Cadmium stress in cotton seedlings: Physiological ...

South African Journal of Botany 104 (2016) 61–68

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Cadmium stress in cotton seedlings: Physiological, photosynthesis and oxidative damages alleviated by glycinebetaine M.A. Farooq a, S. Ali a,⁎, A. Hameed b, S.A. Bharwana a, M. Rizwan a, W. Ishaque b, M. Farid c, K. Mahmood b, Z. Iqbal b a b c

Department of Environmental Sciences and Engineering, Government College University, Allama Iqbal Road, 38000, Faisalabad, Pakistan Nuclear Institute for Agriculture and Biology (NIAB), P.O. Box 128, Jhang Road, Faisalabad, Pakistan Department of Environmental Sciences, University of Gujrat, Hafiz Hayat Campus, Gujrat, Pakistan

a r t i c l e

i n f o

Article history: Received 9 April 2015 Received in revised form 8 September 2015 Accepted 2 November 2015 Available online xxxx Edited by M Vaculik Keywords: Antioxidant enzymes Cadmium Cotton Glycinebetaine Oxidative damages

a b s t r a c t Cadmium (Cd) level is continuously increasing in agricultural soils mainly through anthropogenic activities. Cadmium is one of the most phytotoxic metals in soils. The present study investigates the possible role of exogenously applied glycinebetaine (GB) in alleviating Cd toxicity in cotton (Gossypium hirsutum L.) plants in a hydroponic system. Three concentrations of Cd (0, 1.0, and 5.0 μM) were tested with and without foliar application of GB (1.0 mM). Cadmium toxicity caused a significant decrease in plant height, root length, number of leaves per plant, fresh and dry weights of leaf, stem and root and intensively increased Cd concentration in different plant parts. Cadmium toxicity also decreased photosynthetic pigments and gas exchange characteristics in leaves. Superoxide dismutase (SOD), guaiacol peroxidase (POD), catalases (CAT) and ascorbate (APX) activities increased under lower Cd stress (1.0 μM) while decreased under higher Cd stress (5.0 μM). Cadmium toxicity increased the concentration of reactive oxygen species (ROS) as indicated by the increased production of malondialdehyde (MDA), hydgrogen peroxide (H2O2) and electrolyte leakage in both leaves and roots. Application of GB decreased Cd concentration in different plant parts, alleviated Cd-induced inhibition in plant growth and biomass and led to a significant increase in photosynthetic pigments, protein contents and antioxidant enzymes. Glycinebetaine application alleviated the oxidative damage as evidenced by the decreased production of electrolyte leakage, H2O2 and MDA contents. These results revealed that GB might alleviate Cd toxicity in cotton plants through lowering Cd concentrations and regulating Cd induced oxidative stress in different plant parts possibly by increasing the performance of the antioxidant enzymatic system. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Environmental pollutants, released by human activities including noxious gasses, pesticides and heavy metals, have threatened existence of biota worldwide (Wagner, 1993; Sharma and Pandey, 2014; Adrees et al., 2015a; Rizwan et al., 2015a). Among these pollutants, heavy metal contamination of agricultural soils is a serious environmental threat that affects many physiological and metabolic processes in plants and finally decreased plant growth, photosynthesis and biochemical activities (Ali et al., 2013a, 2013b; Keller et al., 2015; Adrees et al., 2015b; Rizwan et al., 2015b). Among heavy metals, cadmium (Cd) is one of the most phytotoxic elements and has no known biological function in plants and animals (Rizwan et al., 2012; Khaliq et al., 2015; Rehman et al., 2015). Cadmium stress also decreased the uptake and distribution of essential elements in plants (Ahmad et al., 2011; Hediji et al., 2015). Although Cd is not a redox active metal, it causes oxidative stress in ⁎ Corresponding author. Tel.: +92 41 9201566; fax: +92 41 9200671. E-mail address: [email protected] (S. Ali).

http://dx.doi.org/10.1016/j.sajb.2015.11.006 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

plants by the formation of reactive oxygen species (ROS) including superoxide anion (O− 2 ) and hydrogen peroxide (H2O2) etc. (Zhang et al., 2009). Under Cd stress, overproduction of ROS may cause physiological disorders in plants which results in growth and biomass reduction (Ahmad et al., 2011; Saidi et al., 2013; Arshad et al., 2015). In order to avoid the deleterious effect of oxidative stress, plants have evolved well developed ROS scavenging enzymatic apparatus such as superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (POD), and catalase (CAT) (Yin et al., 2008). It has been shown that the activities of antioxidant enzymes increased up to a certain level of Cd stress and then decreased under higher Cd stress (Saidi et al., 2013; Hediji et al., 2015). This showed that under severe Cd stress conditions, the antioxidant enzymatic capacity of plants might not be sufficient to prevent toxic effects of metal (Hossain et al., 2010; Gill et al., 2015). In critical conditions, plants have adopted different protective processes to respond to the heavy metal stress, including Cd stress. One such adaptive mechanism under stressful conditions is the accumulation of compatible solutes including glycinebetaine (GB) and

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M.A. Farooq et al. / South African Journal of Botany 104 (2016) 61–68

proline (Chen and Murata, 2011). Glycinebetaine is one of the most abundant quaternary ammonium compounds produced in higher plants under stressful environments (Yang et al., 2003). Glycinebetaine is involved in the protection of plants against many stresses such as drought (Iqbal et al., 2009; Raza et al., 2014), salinity (Hossain et al., 2010; Hasanuzzaman et al., 2014) and heavy metal stress (Chen and Murata, 2011; Cao et al., 2013; Ali et al., 2015; Jabeen et al., 2015). It has been shown that GB also provides protection against oxidative stress in many plant species under stressful conditions (Hossain et al., 2010; Hasanuzzaman et al., 2014). However, natural production of GB is not enough to protect plants under severe stress conditions. Under such conditions, exogenous application of GB might be a useful strategy to overcome abiotic stresses in plants (Ali et al., 2015). It has been reported that exogenous GB enhanced salt tolerance in rice seedlings by enhancing the activities of antioxidant enzymes (Hasanuzzaman et al., 2014) and enhanced drought tolerance in wheat by improving gas exchange characteristics (Raza et al., 2014). However, its response varies with plant species and genotypes (Chen and Murata, 2011). Exogenous GB also reduced heavy metal toxicity in many plant species (Bharwana et al., 2014; Ali et al., 2015). However, little information is available behind the mechanisms of GB-mediated alleviation of metal toxicity in plants. Thus, based upon the above discussion, this study was designed to test whether exogenous GB application is capable to improve Cd tolerance in cotton plants, an important cash crop in Pakistan and worldwide, either through a reduced Cd uptake or by affecting growth, photosynthesis and antioxidant enzymes activities under Cd stress. For this purpose, a hydroponic experiment was conducted with three concentrations of Cd (0, 1.0 and 5.0 μM) without and with 1.0 mM of GB. After harvesting, various morphological and physiological parameters were determined namely biomass, shoot and root lengths, number of leaves per plant, photosynthetic pigments and gas exchange characteristics, protein contents and Cd concentrations in different plant parts. Oxidative stress, malondialdehyde (MDA), H2O2 and electrolyte leakage (EL), and activities of key antioxidant enzymes, SOD, POD, APX and CAT were measured in different plant parts to evaluate the role of GB in reducing oxidative stress by affecting antioxidant enzymes activities. 2. Materials and methods 2.1. Growth conditions Healthy seeds of cotton genotype MNH 886 were taken from Ayub Agricultural Research Institute (AARI) and immersed in concentrated sulfuric acid solution approximately 15 min just to remove the short fiber on the surface of the seed. Seeds were then rinsed with distilled water thoroughly and sown in 2'' layers of sterilized quartz sand trays in a growth chamber with a photoperiod of 16 h light/8 h dark with light intensity of 400 ± 25 μmolm⁻ 2 s⁻ 1. The light/dark temperature was set at 30 °C/25 °C with relative humidity at 85%. After 2 weeks, uniform seedlings were wrapped with foam at a root shoot junction, and transplanted in thermopore sheets having evenly spaced holes floating on 40 L capacity iron tubs, lined with polyethylene sheet containing modified Hoagland's solution. Continuous aeration was given through an air pump in the nutrient solution by making bubbles. The solution was changed on weekly basis. Complete randomized design (CRD) was applied. Two weeks after transplanting, Cd levels (control (0 μM), 1.0 μM, and 5.0 μM) distributed as CdCl2 and two levels of GB (control and 1 mM) with five replicates were applied. Solution pH was maintained at 6.0 ± 0.1 by adding 1 M H2SO4 or NaOH solution. 2.2. Measurements of plant growth and biomass Plants were harvested after 6 weeks of growth under Cd stress. After measuring shoot and root lengths, plants were separated into leaves, stem and roots, washed thoroughly with distilled water, wiped the plant material and fresh weight of these plant parts was determined.

After this, samples were oven dried at 70 °C for about 72 h and then weighed. 2.3. Gas exchange parameters and chlorophyll contents After 6 weeks of growth with Cd and GB treatments, photosynthetic rate (A), stomatal conductance (gs), transpiration rate (E), water use efficiency (A/E) of a fully expanded youngest leaf of each plant were determined by using infrared gas analyzer (IRGA) (Analytical Development Company, Hoddesdon, England). These measurements were taken from 10:00 am to 11:30 am with the growth conditions as describe above (Section 2.1). Chlorophyll a, chlorophyll b, total chlorophyll and carotenoids were determined by spectrophotometrically (Metzner et al., 1965). After 6 weeks of treatment, the topmost fully expanded fresh leaves were weighed and dipped overnight in 85% (v/v) acetone for the extraction of the chlorophyll pigments. Supernatant taken was centrifuged at 4000 rpm for 10 min and diluted with 85% acetone to the suitable concentration for spectrophotometric measurements. The disappearance was calculated at absorbance of 452.5, 644 and 663 nm alongside blank of 85% liquid acetone. Chl a, b, total chlorophyll and carotenoids were determined by spectrophotometer (Halo DB-20/DB-20S, Dynamica Company, London, UK). The chlorophylls and carotenoids contents were calculated by using the adjusted extinction coefficients and equations (Lichtenthaler, 1987). 2.4. Determination of H2O2, MDA and electrolyte leakage The H2O2 content was colorimetrically determined as described by Jana and Choudhuri (1981). H2O2was extracted by homogenizing 50 mg leaf or root tissues with 3 ml of phosphate buffer (50 mM, pH 6.5). To measure H2O2 content, 3 ml of extracting solution was mixed with 1 ml of 0.1% titanium sulfate in 20% (v/v) H2SO4 and the mixture was centrifuged at 6000 × g for 15 min. The intensity of the yellow color of the supernatant was measured through spectrophotometer at 410 nm. H2O2 content was computed by using the absorbance coefficient of 0.28 μmol−1 cm−1. The level of lipid peroxidation in the leaf tissue was measured in terms of malondialdehyde content (MDA, a product of lipid peroxidation) determined by the thiobarbituric acid (TBA) reaction using the method of Heath and Packer (1968), with minor modifications as described by Zhang and Kirham (1994). A 0.25 g leaf sample was homogenized in 5 ml 0.1% TCA. The homogenate was centrifuged at 10 000×g for 5 min. To 1 ml aliquot of the supernatant, 4 ml of 20% TCA containing 0.5% TBA was added. The mixture was heated at 95 °C for 30 min and then quickly cooled in an ice bath. After centrifugation at 10 000×g for 10 min, the absorbance of the supernatant at 532 nm was read and the value for the nonspecific absorption at 600 nm was subtracted. The MDA content was calculated by using an absorbance coefficient of 155 mM−1 cm−1. Electrolyte leakage was estimated by using the method of DionisioSese and Tobita (1999). After treatment for 6 weeks, leaves samples were cut into small parts of 5 mm length and put in test tubes containing 8 ml deionized and distilled water. The tubes were placed in a water bath at 32 °C for two hours. Initial electrical conductivity of the medium (EC1) was assessed (Conductivity Model 720, INCO -LAB Company, Kuwait). For second electrical conductivity (EC2), samples were placed in autoclave at 121 °C for 20 min to expel all electrolytes. Samples were cooled at 25 °C. Total electrolyte leakage was calculated by using the following formula: EL ¼ ðEC1 =EC2 Þ 100: 2.5. Assay of antioxidant enzymes and soluble protein contents Antioxidant enzymes such as SOD, POD, CAT and APX in roots and leaves were determined by spectrophotometrically. After 6 weeks of


treatment fresh samples (0.5 g) of leaves and roots were grounded with the help of a mortar and pestle and homogenized in 0.05 M phosphate buffer (pH 7.8) under chilled condition. The homogenized mixture was filtered through four layers of muslin cloth and centrifuged at 12 000×g for 10 min at 4 °C. The soluble protein content was analyzed according to Bradford (1976), using Coomassie Brilliant Blue G-250 as dye and albumin as a standard. Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined with the method of Zhang (1992) following the inhibition of photochemical reduction due to nitro blue tetrazolium (NBT). The reaction mixture was comprised of 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 μμ NBT, 2 μμ riboflavin, 0.1 mM EDTA and 100 μl 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. Peroxidase (POD, EC1.11.1.7) activity was assayed by Zhou and Leul (1999) with some modifications. The reactant mixture contained 50 mM potassium phosphate buffer (pH 7.0), 1% guaiacol, 0.4% H2O2 and 100 μl enzyme extract. Variation due to guaiacol in absorbance was measured at 470 nm. Catalase (CAT, EC 1.11.1.6) activity was determined by the method of Aebi (1984). The assay mixture (3.0 ml) was comprised of 100 μl enzyme extract, 100 μl H2O2 (300 mM) and 2.8 ml 50 mM phosphate buffer with 2 mM EDTA (pH 7.0). The CAT activity was assayed by monitoring the decrease in the absorbance at 240 nm as a consequence of H2O2 disappearance (ε = 39.4 mM−1 cm−1). Ascorbate peroxidase (APX, EC 1.11.1.11) activity was assayed according to the method of Nakano and Asada (1981). The reaction mixture consisted of 100 μl enzyme extract, 100 μl ascorbate (7.5 mM), 100 μl H2O2 (300 mM) and 2.7 ml 25 mM potassium phosphate buffer with 2 mM EDTA (pH 7.0). The oxidation of ascorbate was determined by the change in absorbance at 290 nm (ε = 2.8 mM−1 cm−1). 2.6. Determination of GB contents The contents of GB were estimated according to Grieve and Grattan (1983). Leaf glycinebetaine was extracted from plant material with warm distilled water (70 °C). The extract (0.25 ml) was mixed with 0.25 ml of 2 N HCl and 0.2 ml of potassium tri-iodide solution. The contents were shaken and cooled in an ice bath for 90 min. Then 2.0 ml of ice cooled distilled water and 20 ml of 1–2 dichloromethane (cooled at −10 °C) were added to the mixture. The two layers were formed in the mixture. The upper aqueous layer was discarded and optical density of the organic layer was measured at 365 nm. The concentrations of glycinebetaine were calculated on fresh weight basis.

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2.8. Statistical analysis All values reported in this experiment are mean of five replicates. Analysis of variance (ANOVA) was done by using a statistical package, SPSS version 16.0 (SPSS, Chicago, IL) followed by Tukey test between the means of treatments to determine the significant differences. 3. Results 3.1. Effects of glycinebetaine and Cd on plant growth and biomass Results related to plant growth parameters, plant height, root length and number of leaves per plant, and biomass, fresh and dry weights of leaf, stem and roots, have been reported in Table 1. Cadmium concentrations in the growth medium caused a significant decrease in plant height as compared to control. As compared to control, this decrease in plant height was 28.19% and 61.17% with 1.0 and 5.0 μM Cd concentrations respectively. Application of GB caused a significant increase in plant height in Cd-stressed cotton seedlings. Cd-stressed plant treated with GB exhibited 12.74%, 23.91% and 30.52% increase in plant height under 0, 1.0 and 5.0 μM Cd respectively as compared to the respective treatments without GB. Root length and number of leaves per plant significantly decreased with Cd treatments as compared to control. The maximum reduction in root length and number of leaves per plant was recorded under higher Cd (5.0 μM) treatments, which caused 63.41% and 72.12% reduction in root length and number of leaves per plant respectively as compared to control. Addition of GB under Cd stress significantly increased the root length and number of leaves per plant as compared to respective treatments without GB application and the results are significant especially with higher Cd levels in the growth medium. A significant reduction in fresh and dry weights of leaves, stem and roots was observed in Cd-stressed cotton plants (Table 1). Compared with control, Cd stress caused a significant decrease in fresh weights of leaves, stem and roots. Addition of GB in the growth medium along with Cd caused a significant increase in fresh biomass of different plants parts as compared to respective Cd-only treated plants. Dry weights of these plant parts also decreased under Cd stress as compared to control in a dose dependent manner. Compared to control, 1.0 μM Cd stress decreased 41.56%, 46.54% and 50% dry weights of leaves, stem and roots respectively as compared to control while 5.0 μM Cd stress decreased 65.4%, 70% and 70.51% dry weight of leaves, stem and roots as compared to control respectively. Application of GB, at both Cd levels, significantly increased dry weight of different plant parts as compared to Cd-only treatments. Plants treated with 5.0 μM Cd + GB increased the dry weight of leaves, stem and roots by 30%, 21.21% and 28.13% respectively as compared to 5.0 μM Cd stress.

2.7. Determination of Cd concentration Each sample (0.5 g) was dry-ashed, extracted with HCl and centrifuged at 3600 rpm for 15 min. Concentrations of Cd in root, stem and leaves were determined by flame atomic absorption spectrometry (novA A400 Analytik Jena, Germany).

3.2. Effects of glycinebetaine and Cd on chlorophylls and gas exchange attributes Results related to photosynthetic pigments, chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophylls and carotenoids, and gas

Table 1 Effect of different concentration of Cd (0, 1 and 5 μM) and glycinebetaine (GB) (0 and 1 mM) on plant growth parameters and biomass of cotton plants. Values show the means of five replicate ± SE. Different letters indicate that values are significant different at P b 0.05. Treatments

Control GB Cd1 (1 μM) Cd1 + GB Cd5 (5 μM) Cd5 + GB

Fresh Weight (g)

Dry Weight (g)

Plant height (cm)

Root length (cm)

Number of leaf plant−1

Leaf

Stem

Root

Leaf

Stem

Root

35.22 ± 1.01b 40.36 ± 1.24a 25.29 ± 1.20d 33.25 ± 1.63c 13.67 ± 0.88f 19.69 ± 0.33e

32 ± 0.58a 35 ± 1.15a 20.33 ± 0.88c 26 ± 1.15b 11.71 ± 1.20d 17.33 ± 0.88c

12.59 ± 0.30a 12.88 ± 0.46a 6.58 ± 0.23bc 8.21 ± 0.34b 3.51 ± 0.097d 5.18 ± 0.43c

12.59 ± 0.302b 14.89 ± 0.661a 6.59 ± 0.229d 8.22 ± 0.338c 3.51 ± 0.097ef 5.18 ± 0.428e

11.62 ± 0.101a 11.31 ± 0.176a 6.27 ± 0.202c 8.52 ± 0.289b 3.43 ± 0.133e 5.03 ± 0.094d

3.69 ± 0.162b 3.84 ± 0.034a 2.19 ± 0.047d 2.91 ± 0.052c 1.25 ± 0.068f 1.76 ± 0.076e

2.43 ± 0.028a 2.45 ± 0.033a 1.42 ± 0.037c 1.73 ± 0.034b 0.84 ± 0.035d 1.2 ± 0.115c

2.60 ± 0.078a 2.67 ± 0.097a 1.39 ± 0.035c 1.88 ± 0.018b 0.78 ± 0.122e 0.99 ± 0.052d

0.78 ± 0.023b 0.85 ± 0.023a 0.39 ± 0.043c 0.52 ± 0.012b 0.23 ± 0.017d 0.32 ± 0.012 cd

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Table 2 Effect of different concentration of Cd (0, 1 and 5 μM) and glycinebetaine (GB) (0 and 1 mM) on photosynthetic pigments [Chl a, b, total Chl and carotenoids ((mg g−1 (FW)] and gas exchange parameters such as net photosynthetic rate (μmol CO2 m−2 s−1), stomatal conductance (mol H2O m−2 s−1) and transpiration rate (mmol H2O m−2 s−1) of cotton plants. Values show the means of five replicate ± SE. Different letters indicate that values are significant different at P b 0.05. Treatments Chl a

Chl b

Total Chl

Total carotenoids Net photosynthetic rate Transpiration rate Water use efficiency Stomatal conductance

Control GB Cd1 (1 μM) Cd1 + GB Cd5 (5 μM) Cd5 + GB

0.29 ± 0.008a 0.33 ± 0.012a 0.15 ± 0.008bc 0.19 ± 0.009b 0.08 ± 0.002d 0.11 ± 0.015 cd

0.86 ± 0.020a 0.93 ± 0.018a 0.50 ± 0.009c 0.63 ± 0.020b 0.26 ± 0.01e 0.38 ± 0.012d

0.24 ± 0.009a 0.25 ± 0.005a 0.13 ± 0.003c 0.18 ± 0.005b 0.06 ± 0.005d 0.11 ± 0.006c

9.08 ± 0.395b 13.03 ± 0.702a 5.09 ± 0.035d 6.63 ± 0.259c 2.03 ± 0.030f 3.71 ± 0.173e

exchange characteristics, net photosynthetic rate (Pn), transpiration rate (E), water use efficiency (Pn/E) and stomatal conductance (gs), are summarized in Table 2. Cadmium stress caused a significant decrease in all pigments concentrations as compared to control. Higher Cd stress (5.0 μM) decreased the Chl a, Chl b, total chlorophylls and carotenoids contents by 68.42%, 72.40%, 69.77% and 75% respectively as compared to control (without Cd and GB). Addition of GB in the growth medium under Cd stress caused a significant increase in all

A

Leaf SOD (Units g-1 FW)

120

a a

80

b

c d d

C

a b c

40

d d

20 0 160

E

c b

ab a d d

40

c d

80

e

e

D

8

a

a b b

6 4

c c

b

e e

b 120

d

100 50

c

a

c c

d

d

80

0 6000

a Root CAT (Units g-1 FW)

G

F

40

0

Leaf CAT (Units g-1 FW)

b 120

0 160

80

150

a

2

120

200

2.86 ± 0.029a 2.84 ± 0.028a 1.38 ± 0.009c 1.84 ± 0.027b 0.73 ± 0.094e 1.12 ± 0.020d

B

160

0 10

b

60

3.81 ± 0.075b 5.12 ± 0.081a 3.74 ± 0.062c 3.78 ± 0.088c 2.81 ± 0.024e 3.56 ± 0.073d

40

Root APX (Units g-1 FW)

Leaf POD (Units g-1 FW)

0 80

200

GB

160

40

Leaf APX (Units g-1 FW)

0

Root SOD (Units g-1 FW)

200

2.38 ± 0.096a 2.54 ± 0.071a 1.36 ± 0.050bc 1.75 ± 0.148b 0.72 ± 0.029d 1.04 ± 0.036 cd

pigment concentrations, except Chl b, as compared to respective Cd treatments alone. At 5.0 μM Cd + GB, the increase in Chl a, total chlorophylls and carotenoids contents was about 33.31%, 31.56% and 45.41% respectively as compared to 5.0 μM Cd stress alone. Gas exchange characteristics, Pn, E, gs and Pn/E, in cotton plants decreased noticeably under cadmium stress (Table 2). The inhibitory effect was more prominent at 5.0 μM Cd stress which decreased the Pn, E, gs and Pn/E by 77.64%, 69.71%, 74.42% and 26.22% respectively as

Root POD (Units g-1 FW)

0.57 ± 0.018a 0.60 ± 0.005a 0.35 ± 0.015c 0.44 ± 0.010b 0.18 ± 0.008e 0.27 ± 0.011d

H

5000

4000 3000

a b

e de

d

c

2000 1000

0

0 0 1 5 Cd cocentration (µM)

0 1 5 Cd concentration (µM)

Fig. 1. Effect of different concentrations of cadmium (Cd) (0, 1 and 5 μM) and glycinebetaine (GB) (0 and 1 mM) on activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT) in leaves and roots of cotton plants grown under hydroponic conditions. Values show the means of five replicate ± SE. Means followed by same small letters are not significant different at P b 0.05 by using the Tukey test.


compared to control. Addition of GB under Cd stress improved the gas exchange characteristics of cotton plants as compared to Cd-only treatments except E and Pn/E in the presence of 1.0 μM Cd. Under 5.0 μM Cd + GB treatment, the increase in gas exchange characteristics was significant except transpiration rate as compared to 5.0 μM Cd-only treatment. 3.3. Effects of Cd and glycinebetaine on antioxidant enzymes activities in cotton Results related to key antioxidant enzymes activities (SOD, POD, CAT, and APX) in leaves and roots of cotton plants are summarized in Fig. 1. The activities of SOD, POD and CAT were higher in both leaves and roots under Cd stress as compared to control except CAT in roots under 5.0 μM Cd. APX activity was lower in both leaves and roots at 5.0 μM Cd as compared to control. Under Cd only stress, the activities of all studied antioxidant enzymes were higher at lower Cd stress (1.0 μM) as compared to higher Cd stress (5.0 μM). Application of GB further enhanced the activities of these antioxidant enzymes as compared with their respective Cd-only treatments. However, there was no significant difference in leaves SOD and APX activities at 1.0 μM Cd and 1.0 μM Cd + GB and root POD and APX activities at 5.0 μM Cd and 5.0 μM Cd + GB. Furthermore, there was no significant increase in antioxidant enzyme activities in leaves and roots of plants treated with GB only, except APX in leaf, as compared to control. 3.4. Soluble protein and GB contents in plant under Cd stress Soluble protein contents significantly decreased in both leaves and roots under Cd stress as compared to control in a dose dependent manner (Fig. 2A, B). Addition of GB under Cd stress increased the soluble protein contents, except in leaf of control plants, as compared to respective Cd-only treatments and the results were only significant in roots

0

Leaf protein (µg g-1 FW) Leaf GB contents (µmol g-1)

a b b

40 c

c

20

0 160

C

140 120 100 a

80 60 40

c

c

Cadmium treatments significantly increased the MDA, electrolyte leakage, and H2O2 contents in both leaves and roots as compared to control in a dose dependent manner (Fig. 3). GB alone did not significantly affect the MDA, electrolyte leakage, and H2O2 contents in both leaves and roots as compared to control. By contrast, GB application in Cd-stressed plants significantly decreased the production of MDA and H2O2 and electrolyte leakage in both leaves and roots as compared to respective Cd only treatments. 3.6. Cadmium concentration in cotton under glycinebetaine Cadmium concentrations in root, stem and leaves of cotton significantly increased with increasing Cd levels in the nutrient solution in a dose dependent manner (Fig. 4). Roots accumulated highest Cd concentrations followed by stem and leaves respectively. Exogenous application of GB significantly decreased Cd concentrations in all parts of the plant as compared to respective Cd-only treatments. 4. Discussion In the present study, plant growth characteristics, biomass, and photosynthetic parameters decreased under Cd stress (Tables 1, 2). Cadmium-induced inhibition in plant growth and biomass has already

80

GB

a 60

3.5. Effects of glycinebetaine and Cd on MDA, H2O2, and electrolyte leakage

b

d d

20 0 0 1 5 Cd concentration (µM)

Root protein (µg g-1 FW)

A

treated with 5.0 μM Cd + GB as compared to respective Cd-only treatments. In plants without GB application, GB concentrations significantly increased in both leaves and roots with increasing Cd levels in the growth medium as compared to control (Fig. 2C, D). Exogenous application of GB in Cd-stressed plants further increased the GB concentrations in leaves and roots as compared to respective Cd treatments without GB.

Root GB contents (µmol g-1)

80

65

B

60

40

a

a

b b c

20

0 160

d

a

D

140 b

120 100

c

80 60

d

c

d

40 20 0 0

1 5 Cd concentration (µM)

Fig. 2. Effect of different concentrations of cadmium (Cd) (0, 1 and 5 μM) and glycinebetaine (GB) (0 and 1 mM) on protein and GB contents of leaf and root of cotton plants. Values show the means of five replicate ± SE. Means followed by same small letters are not significant different at P b 0.05 by using the Tukey test.

66


25

A

0

25

GB a

20

15

b c

10 d d

5 0 70

C a

50 b

40 30

b c

d

d

10

0 350

E

250 200 a

150

b

100

c d

e e

50

c d d

D a

60 50

b

40

0

c d

30 e e

20 10

0 350

300

bc

b

10

0 70

60

20

a 15

5

Root Electrolyte leakage (%)

Leaf Electrolyte leakage (%)

Root MDA (µM g-1 FW)

b

Root H2O2 (µM g-1 FW)

Leaf MDA (µM g-1 FW)

20

Leaf H2O2 (µM g-1 FW)

B

a

F

300

b

250

c d

200

150 e e

100 50 0



Fig. 3. Effect of different concentrations of cadmium (Cd) (0, 1 and 5 μM) and glycinebetaine (GB) (0 and 1 mM) on malondialdehyde (MDA), electrolyte leakage (EL) and hydrogen peroxide (H2O2) in leaf and root of cotton plants. Values show the means of five replicate ± SE. Means followed by same small letters are not significant different at P b 0.05 by using the Tukey test.

0

A - Root

GB a

8 b

6

c 4

d

2 e

3

toxicity on photosynthetic apparatus (Saidi et al., 2013) and/or structural alterations in plants (Ali et al., 2014). Decrease in plant growth and biomass might also be due to oxidative damage and reduction in antioxidant enzymes activities (Saidi et al., 2013) and/or reduction in mineral nutrients uptake by plants (Hediji et al., 2015). It might be assumed that

B - Stem a

2.5 2

b b

1.5 1

c

0.5


3

C - Leaf

2.5 2 1.5

a b

1

b c

0.5 0

0

0

Leaf Cd concentration (mg kg-1 DW)

10

Stem Cd concentration (mg kg-1DW)

Root Cd concentration (mg kg-1 DW)

been reported in many plant species such as wheat (Rizwan et al., 2012, 2015b; Rehman et al., 2015), rice (Nahakpam and Shah, 2011), maize (Vaculik et al., 2015), Brassica napus (Ali et al., 2014; Ehsan et al., 2014), tomato (Hediji et al., 2015) and bean plants (Saidi et al., 2013). Decrease in plant growth and biomass might be due to Cd-induced


0

1

5

Cd concentration (µM)

Fig. 4. Effect of different concentrations of cadmium (Cd) (0, 1 and 5 μM) and glycinebetaine (GB) (0 and 1 mM) on concentrations of Cd in roots, stem and leaves of cotton plants. Values show the means of five replicate ± SE. Means followed by same small letters are not significant different at P b 0.05 by using the Tukey test.


this decrease in the growth of the plant could be due to the reduced cell expansion (Daud et al., 2013; Dias et al., 2013). The decrease in photosynthesis could be due to the inhibition of the activities of key enzymes of the Calvin cycle and the photosynthetic electron-transport chain. Reduction in photosynthesis might also be due to Cd-induced inhibition in gas exchange chacacteristics of plants (Astolfi et al., 2005). In the present investigation, it was found that GB markedly alleviated Cd-induced reduction in growth, biomass and photosynthetic parameters in cotton plants (Tables 1, 2). It has been reported by many researchers that exogenous application of GB enhanced the plant tolerance to heavy metals (Cao et al., 2013; Ali et al., 2015) and other stresses such as drought, salinity, heat and cold (Yang et al., 2008; Iqbal et al., 2009; Islam et al., 2009; Chen and Murata, 2011). GB-mediated increase in biomass might be due to increase in the uptake of plants nutrients under stressful conditions (Shahbaz et al., 2011). GB may maintain the photosynthetic capacity of plants by increasing stomatal conductance and maintaining rubisco activity and chloroplast ultra-structure under drought stress (Nomura et al., 1998). In the present study, GB-mediated increase in plant biomass under Cd stress might be due to increase in photosynthetic pigments and gas exchange characteristics of cotton plants (Table 2). Many studies have verified that the improvement of photosynthesis by GB in stressed plants might be due to enhancement in the photochemical activity of photosystem II (PSII) (Holmstrom et al., 2000), because GB protects the PSII complex by stabilizing the association of the extrinsic PSII complex proteins under stress condition (Sakamoto and Murata, 2002). GB is mainly localized in chloroplasts and protects the thylakoid membranes, thereby maintaining photosynthetic efficiency and plasma membrane integrity (Yokoi et al., 2002; Yang and Lu, 2005). According to Murata et al. (1992), GB protects PSII by stabilizing the link of the extrinsic PS II complex proteins where it would contribute to improve stress tolerance. Cadmium inhibited soluble protein contents in both root and leaf parts of cotton plants (Fig. 2A, B). It might be the result of more oxidative stress under metal stress (Ali et al., 2015). Increased content of GB was observed in our experiments in Cd treated cotton seedlings compared to respective Cd-only treatments (Fig. 2C, D). Accumulation of GB in response to environmental stresses including metal toxicity has been reported by previous researchers (Sharma and Dubey, 2005). This increase in the GB contents under the cadmium stress alone may protect the proteins via a chaperon-like action on protein folding and may act as a signal molecule to tolerate the stress response (Rontein et al., 2002). However exogenous application of GB may reduce the adverse effects of environmental stresses (Yang and Lu, 2005) and improve the protein contents and GB status of cotton plants. The activities of antioxidant enzymes increased under lower Cd (1.0 μM) stress while decreased under higher Cd stress (5.0 μM) (Fig. 1). Increase in antioxidant enzymes activities at lower Cd stress might be due to the activation of plant defense mechanism under metal stress (Ali et al., 2014; Ehsan et al., 2014; Adrees et al., 2015b). Antioxidant enzymes play an important role in reducing the MDA contents under metal stress (Farooq et al., 2013; Ali et al., 2014). However, reduction in antioxidant enzymes activities at higher Cd stress might be due to the breakdown of ROS homeostatic balance and as a result, H2O2, MDA contents and electrolyte leakage increased (Fig. 3). Similar effect of Cd on antioxidant enzymes activities and ROS production has been previously reported in many plant species (Najeeb et al., 2011; Saidi et al., 2013; Ehsan et al., 2014). However, the application of GB further increased antioxidant enzyme activities and led to a decrease in oxidative stress as evidenced by the decreased production of MDA level and H2O2 contents and electrolyte leakage both in roots and leaves under Cd stress (Figs. 1 and 3). Similar effect of GB on antioxidant enzymes activities has also been observed in plants under various stresses (Cao et al., 2013; Saidi et al., 2013; Ali et al., 2015). The GB-induced inhibition of electrolyte leakage, MDA and H2O2 contents indicates that GB could significantly alleviate the harmful effects of Cd stress in cotton plants. In present study, increase in antioxidant enzymes activities with GB

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might also be due to lower Cd concentrations in different plant parts (Fig. 4). The present study suggests that GB-mediated stimulation of antioxidant enzymes activities and decreased ROS production might be one of the possible tolerance mechanisms of plants against Cd-induced oxidative stress in cotton plants. Plants have developed numerous ways and mechanisms to maintain and regulate cellular metal homeostasis by preventing uptake and accumulation of high concentrations of free heavy metal ions (Saidi et al., 2013; Ali et al., 2015; Rehman et al., 2015). All Cd treatments caused a significant increase in Cd concentrations in roots followed by stem and leaves of cotton plants (Fig. 4). Higher Cd concentrations in roots under Cd stress might be a strategy of plants to cope with metal stress as suggested by many researchers (Arshad et al., 2015; Hediji et al., 2015; Khaliq et al., 2015; Rizwan et al., 2015b). Addition of GB in Cd-stressed cotton plants significantly decreased Cd concentration in roots, stem and leaves as compared to respective Cd-only treatments (Fig. 4). GB-mediated decrease in heavy metal concentrations has already been reported in other plant species (Islam et al., 2009; Cao et al., 2013; Ali et al., 2015). GB-mediated decrease in Cd concentration in different plant parts might be due to protective role of GB in the cell membranes and as a result Cd concentration decreased in cotton plants (Giri, 2011). 5. Conclusion In conclusion, addition of GB significantly alleviated Cd-induced inhibition of cotton plant growth, biomass, chlorophyll contents, and gas exchange attributes and reduced Cd concentrations in roots, stem and leaves. Addition of GB markedly reduced Cd-induced MDA, H2O2 accumulation and electrolyte leakage. GB also increased the activities of key antioxidant enzymes activities in roots and shoots as compared to respective Cd-only treatments. The overall results from this research suggest that GB has the potential to overcome the deleterious effects of Cd on cotton by enhancing plant growth attributes, and by lowering uptake and accumulation of Cd, MDA, H2O2 and electrolyte leakage by up regulating the antioxidant enzymes. Thus, GB might be an important osmolyte that enables the plants to tolerate abiotic stress such as Cd stress in cotton seedlings. Acknowledgement Thanks to Higher Education Commission of Pakistan for financial support. The results presented in this paper are a part of M.Phil studies of Muhammad Ahsan Farooq. References Adrees, M., Ali, S., Iqbal, M., Bharwana, S.A., Siddiqi, Z., Farid, M., Ali, Q., Saeed, R., Rizwan, M., 2015b. Mannitol alleviates chromium toxicity in wheat plants in relation to growth, yield, stimulation of anti-oxidative enzymes, oxidative stress and Cr uptake in sand and soil media. Ecotoxicology and Environmental Safety 122, 1–8. Adrees, M., Ali, S., Rizwan, M., Ibrahim, M., Abbas, F., Farid, M., Rehman, M.Z., Irshad, M.K., Bharwana, S.A., 2015a. The effect of excess copper on growth and physiology of important food crops: A review. Environmental Science and Pollution Research 2, 8148–8162. Aebi, H., 1984. Catalase in vitro. Methods in Enzymology 105, 121–126. Ahmad, P., Nabi, G., Ashraf, M., 2011. Cadmium-induced oxidative damage in mustard [Brassica juncea (L.) Czern. & Coss.] plants can be alleviated by salicylic acid. South African Journal of Botany 77, 36–44. Ali, B., Qian, P., Jin, R., Ali, S., Khan, M., Aziz, R., Tian, T., Zhou, W., 2014. Physiological and ultra-structural changes in Brassica napus seedlings induced by cadmium stress. Biologia Plantarum 58, 131–138. Ali, S., Chaudhary, A., Rizwan, M., Anwar, H.T., Adrees, M., Farid, M., Irshad, M.K., Hayat, T., Anjum, S.A., 2015. Alleviation of chromium toxicity by glycinebetaine is related to elevated antioxidant enzymes and suppressed chromium uptake and oxidative stress in wheat (Triticum aestivum L.). Environmental Science and Pollution Research 22, 10669–10678. Ali, S., Farooq, M.A., Hussain, S., Yasmeen, T., Abbasi, G.H., Zhang, G.P., 2013b. Alleviation of chromium toxicity by hydrogen sulfide in barley. Environmental Toxicology and Chemistry 32, 2234–2239.

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