochemical efficiency of PS II and is used as basic tool in plant photosynthetic research for stress studies. In order to measure these parameters, expanded leaves were first dark adapted for 15 min. And all measurements were taken from the same leaf. There were three replications and in each replication, three leaves were randomly selected from three different plants. Measurements for each parameter on a single leaf were done at five different locations and their means were calculated. Thus for every replication, the means were calculated for 15 different locations of the three different leaves. 2.6. Total soluble proteins and antioxidants status In order to determine the total soluble proteins and antioxidative metabolism status in leaves of upland cotton under Cd stress, 0.5 g leaves’ sample was homogenized in 8 ml of 50 mM potassium phosphate buffer (pH7.8) under chilled conditions. The crude extract was centrifuged at 14 000 g for 15 min at 4 °C and the supernatant was used for the determination of total soluble protein and various enzymatic and non-enzymatic antioxidants. Total soluble protein content was determined using the method of Bradford (1976) and bovine serum albumin was used as a standard. Superoxide dismutase (SOD) (EC1.15.1.1) activity was determined according to Zhang et al. (2008) following the inhibition of photochemical reduction due to nitro blue tetrazolium (NBT). The reaction mixture consisted of 50 mM potassium phosphate buffer (pH7.8), 13 mM methionine, 75 mM NBT, 2 mM riboflavin,0.1 mM EDTA-Na2 and 100 ll of enzyme extract in a 3 ml volume. One unit of SOD activity was measured as the amount of enzyme required to cause 50% inhibition of the NBT reduction measured at 560 nm. Ascorbate peroxidase (APX) (EC 1.11.1.11) activity was measured in a reaction mixture of 3 ml containing 50 mM potassium phosphate buffer (pH7.8), 0.1 mM EDTA-Na2, 0.3 mM ascorbic acid, 0.06 mM H2O2 and 100 ll enzyme extract. The change in absorption was taken at 290 nm 30s after addition of H2O2 (Nakano and Asada, 1981). Catalase (CAT) (EC1.11.1.6) activity was measured according to Aebi (1984) with the use of H2O2 (extinction coefficient 39.4 mM cm1) for 1 min at A240 in 3 ml reaction mixture containing 50 mM potassium phosphate buffer (pH7.8), 2 mM EDTA-Na2, 10 mM H2O2 and 100 ll enzyme extract. Peroxidase (POD) (EC1.11.1.7) activity was assayed by Zhou and Leul (1998) with some modifications. The reactant mixture was consisted of 50 mM potassium phosphate buffer (pH7.8), 1% guaiacol,0.4% H2O2 and 100 ml enzyme extract. Variation due to guaiacol in absorbance was measured at 470 nm. Glutathione reductase (GR) (EC1.6.4.2) activity was assayed according to Jiang and Zhang (2002) with the oxidation of NADPH at 340 nm (extinction coefficient 6.2 mM cm1) for 1 min. The reaction mixture was contained of 50 mM potassium phosphate buffer (pH7.8), 2 mM EDTA-Na2, 0.15 mM NADPH, 0.5 mM GSSG and 100 ll enzyme extract in a 1 ml volume. 2.7. Ultra-morphological and microlocalization studies In order to observe the Cd-induced ultrastructural modifications in leaf mesophyll cells, 1 mm2 leaf segments without veins were taken (n = 3) and were fixed overnight in 2.5% glutaraldehyde

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(v/v) in 100 mM PBS (Sodium Phosphate Buffer, pH 7.4) after vacuum infiltration for 15 min in the same solution. Samples were then processed for ultramicroscopic (transmission, scanning) and Cd microlocalization studies using manufacturers standard protocols (JEOL TEM-1230EX for TEM, Hitachi Model TM-1000 SEM for SEM and EDAX GENESIS XM2 30TEM for EDX). 2.8. Protein extraction, visualization and identification studies 2.8.1. Soluble protein extraction and 2-DE analysis For proteomic study, two biological independent replicates were used throughout the experiment. Total soluble proteins were extracted according to Carpentier et al. (2005) with minor modification using phenol extraction method. Concentration was determined by Bradford (1976) method. Proteins were separated by two-dimensional gel electrophoresis (2-DE), and the protein spots in analytical gels were visualized by silver staining method. 2.8.2. Protein visualization, image analysis, and quantification Protein visualization, image analysis, and quantification were done according to Bah et al. (2010). Briefly, PowerLook1100 scanner (UMAX) was used for scanning and calibration of the protein spots. Only those with significant and reproducible changes (P 6 0.05) were considered to be differentially accumulated proteins. The target protein spots were automatically excised from the stained gels and digested with trypsin using a Spot Handling Workstation (Amersham Biosciences). Peptides gel pieces were placed into the EP tube and washed with 1:1 mixture of 50 lL of 30 mM K3Fe(CN)6 and 100 mM NaS2O3 for 10–15 min until completely discolored then washed with 200 lL bi-distilled water (two times for 5 min each). The washed solution was drained and washed with 50% CAN (acetonitrile, Fisher A/0626/17) and 100% ACN rotationally, and then incubated in 25 mM NH4HCO3 (Sigma A6141) for 5 min at 37 °C. After trickling down of the incubation solvent, 50% ACN and 100% ACN was rotationally added and dried at 40 °C for 5 min respectively. Trypsin digestion was carried out as follows: sequencing-grade porcine trypsin (Promega, Madison, WI, USA) was suspended in 25 mM NH4HCO3 at a concentration of 12.5 ng ll1 to rehydrate the dried gel pieces. The trypsin digestion was carried out for 16 h at 37 °C. Peptides were extracted from the digest as follows for three times: 10 lL of 50% ACN containing 0.1% TFA (trifluoroacetic acid, GE HealthCare) was added to each tube and incubated for 5 min at 37 °C and transferred the supernatants to new EP tube. The extracts were pooled and then vacuum concentrated for about 2 h. A solution of peptides was filtrated via Millipore (Millipore ZTC18M096) and mixed with the same volume of a matrix solution consisting of saturated a-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN containing 0.1% TFA. After the peptides were co-crystallized with CHCA by evaporating organic solvents, tryptic-digested peptide masses were measured using a MALDI-TOF–TOF mass spectrometer (ABI4700 System, USA). All mass spectra were recorded in positive reflector mode and generated by accumulating data from 1000 laser shots. The following threshold criteria and settings were used: detected mass range of 700–3200 Da (optimal resolution for the quality of 1500 Da), using a standard peptide mixture (des-Argl-Bradykinin Mr904.468, Angiotensin I Mr1296.685, Glul-Fihrinopeptide B Mr1570.677, ACTH (1–17) Mr2093.087, ACTH (18–39) Mr2465.199; ACTH (7–38) Mr3657.929) as an external standard calibration, with laser frequency of 50 Hz, repetition rate of 200 Hz, UV wavelength of 355 nm, and accelerated voltage of 20 000 V. Peptide mass fingerprint data were matched to the NCBInr database using Profound program under 50 ppm mass tolerance.

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2.8.3. Peptide and protein identification by database search Data were processed via the Data Explorer software and proteins were unambiguously identified by searching against a comprehensive non-redundant sequence database using the MASCOT software search engine (http://www.matrixscience.com/cgi/ search%20form.pl?FORMVER=2&SEARCH=MIS). Moreover, in order to evaluate protein identification, we considered the percentage of sequence coverage, the observation of distribution of matching peptides (authentic hit is often characterized by peptides that are adjacent to one another in the sequence and that overlap), the distribution of error (distributed around zero), the gap in probability and score distribution from the first to other candidate; only matches with over 90% sequence identity and a maximum e-value of 1010 were considered.

2.9. Statistical analyses The data were subjected to one-way analysis of variance (ANOVA) using STATIX9 for statistical significance at P < 0.05. All

the results were the mean ± SE of three replications. Means were separated by Least Significant Difference (LSD) test at 5% level of significance.

3. Results 3.1. Cd disturbs physiology and chlorophyll fluorescence of leaves and enhances the MDA and ROS contents Mean data regarding physiological traits showed variable responses (Table 1). As a whole, statistical significant inhibition (P < 0.05) was found in chlorophyll a and b, total chlorophyll contents and carotenoids over their respective controls. At 500 lM Cd, Cd contents were higher in roots (2.3 mg g1 DW) than leaves (0.55 mg g1 DW). The quantification and localization studies of MDA and ROS were also done (Fig. 1(A–J)). Taken together, Fig. 1(I) reveals a significant enhancement in the levels of MDA and ROS at 5% probability level. MDA contents increased by 64%, while O2A and

Table 1 Biomass, Cd concentration and photosynthetic pigment quantification (mg/g FW) in leaves of both Cd non-treated and treated upland cotton seedlings. Values are the means ± SE of three replications. Variants possessing same letters are not statistically significant at 5% probability level. Cd Treatment

FW

DW

Root Cd contents (mg/g dw)

Leaf Cd contents (mg/g dw)

0 lM 500 lM

1.92 ± 0.06a 1.88 ± 0.13a

0.19 ± 0.01a 0.16 ± 0.01a

0.036 ± 3.58b 2.3 ± 68.49a

0.002 ± 0.26b 0.55 ± 36.74a

Cd Treatment

Chl a (mg/g FW)

Chl b (mg/g FW)

Chl a + b (mg/g FW)

Carotenoids (mg/g FW)

0 lM 500 lM

0.72 ± 0.13a 0.31 ± 0.05b

0.49 ± 0.02a 0.22 ± 0.27b

1.21 ± 0.55a 0.53 ± 1.05b

0.044 ± 0.00a 0.021 ± 0.01b

Fig. 1. (A–H) In situ localization of both O2A and H2O2 in presence (A, D, E, H) and absence (B, C, F, G) of inhibitors in control (A,B & E,F) and Cd treated (C,D & G,H) leaves of cotton. Blue and brown spots show the presence of O2A and H2O2 respectively. (I, J). Left quantitative estimation of MDA and ROS in both control and Cd-stressed leaves (I). Right chlorophyll fluorescence parameters in both control and Cd-stressed cotton leaves (J). Y bars show the mean values of the studied parameters. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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H2O2 contents respectively increased by 558% and 140% over their related controls. Blue and brown spots showed the presence of superoxide radical and hydrogen peroxide in both Cd non-adapted and adapted leaves (Fig. 1(A–H)), respectively. They were almost higher in Cd-treated leaves as compared to their respective positive and negative controls. Various photosynthetic quenching parameters such as F0, Fm, F 0m and Fv/Fm were also studied an independent experiment (Fig. 1(J)). As compared to controls, 17%, 32%, 30% and 19% relative decrease were noticed in F0, Fm, F 0m and Fv/Fm at 500 lM Cd. 3.2. Cd stress greatly perturbs soluble proteins and oxidative metabolism levels in leaves, while variably affects ultramorphology of leaves Fig. 2 shows that the total soluble proteins significantly reduced (37%) at 500 lM Cd over the control, while SOD activity enhanced (344 U mg1 protein) at 500 lM Cd. In case of CAT, POD and GR activities, upregulation at 500 lM Cd was noticed, which were only statistically significant regarding POD and GR activities. The transmission electron microscopy micrographs of both whole leaf mesophyll cells and chloroplast showed fewer alterations in Cd-stressful environment (Fig. 3(A–D)). Under nonstressed conditions (Fig. 3(A and B)), whole mesophyll cells showed a clean and thin cell with well-shaped nucleus and a number of lipid bodies. The chloroplasts were of elliptical-shaped possessing properly arranged thylakoids with few starch granules. In Cd treated cells (Fig. 3(C and D)), there was an increase in number of lipid bodies and nucleoli. Chloroplasts were misshaped with little swollen and disrupted thylakoids. While there was an increase in number of starch granules and plastoglobuli. The electron dense granules were observed in the vacuolar, cytosolic regions as well as attached to the cellular walls in Cd treated leaf mesophyll cells. The scanning electron micrographs of cotton leaf

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showed almost smooth surface (Fig. 3(E and F)). Stomatal closure was roughly absent in Cd stressed leaves and there was an insignificant increase in the number of stomata (Fig. 3(F)). EDX spectra confirmed the presence of Cd dense precipitates in regions of cell wall, vacuoles and intracellular spaces (Fig 3 (A–F below)). 3.3. Cd stress induces variable proteomic changes Fig. 4(A-F) shows proteomic changes in both Cd non-stressed and stressed leaves. Based on 1.5-fold quantitative change criteria for expression, total thirty-six protein spots showed differential expression between Cd treated and non-treated leaf proteomes of cotton (Fig. 4(E and F)). Nineteen spots contained up-regulated proteins (A01-A19) and seventeen spots showed down-regulated ones (B01-B17). Out of thirty-six differentially regulated proteins in response to Cd stress, fifteen (42%) showed no matches, while twenty-one (58%) could be identified by proteomics analysis (Tables 2 and 3). Out of 21 identified reproducible protein spots, 9 proteins (43%) were induced and 12 proteins (57%) were suppressed. Out of fifteen unmatched proteins, 67% proteins were from up-regulated category and the rest (33%) was from down-regulated category. Spots containing induced proteins showed relative intensities in the range of 1.51–1 000 000, while those having suppressed proteins possessed relative intensities range of 1.5–12.5. Moreover, there were only two predicted proteins (9.5%) and two putative proteins (9.5%) one each from both categories. There were two proteins (Spots # A05; A19) in the up-regulated proteins category, which were sharing the same set of 20 peptides when they were compared with their other matches. Most of the identified proteins belonged to Gossypium spp., which was 38% of the other total spp. Out of the total identified protein belonging to Gossypium spp., 57% of them belonged to Gossypium raimondii and other 43% belonged to G. hirsutum.

Fig. 2. Total soluble proteins and ROS scavenging enzymatic and non-enzymatic antioxidants status in cotton leaves under Cd stress. Legends possessing same letters are not statistically significant at 5% probability level. Y bars depict mean activity levels of the studied parameters.

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Fig. 3. Above: Electron micrograhps of transmission (A–D) and scanning (E and F) of cotton leaf mesophyll cell in both Cd non-stressed and Cd-stressed conditions. CW = cell wall, PM = plasma membrane, ER = endoplasmic reticulum, Thy = thylakoids, PG = plastoglobuli, N = nucleus, Nu = nucleolus, Chl = chloroplast, S = starch granules, Chl = chloroplast, St = stomata, LB = lipid bodies. Below: EDX spectra of precipitates in different parts of the cotton leaf mesophyll cells (A–F). Spectra show the presence of Cd in vacuoles and intracellular spaces of the cells at various spots of micrographs. Spots #1-3 indicate the presence of Cd in vacuoles (V), while spot # 4 shows the accumulation of Cd in intercellular spaces (IS). Y bars reveal the peak counts of the detected heavy metals present in the analyzed spots of electron micrographs.

4. Functional distribution of the identified proteins Out of thirty-six detected proteins, twenty-one Cd-responsive proteins were identified from the leaf. They were involved in a nine different biological functions and pathways during the cellular adaptation to Cd stress. These pathways and biological functions are such as protein synthesis and regulation, photosynthesis/CO2

assimilation, energy and carbohydrate metabolism, redox homeostasis, cell rescue/defense, chaperones and stress related protein and seed storage proteins. All the identified proteins lay under eight functional classes on the basis of their putative functions (Tables 2 and 3). Proteins involved in protein synthesis/regulation (5%, designated as A01), photosynthesis (10% designated as A02, B03), redox homeostasis

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Fig. 4. (A–D). Master gels from a 2D electrophoresis using 4-7 pH IGP strips showing differentially expressed proteins in leaves of upland cotton under normal (A) and 500 lM Cd (B) treated conditions along with protein marker. Differentially regulated protein spots are indicated by green slashes (C and D). Nineteen up-regulated spots (A1-A19 (C) and seventeen down-regulated spots (B1-B17) (D) are indicated on the map. The spot view of upregulated proteins (E) with A lettering and downregulated proteins (F) with B lettering in leaves of upland cotton under Cd non-stressed and stressed conditions are also shown in Fig. 4 (E and F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(5%, designated as A06), carbohydrate metabolism (5%, designated as A07), cell rescue/defense (10%, designated as A13, A14), energy pathway (10%, designated as A17, B01), chaperones and stressrelated proteins (19%, designated as B07, B10, B11, B13) and seed storage proteins (19%, designated as B14-B17). Proteins with unknown function constitute 14% of the total identified proteins. There was also found one suppressed hypothetical protein (gi|147809607). Among the identified induced proteins, abundant proteins were chitinases (having same gi/1729760), which were involved in cell rescue/defense. Other proteins such as methionine synthase (Sal k 3) (gi/ 225810599), ribulose 1,5-bisphosphate carboxylase (gi/4206520), apoplastic anionic guaiacol peroxidase (gi/19569160), glyceraldehydes-3-phosphate dehydrogenase; chloroplastic isoform B (gi/225457604), ATP synthase D chain, mitochondrial (gi/255577651) were active in protein synthesis, CO2 assimilation/photosynthesis, redox homoeostasis, carbohydrate metabolism and energy pathway respectively. Up-regulated proteins with unknown functions were Os070677600 (gi/11547406) and un-named protein product (gi/296087928). Regarding the suppressed identified proteins, greater numbers of protein belonged to chaperones and stress related proteins as well as seed storage proteins. In our present experiment, identified chaperons and stress related were hsp70 (AA6-651) (gi/20559), two chaperonin-60 alpha subunit (gi/10697184) and putative protein disulfide isomerase (gi/133902301). And the identified suppressed seed storage proteins were seed storage protein vicilin B, partial (gi/346426328), two seed storage protein legumin A, partial (gi/346426300) and one seed storage protein legumin B (gi/ 346426306). All of these seed storage belong to Gossypium raimondii except seed storage protein legumin B, which belong to G. hirsutum. Other identified suppressed proteins such as ATP-dependent Clp protease ATP-binding subunit ClpC (gi/184232), ribulose-1,5bisphophate carboxylase/oxygenase large subunit (gi/291010934) were respectively involved in energy pathway and CO2

assimilation/photosynthesis. While the function of predicted protein (gi/224074699) belonging to Populus trichocarpa could not be found in the Cd-stressful environment. 5. Discussion 5.1. Physiological, biochemical and ultrastructural responses of leaves towards Cd stress Cd is a toxic trace pollutant and can cause anatomical and structural changes in roots and leaves (Küpper et al., 2000; Najeeb et al., 2011). In present study, Cd inhibited the plant growth in terms of plant biomass. Same trend was observed by Pál et al. (2006) in maize, Jin et al. (2008) in Sedum alfredii, Daud et al. (2009a) in cotton and Kieffer et al. (2009) in poplar cultivars under different heavy metal stresses. Such reduction might be due to the blockage of essential nutrients (such as Fe, Ca and Mg) in roots by interaction of Cd with these nutrients. In the present study, Cd uptake by roots and leaves was increased at 500 lM Cd over the control. Similar findings were obtained in cotton by Wu et al. (2004) and Daud et al. (2009a) and Daud et al. (2009b) and in Sedum alfredii by Jin et al. (2008). Cd also decreased photosynthetic pigments by decreasing the levels of Chl a, Chl b, total chlorophyll contents and carotenoids. They are line with the findings of Ali et al. (2013). Such reduction of chlorophyll or inhibition of its biosynthesis might be the key factor for reduced photosynthesis and growth (Bazzaz et al., 1992) by Cd. In the present experiment, there was a significant enhancement in the production of MDA and ROS (superoxide and H2O2). ROS produced in greater amount as compared to MDA. Also the tissue localization of both O2 and H2O2 revealed that their production was higher in Cd treated leaves as compared with their related control. Such enhancement in MDA and ROS contents was also observed in Sedum alfredii (Jin et al., 2008), J. effuses (Najeeb

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Fig. 4 (continued)

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M.K. Daud et al. / Chemosphere 120 (2015) 309–320 Table 2 List of differentially induced proteins in leaves of upland cotton under cadmium stress. Spot ID

Acc. No.

Homologous protein name

Identified Sp.

Mascot score

Mol. mass (Da)

Peptide matches

Fold increase

Peptides

Ions No.

Functional category

A01

gi|225810599

Methionine synthase(Sal k 3)

Salsola kali

181

83800

5

1 000 000

K.YLFAGVVDGR.N

40

A02

gi|4206520

Ribulose 1,5-bisphosphate carboxylase

Severinia buxifolia

136

50191

12

1.54

R.EGNEIIR.E

–

Regulation/ Protein synthesis CO2 assimilation/ Photosynthesis

A03 A04 A05

NI NI gi|11547406

Os07g0677600

1.93 1.68 3.47

K.MGNISPLTGDDGEIR.E

– – –

A05

gi|125559602

OsI_27331 (Hypothetical)

K.MGNISPLTGDDGEIR.E

–

A06

gi|19569160

A07

gi|225457604

Apoplastic anionic gaiacol peroxidase Glyceraldehyde-3-phosphate dehydrogenase; chloroplastic isoform B (Predicted)

A08 A09 A10 A11 A12 A13

NI NI NI NI NI gi|1729760

Chitinase

A14

gi|1729760

Chitinase

A15 A16 A17

NI NI gi|255577651

A18 A19 A19

Oryza sativa Japonica Oryza sativa Indica Gossypium hirsutum Vitis vinifera

Gossypium hirsutum Gossypium hirsutum

70

34813

1

70

34827

1

313

37955

6

3.26

R.IGASLIR.L

–

297

47619

9

1.53

K.LNGIALR.V

–

R.YCDILK.V

– – – – – –

K.GSNPQVEDR.I

46

167

29187

5

1.50 1.51 1.59 2.54 1.57 2.38

77

29194

3

1.63

K.EFATLR.R

– – –

R.VTNTATGTQATVR.I

– –

R.VTNTATGTQATVR.I

–

ATP synthase D chain, mitochondrial (Putative)

Ricinus communis

106

19728

6

1.63 1.52 1.55

NI gi|296087928

Unnamed protein product

61

17427

1

1.54 1.61

gi|297833160

PR4-type protein

Vitis vinifera Arabidopsis lyrata subsp. lyrata

61

16215

1

et al., 2011) and Brassica (Ali et al., 2013). This type of oxidative deterioration is an inherent feature of senescence process in leaves of plants (del Rio et al., 1998). Moreover, the mean values of photosynthetic quenching parameters remarkably reduced at 500 lM Cd in comparison with control. Almost similar trend was observed by Ali et al. (2011) in barley and Ahammed et al. (2012) in tomato under various heavy metal stresses. Also Pagliano et al. (2006) found a decrease in chlorophyll and carotenoids. This decline might be due to either a decrease in chlorophyll content, overproduction of ROS, decreased protein, or disturbed PSII photochemistry (Ahammed et al., 2012). Under stressful conditions, total soluble proteins of leaf significantly reduced in this experiment. Such decline can be correlated with pigment loss, reduction in the photosynthetic efficiency, decreased RNA levels etc. (Masood et al., 2012). The mechanism of Cd-induced oxidative stress shows contrasting reports about Cd effects on antioxidative enzymes. SOD activity showed a significant increase in Cd related experiment about cotton leaf. Such enhancement is in line with the findings of Jin et al. (2008). APX detoxify hydrogen peroxide into water and oxygen. Upon Cd exposure, APX activities in the leaf tissue decreased at 500 lM as compared to control. A marked reduction in APX activity was also found in roots of Sedum alfredii by Jin et al. (2008). CAT is indispensable for ROS detoxification (Gill and Tuteja, 2010). Its activity was up-regulated in leaves at 500 lM Cd. Such findings were also noticed by Mobin and Khan (2007) under Cd stress. However, in Phragmites australis (Iannelli et al., 2002), and Capsicum annuum (Leon et al., 2002), CAT activity decreased. This upregula-

Unknown function Redox homeostasis Carbohydrate metabolism

Cell rescue/ defense Cell rescue/ defense

Energy pathway Unknown function Pathogen stress related protein

tion could be due to the downregulation of ATP protease as revealed by proteomic data. Since some proteases being present in peroxisomes may induce senescence (Distefano et al., 1999) that is why upregulation of CAT activity and downregulation of ATP protease caused little alterations in the ultramorphology of chloroplast. Furthermore, the GR activity significantly enhanced in leaves. Similar findings were observed by Masood et al. (2012) in mustard under Cd stress. Ultrastructural observations of both whole leaf mesophyll cells and chloroplast showed less alteration in Cd-stressful environment. There were few lipid bodies and starch granules. Chloroplasts were misshaped with little swollen and disrupted thylakoids as well as there was less increase in number of starch granules and plastoglobuli. The scanning electron microscopy of leaf showed smooth surfaces. Almost such observations have been made by various researchers (Jin et al., 2008; Ali et al., 2013). Accumulation of starch in leaves either in case of our study or that of Sandalio et al. (2001) might be due to either nutrient deficiencies (Vazquez et al., 1987), decrease of the sink force or disturbed vein loading system (Rauser and Samarakoon, 1980). However, Ouzounidou et al. (1997) obtained different results during their studies on wheat under Cd stress. Our scanning microscopic observations are against the findings of those Sandalio et al. (2001). Microlocalization of Cd was done using TEM-EDX technology in order to analyze the observed electron dense precipitates. EDX spectra revealed the presence of Cd dense precipitates in cell wall, vacuoles and intracellular spaces. This showed that Cd was mostly accumulated in the dead parts as well as cytosol of the cell. Greater

318

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Table 3 List of differentially suppressed proteins in leaves of upland cotton under cadmium stress. Spot ID

Acc. No.

Homologous protein name

Identified Sp.

Mascot score

Mol. mass (Da)

Peptide matches

Fold increase

Peptides

Ions No.

Functional category

B01

gi|184232

ATP-dependent Clp protease ATP-binding subunit ClpC

Arabidopsis thaliana

287

103 620

33

1.51

R.GEFEER.L

Energy pathway

B 02 B 03

NI gi|291010934

Pterocymbium beccarii

222

51 890

15

1.80 2.57

R.EGNEIIR.A

CO2 assimilation/ photosynthesis

B 04

gi|224074699

Ribulose-1,5bisphosphate carboxylase/oxygenase large subunit Predicted protein

Populus trichocarpa

253

75 321

21

2.85

K.TKADVDK.M

Unknown function

B 05 B 06 B 07

Ni Ni gi|20559

K.DISGNPR.A

Chaperon and stress protein

B 08 B 09

NI gi|147809607

B 10

gi|10697184

B 11

gi|10697184

B 12 B 13

NI gi|133902301

B 14

gi|346426328

B 15

gi|346426300

B 16

gi|346426300

B 17

gi|346426306

HSP70 (AA 6–651)

Petunia hybrida

314

71 144

13

1.71 3.86 3.91

Hypothetical protein VITISV_000418 Chaperonin-60 alpha subunit Chaperonin-60 alpha subunit

Vitis vinifera

276

74 544

15

1.94 1.61

R.FTAVGAR.I

Avicennia marina Avicennia marina

247

35 599

9

2.51

K.VVNDGVTIAR.A

Chaperone

131

35 599

2

1.95

K.LADAVGLTLGPR.G

Chaperone

Putative protein disulfide isomerase Seed storage protein vicilin B, partial Seed storage protein legumin A, partial Seed storage protein legumin A, partial Seed storage protein legumin B

Gossypium raimondii Gossypium raimondii Gossypium raimondii Gossypium raimondii Gossypium hirsutum

83

5851

6

1.50 3.2

R.GYPTVYFR.S

255

64 728

19

4.43

R.GINEFR.L

300

56 290

13

2.3

R.ISTLNR.F

345

56 290

12

12.55

R.ISTLNR.F

332

56 599

2

5.00

R.HQSQCQLQNLNALQPK.H

Stress related protein Seed storage protein Seed storage protein Seed storage protein Seed storage protein

accumulation was observed in vacuoles conveying the message that there would have been strong complexation of Cd with phytochelatins. 5.2. Functional characterization of various leaf proteins 5.2.1. Protein synthesis and regulation Methionine synthase (MS) is responsible to enhance the lignin biosynthesis in vascular plants as well as the mechanical rigidity of cell wall. Methionine synthase (MS) was up-regulated protein during our 24 h Cd stress regime. Similar trend was found by Sarry et al. (2006) in Arabidopsis thaliana and Kieffer et al. (2008) in poplar under Cd stress. Thus, its upregulation might have caused increased deposition of lignin in the vascular plant such as cotton. 5.2.2. Photosynthesis/CO2 assimilation Heavy metals can adversely affect the photosynthetic pathway (Ahsan et al., 2009) by depressing proteins involved in carbon fixation such as rubisco proteins LSU and SSU. In the present study, rubisco proteins were both upregulated and downregulated. Since the downregulation was more, it conveys the idea that the Calvin cycle was slowed down, although there was less impairment of the photosynthetic process as revealed by the chlorophyll pigment quantification and chlorophyll fluorescence parameters. Its inhibition may be due to reduced photosynthetic efficiency and chlorophyll pigments of the cotton leaves in Cd stressed conditions as has been concluded by Ahsan et al. (2009) in their review. Reasons behind the upregulation of rubisco carboxylase can be better explained having a look on micrographs of scanning microscopy. According to SEM micrographs, there was less number of closed stomata, which reveals that there was no significant drop in internal CO2 concentration. This insignificant closure of stomata

20

and drop of CO2 concentration can be correlated with upregulation of rubisco carboxylase. 5.2.3. Redox homeostasis Only one protein related to apoplastic anionic guaiacol peroxidase was found to be upregulated in leaves of Cd treated seedlings. Similar upregulation has been found in poplar by Kieffer et al. (2008) and Phytolacca americana by Zhao et al. (2011) in response to Cd stress. It corresponds that only one enzyme was likely to be involved in the apoplastic oxidative burst. Its increase in abundance may increase lignification. This could be the reason that Cd did not influence the ultramorphology of the cell walls in the leaf mesophyll cells of Cd stress plants. 5.2.4. Carbohydrate metabolism Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) is a main enzyme of glycolysis, which was upregulated. This shows that Cd caused a lesser oxidative stress in chloroplastic regions. This upregulation suggest the glycolytic pathway was upregulated during Cd-induced oxidative in order to utilize carbohydrate for energy production and to cope with Cd stress. Similar findings were obtained by Ahsan et al. (2010) in rice during As stress. A high reducing power is needed to combat Cd stress, which is provided by an upregulation of mitochondrial respiration. Thus upregulation of GAPDH indicated that part of the photoassimilates from the chloroplast might have been used in order to facilitate mitochondrial respiration (Kieffer et al., 2009). This hypothesis can be based upon the subsequent upregulation of ATP synthase in the present experiment. And because of this coordinated upregulation, the Cd stressful effects were not so much apparent as revealed by physiological, ultramorphological and biochemical studies.

M.K. Daud et al. / Chemosphere 120 (2015) 309–320

5.2.5. Cell rescue/defense and energy pathway Chitinases are important class of PR (pathogen-responsive) proteins. Two chitinases of class 1, were found to be upregulated. Similar to our findings, Fecht-Christoffers et al. (2003) in cowpea and Kieffer et al. (2008) in poplar analyzed the upregulation of PR proteins under heavy metal stresses. ATP synthase plays key roles in energy transduction in chloroplasts and mitochondria. Its expression increased under Cd treatment. This increase suggests the need of cotton seedlings for greater production of ATP to cope with Cd stressful environment as is also evident from the findings of Joëlle et al. (2009) in black root rot fungi. Thus, plant might have nitrogen starvation and has to boost up ATP synthesis process by increasing mitochondrial respiration rate. Such upward trend of ATP was also observed by Zhang et al. (2008) in Arabidopsis, Kottapalli et al. (2009) in peanut and Deeba et al. (2012) in cotton under different environmental stresses. 5.2.6. Chaperones and stress related proteins Molecular chaperones (e.g. HSPs and protein disulfide isomerase (PDI)) are protein repairing (Ahsan et al., 2009) bodies, which are active in cells under normal and adverse conditions (Wang et al., 2004). In the present experiment, molecular chaperones were suppressed therefore; the deregulation of these molecular chaperones might have caused irreversible protein denaturation or damage to the membranes of leaf cells during Cd stress. That is why there was an increase in MDA contents and a decrease in total soluble proteins. 5.2.7. Seed storage proteins Seeds have stored proteins in significant quantities, which are rapidly hydrolyzed upon germination to provide N and C for the early stages of seedling growth (Duranti et al., 2008). In the present study, seed storage proteins such as legumin and vicilin were down-regulated. However, this downregulation did not put significant impact on C and N sources which can be examined by biomass production and lack of senescence in leaves. Contrary to this finding, Labra et al. (2006) in maize and Ahsan et al. (2007) in rice found upregulation of globulins under Cd and Cr stresses, respectively. 6. Conclusion It can be concluded that Cd induced fewer modifications in leaves. Greater involvement of ROS-scavenging enzymes as well as significant upregulation of oxidative stress and carbohydrate metabolism related proteins showed that they may play counteractive role against Cd transport in yielding part of plant that is boll. Thus, all these inter-related studies convey a single message about the Cd resistant nature of ZMS-49. Acknowledgments The project was financially supported by 973 Project of National Natural Science Foundation of China and the National High Technology Research and Development Program of China. The published research work is a part of the first author postdoctoral research project titled Metabolomic, Proteomic, and Transcriptomic Changes in Upland Cotton under Heavy Metal Stresses. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2014.07.060.

319

References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Ahammed, G.J., Choudhary, S.P., Chen, S., Xia, X., Shi, K., Zhou, Y., Yu, Y., 2012. Role of brassinosteroids in alleviation of phenanthrene–cadmium co-contaminationinduced photosynthetic inhibition and oxidative stress in tomato. J. Exp. Bot. 63, 695–709. Ahsan, N., Lee, S.H., Lee, D.G., Lee, Y., Lee, S.W., Bahk, J.D., Lee, B.H., 2007. Physiological and protein profiles alternation of germinating rice seedlings exposed to acute cadmium toxicity. Plant Biol, pathol./Biologie et pathologie végétales. C.R. Biologies 330, 735–746. Ahsan, N., Renault, J., Komatsu, S., 2009. Recent developments in the application of proteomics to the analysis of plant responses to heavy metals. Proteomics 9, 2602–2621. Ahsan, N., Lee, D.G., Kim, K.H., Alam, I., Lee, S.H., Lee, K.W., Lee, H., Lee, B.H., 2010. Analysis of arsenic stress-induced differentially expressed proteins in rice leaves by two-dimensional gel electrophoresis coupled with mass spectrometry. Chemosphere 78, 224–231. Ali, S., Zeng, F., Qiu, L., Zhang, G., 2011. The effect of chromium and aluminum on growth, root morphology, photosynthetic parameters and transpiration of the two barley cultivars. Biol. Plant. 55, 291–296. Ali, B., Huang, C.R., Qi, Z.Y., Ali, S., Daud, M.K., Geng, X.X., Liu, H.B., Zhou, W.J., 2013. 5-Aminolevulinic acid ameliorates cadmium-induced morphological, biochemical, and ultrastructural changes in seedlings of oilseed rape. Environ. Sci. Pollut. Res. 20 (10), 7256–7267. Bah, A.M., Sun, H., Chen, F., Zhou, J., Dai, H., Zhang, G., Wu, F., 2010. Comparative proteomic analysis of Typha angustifolia leaf under chromium, cadmium and lead stress. J. Hazard. Mater. 184, 191–203. Bazzaz, F.A., Rolfe, G.L., Carlson, R.W., 1992. Effect of cadmium on photosynthesis and transpiration of excised leaves of corn and sunflower. Physiol. Plant. 32, 373–377. Bernstein, N., Shoresh, M., Xu, Y., Huang, B., 2010. Involvement of the plant antioxidative response in the differential growth sensitivity to salinity of leaves vs roots during cell development. Free Radical Biol. Med 49, 1161–1171. Bradford, N.M., 1976. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem. 72, 248–254. Carpentier, S.C., Witters, E., Laukens, K., Deckers, P., Swennen, R., Panis, B., 2005. Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 5, 2497–2507. Daud, M.K., Sun, Y., Dawood, M., Hayat, Y., Variath, M.T., Wu, Y.X., Raziuddin, Mishkat, U., Salahuddin, Najeeb, U., Zhu, S.J., 2009a. Cadmium-induced functional and ultrastructural alterations in roots of two transgenic cotton cultivars. J. Hazard. Mater. 161, 463–473. Daud, M.K., Variath, M.T., Ali, S., Najeeb, U., Jamil, M., Hayat, Y., Dawood, M., Khan, M.I., Zaffar, M., Cheema, S.A., Tong, X.H., Zhu, S.J., 2009b. Cadmium-induced ultramorphological and physiological changes in leaves of two transgenic cotton cultivars and their wild relative. J. Hazard. Mater. 168, 614–625. Daud, M.K., Ali, S., Variath, M.T., Zhu, S.J., 2013a. Differential physiological, ultramorphological and metabolic responses of cotton cultivars under cadmium stress. Chemosphere 93, 2593–2602. Daud, M.K., Mei, L., Ali, B., Chen, Y., Cheng, X., Zhu S.J., 2013b. Cadmium-induced upregulation of lipid peroxidation and reactive oxygen species caused physiological, biochemical and ultra-structural changes in cotton seedlings. Biomed Research Interna. http://dx.doi.org/10.1155/2013/374063. Deeba, F., Pandey, A.K., Ranjan, S., Mishra, A., Singh, R., Sharma, Y.K., Shirke, P.A., Pandey, V., 2012. Physiological and proteomic responses of cotton Gossypium herbaceum L. to drought stress. Plant Physiol. Biochem. 53, 6–18. del Rio, L.A., Pastori, G.M., Palma, J.M., Sandalio, L.M., Sevilla, F., Corpas, F.J., Jimenez, A., Lopez-Huertas, E., Hernandez, J.A., 1998. The activated oxygen role of peroxisomes in senescence. Plant Physiol. 116, 1195–1200. Distefano, S., Palma, J.M., McCarthy, I., Del Río, L.A., 1999. Proteolytic cleavage of plant proteins by peroxisomal endoproteases from senescent pea leaves. Planta 209, 308–313. Duranti, M., Consonni, A., Magni, C., Sessa, F., Scarafoni, A., 2008. The major proteins of lupin seed: characterizations and molecular properties for use as functional and nutraceutical ingredients. Trends Food Sci. Technol. 19, 624–633. Fecht-Christoffers, M.M., Braun, H.P., Lemaitre-Guillier, C., Van Dorsselaer, A., 2003. Effect of manganese toxicity on the proteome of the leaf apoplast in cowpea. Plant Physiol. 133, 1935–1946. Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909–930. Iannelli, M.A., Pietrni, F., Fiore, L., Petrilli, L., Massacci, A., 2002. Antioxidant response to cadmium in Phragmites australis plants. Plant Physiol. Biochem. 40, 977–982. Jiang, M., Zhang, J., 2002. Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and upregulates the activities of antioxidant enzymes in maize leaves. J. Exp. Bot. 53, 2401–2410. Jin, X., Yang, X., Islam, E., Liu, D., Mahmood, Q., 2008. Effects of cadmium on ultrastructure and antioxidative defense system in hyperaccumulator and nonhyperaccumulator ecotypes of Sedum alfredii Hance. J. Hazard. Mater. 156, 387– 397. Joëlle, V.F., Poljak, C.A., Raftery, M.J., Backhouse, D., Pereg-Gerk, L., 2009. Analysis of cotton (Gossypium hirsutum) root proteomes during a compatible interaction with the black root rot fungus Thielaviopsis basicola. Proteomics 9, 335–349.

320

M.K. Daud et al. / Chemosphere 120 (2015) 309–320

Kieffer, P., Dommes, J., Hoffmann, L., Hausman, J.F., Renaut, J., 2008. Quantitative changes in protein expression of cadmium-exposed poplar plants. Proteomics 8, 2514–2530. Kieffer, P., Planchon, S., Oufir, M., Ziebel, J., Dommes, J., Hoffmann, L., Hausman, J.F., Renaut, J., 2009. Combining proteomics and metabolite analyses to unravel cadmium stress-response in poplar leaves. J. Proteome Res. 8, 400–417. Kottapalli, K.R., Rakwal, R., Shibato, J., Burow, G., Tissues, D., Burke, J., Puppala, N., Burow, M., Payton, P., 2009. Physiology and proteomics of the water-deficit stress response in three contrasting peanut genotypes. Plant Cell Environ. 32, 380–407. Küpper, H., Lombi, E., Zhao, F.J., McGrath, S.P., 2000. Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta 212, 75–84. Labra, M., Gianazza, E., Waitt, R., Eberini, I., Sozzi, A., Regondi, S., Grassi, F., Agradi, E., 2006. Zea mays L. protein changes in response to potassium dichromate treatments. Chemosphere 60, 1234–1244. Lage-Pinto, F., Oliveira, J.G., Cunha, M.D., Souza, C.M.M., Rezende, C.E., Azevedo, R.A., Vitória, A.P., 2008. Chlorophyll a fluorescence and ultrastructural changes in chloroplast of water hyacinth as indicators of environmental stress. Environ. Exp. Bot. 64, 307–331. Lannig, G., Cherkasov, A.S., Sokolova, I.M., 2006. Temperature-dependent effects of cadmium on mitochondrial and whole-organism bioenergetics of oysters (Crassostrea virginica). Mar. Environ. Res. 62, S79–S82. Leon, A.M., Palma, J.M., Corpas, F.J., Gomez, M., Romero-Puertas, M.C., Chatterjee, D., Mateos, R.M., Del Rio, L.A., Sandalio, L.M., 2002. Antioxidant enzymes in cultivars of pepper plants with different sensitivity to cadmium. Plant Physiol. Biochem. 40, 813–820. Masood, A., Iqbal, N., Khan, N.A., 2012. Role of ethylene in alleviation of cadmiuminduced photosynthetic capacity inhibition by sulphur in mustard. Plant Cell Environ. 35, 524–533. Mobin, M., Khan, N.A., 2007. Photosynthetic activity, pigment composition and antioxidative response of two mustard (Brassica juncea) cultivars differing in photosynthetic capacity subjected to cadmium stress. J. Plant Physiol. 164, 601– 610. Najeeb, U., Jilani, G., Ali, S., Sarwar, M., Xu, L., Zhou, W., 2011. Insights into cadmium induced physiological and ultra-structural disorders in Juncus effusus L. and its remediation through exogenous citric acid. J. Hazard. Mater. 186, 565–574. Nakano, Y., Asada, K., 1981. Hydrogen peroxide scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physio. 22, 867–880. Nwugo, C.C., Huerta, A.J., 2011. The effect of silicon on the leaf proteome of rice (Oryza sativa L.) plants under cadmium-stress. J. Proteom. Res. 10, 518–528.

Ouzounidou, G., Moustakas, M., Eleftheriou, E.P., 1997. Physiological and ultrastructural effects of cadmium on wheat (Triticum aestivum L.) leaves. Arch. Enviorn. Contam. Toxicol. 32, 154–160. Pagliano, C., Raviolo, M., La Vecchia, F., Gabbrielli, R., Gonnelli, C., Rascio, N., Barbato, R.La., Rocca, N., 2006. Evidence for PSII donor side damage and photoinhibition induced by cadmium treatment on rice (Oryza sativa L.). J. Photochem. Photobio. B 84, 70–78. Pál, M., Horváth, E., Janda, T., Páldi, E., Szalai, G., 2006. Physiological changes and defense mechanisms induced by cadmium stress in maize. J. Plant Nutr. Soil Sci. 169, 239–246. Rauser, W.E., Samarakoon, A.B., 1980. Vein loading in seedlings of Phaseolus vulgaris exposed to excess cobalt, nickel and zinc. Plant Physiol. 65, 578–583. Sandalio, L.M., Dalurzo, H.C., Gómez, M., Romero-Puertas, M.C., Del Río, L.A., 2001. Cadmium-induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 52, 2115–2126. Sarry, J.E., Kuhn, L., Ducruix, C., Lafaye, A., Junot, C., Hugouvieux, V., Jourdain, A., Bastien, O., Fievet, J.B., Vailhen, D., Amekraz, B., Moulin, C., Ezan, E., Garin, J., Bourguignon, J., 2006. The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses. Proteomics 6, 2180–2198. Vazquez, M.D., Poschenrieder, C., Barcelo, J., 1987. Chromium VI induced structural and ultrastructural changes in bush bean plants (Phaseolus vulgaris L.). Ann. Bot. 59, 427–438. Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Plant Sci. 151, 59–66. Wang, W., Vinocur, B., Shoseyov, O., Altman, A., 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 9, 244–252. Wu, F.B., Wu, H.X., Zhang, G.P., Bachir, D.M.L., 2004. Differences in growth and yield in response to cadmium toxicity in cotton genotypes. J. Plant Nutr. Soil Sci. 167, 85–90. Zhang, Z.F., Zhang, F., Raziuddin, Gong, H.J., Yang, Z.M., Lu, L., Ye, Q.F., Zhou, W.J., 2008. Effects of 5-aminolevulinic acid on oilseed rape seedling growth under herbicide toxicity stress. J. Plant Growth Regul. 27, 159–169. Zhao, L., Sun, Y.L., Cui, S.X., Chen, M., Yang, H.M., Liu, H.M., Chai, T.Y., Huang, F., 2011. Cd-induced changes in leaf proteome of the hyperaccumulator plant Phytolacca Americana. Chemosphere 85, 56–66. Zhou, W.J., Leul, M., 1998. Uniconazole-induced alleviation of freezing injury in relation to changes in hormonal balance, enzyme activities and lipid peroxidation in winter rape. J. Plant Growth Regul. 26, 41–47.