Red and blue lights induced oxidative stress tolerance promote cadmium rhizocomplexation in Oryza sativa

Journal of Photochemistry and Photobiology B: Biology xxx (2014) xxx–xxx

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Red and blue lights induced oxidative stress tolerance promote cadmium rhizocomplexation in Oryza sativa Abin Sebastian, M.N.V. Prasad ⇑ Department of Plant Sciences, University of Hyderabad, Hyderabad 500046, India

a r t i c l e

i n f o

Article history: Received 13 September 2013 Received in revised form 9 December 2013 Accepted 10 December 2013 Available online xxxx Keywords: Cadmium Light Photosynthesis Redox regulation Metal chelator Plant nutrient

a b s t r a c t Cadmium (Cd) accumulation and related stress responses have been investigated in red, blue and white lights exposed Oryza sativa L. cv MTU 7029. Cd translocation was reduced significantly by red and blue lights. Increase in amount of organic acids, thiols, and nutrients in the roots that cause Cd rhizocomplexation was the reason for reduction in Cd translocation. These effects were due to higher efficiency to perform photosynthesis and transpiration under red or blue lights compare with white light during Cd stress. Increased photosynthetic assimilate turnover was witnessed as a function of sugar content. Amount of redox regulators such as glutathione and ascorbate were also increased under red and blue light exposure. Together with up regulation of antioxidant enzyme activities, these metabolites ensured redox balance in presence of reactive oxygen species produced due to Cd toxicity. Protection of photosynthesis from Cd inducible oxidative stress ensured supplies of sugar intermediates essential for the synthesis of metal chelators in roots. Therefore, it was inferred that red and blue lights promote Cd rhizocomplexation and ameliorated Cd stress in rice seedlings. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Cadmium (Cd) is known to alter several physiological processes in plants [1,2]. Redox dependant metabolic pathways are the primary sites of Cd toxicity, since Cd2+ ions have redox potential. Photosynthesis being redox dependant process is prone to Cd toxicity [2,3]. Ionic charge play crucial role in reorganization of thylakoid membranes in the chloroplast [4,5]. Hence the presence of charged ions such as Cd2+ affects organization of thylakoid membrane structure which damage photosynthetic electron transport chain. Additionally, proteins in photosynthetic electron transport chain undergo damage due to Cd inducible reactive oxygen species (ROS) [6]. Photooxidative stress mediated ROS production is reported to cancel out under specific wave lengths of light such as blue, red and UV [7]. Cd uptake and transport in plants is reported through metal transporters that are meant for mineral nutrients [8]. It is well known that mineral nutrient uptake in plant is redox regulated and photosynthesis play key role in regulation of redox status of the plant. It is also known that red and blue wavelengths of lights are most effective in photosynthesis. Therefore, as per the information stated supra vide; it is believed that there is a relationship between the uptake of Cd and red or blue light regulation of photosynthesis.

⇑ Corresponding author. Tel.: +91 4023134509; fax: +91 4023010120/145.

Cytotoxic effects of Cd in plants are due to changes in redox potential in the cell that end up in oxidative stress [9,10]. Hence operation of antioxidant system is essential to prevent Cd inducible oxidative stress. Asada–Halliwell pathway is the well known antioxidant pathway that operates in plants during oxidative stress [11]. This pathway detoxifies superoxide produced from photosynthetic electron transport chain in chloroplast. Superoxide formed is converted into hydrogen peroxide either by spontaneous dismutation or superoxide dismutase enzyme (SOD). Hydrogen peroxide is scavenged by ascorbate with help of enzyme ascorbate peroxidase (APX). Ascorbate will regenerate either by monodehydroascorbate reductase (MDHAR) mediated reactions or dehydroascorbate reductase (DHAR) mediated reaction with help of GSH. In this way Asada–Halliwell pathway helps to detoxify hydrogen peroxide which has the potential to cause oxidative stress. Red light and blue lights are known to affect plant physiological process such as photosynthesis and stomatal opening [12,13]. Photoreceptor mediated signal transduction process is elucidated as reason for these effects [14]. Stimulation in the synthesis of stress inducible proteins or over expression of cellular proteins that are part of biosynthetic pathways with help of blue and red lights thus help to overcome stress experienced by plants. Increase of photosynthesis by these lights is postulated to help alleviation of Cd stress in the present study because production of metal chelators such as organic acids depends on sugar metabolism. Effects of these lights on stomatal opening controls water balance as well as nutrients uptake that may also affect Cd uptake. Since millions

E-mail address: [email protected] (M.N.V. Prasad). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.12.011

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of tons of rice are reported to contaminate with Cd, present study is relevant with regard to characterization of essential ecophysiological functions that play important role in reduction of root to shoot translocation of Cd. 2. Materials and methods 2.1. Hydroponics and Cd treatments Seedlings were raised from Oryza sativa (cv MTU 7029) seeds kept in dark for 72 h at 37 °C in germination boxes. Growth media for seedlings was Hoaglands nutrient solution having pH 6.0 [15]. Cd treatment was performed by transferring seedlings in to Hoagland media containing 0, 25 and 50 lM CdCl2 under white, red and blue light respectively. Each group maintained with twenty seedlings in 50 ml media. LED panel was made for each light source and light intensity (100 ± 5 lmol photons m2 s1, ideal for germinating seedlings) controlled with help of voltage regulator unit. Spectral distribution of white light source was 460–700 nm. Peak wavelengths for the blue and red LEDs were 466 and 665 nm respectively. Photoperiod (18 h light/6 h dark), temperature (25 ± 2 °C) and relative humidity (50 ± 10%) were maintained throughout the growth period of 14 days. Low light intensity also helps to avoid overlapping of photo oxidative stress and Cd inducible oxidative stress. 2.2. Chlorophyll fluorescence analyses Quantum efficiency of photosystem II (YII), Linear electron transport rate (ETR) and Non-photochemical quenching (NPQ) were measured using PAM 2500 (Heniz Walz; Germany) in light curve mode. After dark adaptation for 30 min and the maximum fluorescence was monitored by application of a 0.8 s saturating light pulse (6000 lmol photons m2 s1). The steady state fluorescence yield was monitored through exposure of leaf to actinic light range from 8 to 1200 lmol photons m2 s1. 2.3. Photosynthesis rate and plant pigments The photosynthetic light mediated evolution of oxygen of the intact leaves was measured using Oxylab system (Hansatech Instruments, United Kingdom). Leaves were subjected to the instrument with 200 ll 1.0 M bicarbonate buffer under light (1200 lmol photons m2 s1) for a period of 15 min. Rate of photosynthesis was expressed as rate of oxygen evolved per minute. Chlorophyll and carotenoids were estimated from acetone: DMSO (50:50) extract of leaves [16]. Anthocyanin was estimated from methanol/HCL/water (90:1:1) extract of leaf [17]. 2.4. Stomata frequency and transpiration Stomata frequency was calculated as number of stomata present in unit area (mm2) using stereo microscope. Transpiration was measured by using a test tube filled with 20 ml water and inserting single seedlings in it. Oil was poured on the surface of water to prevent loss of water by evaporation. Test tubes were allowed to rest in a small beaker and weighed them together. After standing for 8 h; tube with beaker was again weighed. Difference in weight indicated loss of water due to transpiration. 2.5. SOD inhibitory NADPH dependent superoxide ðO 2 Þ generation Leaves (1 g) were homogenized in 50 mM HEPES–Tris buffer (pH 7.5), 40 mM PMSF, 2% (w/v) PVPP and centrifuged at 13,000g for 20 min at 4 °C to obtain the supernatant. Superoxide generation

was assayed by measuring the rate of SOD-inhibitory cytochrome C reduction in the presence of NADPH [18]. Reaction mixtures in the sample cuvette contained 50 mM HEPES–KOH (pH 7.8), 0.1 mM EDTA, 1 lM K 4Fe (CN)6, 75 lM cytochrome C, 100 lg protein and 50 lM NADPH. The reference cuvette was identical to the sample cuvette except for the addition of 25 lM superoxide dismutase. After 1 min pre-incubation, the reaction was started by the addition of NADPH to both the cuvettes and the absorbance changes at 550 nm were followed for 1 min. Rates of O 2 generation were calculated using an extinction co-efficient of 21.0 mol1 m3 cm1. Superoxide production was expressed in nmol mg1 protein min1. Under similar conditions NADPH oxidation was measured at 340 nm except that cytochrome C was omitted from the reaction mixture. Rates of NADPH oxidation were calculated using extinction co-efficient of 6.2 mol1 m3 cm1. 2.6. Chloroplast dependant superoxide ðO 2 Þ radical production Chloroplast was isolated from leaves at ice cold conditions according to Atal et al. [19]. Levels of superoxide radical were determined by the rate of epinephrine to adrenochrome with 1 mM NADPH as substrate [20]. The absorbance difference (A485–A575, e = 2.96 mM1 cm1) was recorded at room temperature within 5 s after illuminating with white light (700 lmol photons m2 s1) for 8 min. The 3 ml reaction mixture contained 1 mM epinephrine, 1 mM NADPH and chloroplast suspension (50 lg chlorophyll equivalent). 2.7. H2O2 and MDA content H2O2 isolation was made from 2.0 g of the treated plant material in ice-cold 100% acetone. By addition of 5% (w/v) titanyl sulfate and conc. NH4OH solution, the peroxide–titanium complex was precipitated [21]. The precipitate was dissolved in 15 ml of 2 M H2SO4, making the final volume to 20 ml in cold water. The absorbance of the resultant was read at 415 nm. H2O2 content was calculated from a standard curve prepared in similar way. MDA in leaves (100 mg) was determined after extraction with trichloroacetic acid containing 0.5% 2-thio-barbituric acid (TBA) [22]. 2.8. Protein extraction, SDS page and MALDI TOF analysis Tissue homogenized in tris buffer was centrifuged at 10,000g for 20 min at 4 °C and the supernatants were collected for protein estimation according to Lowry et al. [23]. Extracted proteins were precipitated using acetone at 4 °C. Precipitated protein was dissolved in 25 mM Tris HCl buffer having pH 7 and thereafter 80 lg protein which was cooked in sample buffer subjected for SDS Page analysis [24]. After SDS page; the bands were cut for from the gel and processed for MALDI TOF analysis (Bruker Autoflex III smartbeam, Bruker Daltonics, Bremen, Germany) following the method of Shevchenko et al., 1996 [25]. 2.9. Antioxidant enzyme assays MDHAR activity was assayed by monitoring the change in absorbance at 340 nm due to NADPH oxidation (e = 6.2 mM– 1 cm1) using ascorbate oxidase [26]. DHAR activity measured by formation of AsA at 265 nm (e = 14 mM1 cm1) in a reaction mixture containing 0.1 M Na-phosphate buffer (pH 6.2) and 2 mM GSH [27]. Assay of SOD conducted according to Beauchamp and Fridovich [28]. Catalase (CAT) activity was determined by measuring the rate of H2O2 disappearance at 240 nm [29]. Guiacol peroxidase (GPX) assay performed in a reaction mixture contained 50 mM phosphate buffer, 0.2 mM guiacol, 10 mM H2O2 and distilled water

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Sample (100 mg) was ground in double distilled water and filtered. Filtrate subjected to react with antherone reagent and absorbance was read at 620 nm [37]. Total phenols calculated from extract (10 ml from 1 g dried leaf tissue) after reaction with by Folin Ciocalteu reagent according to McDonald et al., 2001 [38]. Total flavonoid contents were measured with the aluminum chloride colorimetric assay as described in Kumar et al. 2008 from methanol extract [39]. 2.15. Nutrient and cadmium analysis Nutrient analysis carried out from dehydrated leaf powder using wavelength dispersive X-ray spectroscopy (WDS) (INCA

B R

6.6 + 0.3a 82.3 ± 1.5b 22.7 ± 1.5a 21.3 ± 2.2e 51.1 ± 4.3c 65.3 ± 1.6d 126.6 ± 5.5c 72.3 ± 4.7c 5.1 ± 0.5a 4.4 ± 0.3b 30.4 ± 2.1b 25.8 ± 3.6b 74.2 ± 2.1c 63.1 ± 5.3b 0.96 ± 0.06a 0.57 ± 0.02e 6.4 + 0.4b 70.3 ± 3.8e 19.3 ± 0.6b 57.2 ± 4.3a 88.6 ± 7.2a 98.4 ± 4.1a 154.6 ± 8.1a 86.6 ± 1.9a 3.2 ± 0.2c 3.5 ± 0.3d 14.7 ± 2.1d 13.3 ± 2.5d 34.1 ± 2.6 h 19.6 ± 3.7e 0.46 ± 0.02d 0.34 ± 0.05f 6.2 + 0.2b 81.7 ± 2.1a 15.0 ± 1.7c 26.4 ± 1.1c 48.7 ± 1.2c 51.3 ± 5.1f 124.6 ± 2.5c 70.6 ± 3.2d 5.05 ± 0.6a 6.7 ± 0.4a 27.3 ± 4.3a 37.3 ± 1.5a 67.2 ± 1.1d 57.6 ± 4.2b 0.84 ± 0.05b 0.88 ± 0.01a 7.1 + 0.2a 86.4 ± 2.1a 15.7 ± 0.6c 18.1 ± 1.3f 49.3 ± 2.5c 77.5 ± 5.8c 128.6 ± 12.5c 68.2 ± 1.6e 4.3 ± 0.4b 4.7 ± 0.5b 33.3 ± 5.1a 35.1 ± 3.6a 83.6 ± 3.7b 69.6 ± 4.1a 1.1 ± 0.12a 0.92 ± 0.06a 5.6 + 0.4d 74.6 ± 1.9d 12.7 ± 0.6d 45.2 ± 2.6b 60.4 ± 5.2b 92.1 ± 3.4b 95.3 ± 3.1d 79.1 ± 2.3b 2.5 ± 0.3e 3.9 ± 0.7c 20.2 + 5.2c 19.8 ± 2.6c 40.6 ± 1.5 g 28.6 ± 3.2d 0.51 ± 0.01d 0.58 ± 0.01e

25 lM CdCl2

B R

Nb: W, R and B indicate white, red and blue lights respectively. Alphabets comes with values indicate result of Duncan’s multiple range test for statistical means separation. Figure not showing a common letter are significantly differ from one another at the 5% error level where letters a, b, c, d, e and f represents first, second, third, fourth, fifth and sixth level of statistical significance respectively.

2.14. Estimation of total soluble sugar, phenols and flavanols

W

Dried powder of plant tissues (25.0 mg) was subjected to form pellet with potassium bromide. Pellet was loaded in to FTIR spectrometer (JASCO FTIR-5300) at room temperature [36]. Spectral wave number ranges of 400–4000 cm1 were recorded. For organic acid; data was expressed in terms of characteristic absorbance of – COOH group per 20 mg tissue.

Parameters

2.13. Analysis of amines, amides and organic acids

Table 1 Biomass, relative water content, oxidative stress modulators and major cellular metabolites.

GSH and GSSG were estimated from sodium phosphate – H3PO3 extract [35]. Final assay mixture (2.0 ml) contained 100 ll of the diluted supernatant, 1.8 ml of phosphate-EDTA buffer and 100 ll of Ophthalaldehyde (1 mg ml1). After thorough mixing and incubation at room temperature for 15 min, the solution was transferred to a quartz cuvette and the fluorescence at 420 nm was measured after excitation at 350 nm. Estimation of GSSG carried out after incubation of extract with 200 ll of 0.04 M N-ethylmaleimide for 30 min to interact with the GSH present in the supernatant.

0 lM CdCl2

2.12. Estimation of glutathione

W

R

The treated plants were homogenized in 0.02 M EDTA under cold conditions. The thiol contents of the homogenates were measured using Ellman’s reagent (5,50 -Dithio-bis-2-nitrobenzoic acid or DTNB) [34]. Aliquots of 5 ml of the homogenates were mixed with 4 ml of distilled water and 1 ml of 50% TCA to separate nonprotein thiols and protein bound thiols. The protein bound thiols were calculated by subtracting the non-protein thiols from total thiols.

4.4 + 0.3e 84.3 ± 1.2a 13.0 ± 1.0d 25.3 ± 0.9c 44.3 ± 1.5d 55.4 ± 3.3e 127.3 ± 4.1c 70.2 ± 2.6e 3.4 ± 0.3c 4.8 ± 0.3 b 25.6 ± 2.2b 35.6 ± 2.2a 93.1 ± 1.2a 66.3 ± 3.7a 0.84 ± 0.03b 0.84 ± 0.01b

50 lM CdCl2

W B

2.11. Measurement of protein bound and non-protein thiols

5.5 + 0.4d 84.9 ± 0.8a 12.3 ± 1.5d 23.2 + 1.1d 46.2 ± 1.2d 64.5 ± 1.7d 129.6 ± 2.1b 66.4 ± 1.3f 2.6 ± 0.1d 4.2 ± 0.3b 32.4 ± 2.1a 38.3 ± 6.9a 93.3 ± 5.5a 60.3 ± 5.5b 0.75 ± 0.09b 0.71 ± 0.03d

Total ascorbate and dehydroascorbate (DHA) were measured according to Ma and Cheng [32]. Ascorbic acid (AsA) was assayed spectrophotometrically at 265 nm (e = 14 mM1 cm1) in 100 mM potassium phosphate buffer (pH 5.6), by subtracting the optical density, before and after 15 min incubation with 5 units of ascorbate oxidase. For total ascorbate, 100 ll of extract was neutralized with 30 ll of 2 mM Na2CO3 and incubated for 30 min at room temperature with equal volume of 20 mM GSH in 100 mM Tricine– KOH (pH 8.5) and Optical density was measured at 265 nm. DHA was calculated as the difference between total ascorbate and AsA. Total antioxidant capacity was determined by the phosphomolybdenum method in terms of ascorbic acid equivalents [33].

6.1 + 0.5c 82.1 ± 4.0c 12.7 ± 1.2d 24.7 ± 0.6c 41.3 ± 1.5d 44.7 ± 3.2f 136.3 ± 4.1b 76.2 ± 2.1c 1.3 ± 0.2f 3.3 ± 0.1e 21.1 ± 3.1b 25.6 ± 5.5b 47.3 ± 2.3f 51.3 ± 4.1c 0.66 ± 0.01c 0.77 ± 0.3c

2.10. Total ascorbate, dehydroascorbate (DHA) and antioxidant activity

Biomass (mg dry wt/plant) Relative water content (%) Stomata frequency (N/50 lm2) NADPH dependant O 2 (nmol/mg protein/min) Rate of NADPH oxidation (nmol/mg protein/min)  Chloroplastic O2 (nmol/50 lg Chlorophyll) H2O2 (nmol/g fresh wt) MDA (lmol/g fresh wt) Phenols (lg/g dry wt) Flavanols (lg/g dry wt) Sugar (mg/g fresh wt) Leaf Root Protein (mg/g fresh wt) Leaf Root Organic acids (A/20 mg dry wt) Leaf Root

in a total volume of 3 ml [30]. Ascorbate peroxidase (APX) was assayed by the method of Nakano and Asada, 1981 [31].

5.9 + 0.3c 79.9 ± 1.8c 22.0 ± 1.0a 28.4 ± 0.5c 53.6 ± 2.8b 58.1 ± 2.5e 137.6 ± 4.2b 75.3 ± 1.5c 5.6 ± 0.3a 6.4 ± 0.6a 28.2 ± 2.3b 27.6 ± 3.7b 55.3 ± 2.6e 52.3 ± 0.6c 0.68 + 0.08c 0.66 ± 0.03d

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instruments) coupled with scanning electron microscope (FSEM, Oxford instruments). For quantitative estimation of Cd, plants were first manually cleaned with deionized water followed by washing using 0.5 M EDTA and dried at 80 °C in an oven until completely dried. After acid digested plant material was solubilzed in 0.1 M HCL and subjected to standard calibrated atomic absorption spectrophotometer (GBC scientific, Australia) [40].

3. Results 3.1. Morphology, biomass, relative water content and plant pigments Red and blue light grown plants achieved more biomass compare with plants grown under white light (Table 1, Supplementary data 1). Stomata frequency found to be highest in red and blue lights treatment (Table 1). Another noticeable feature was the decrease of stomata aperture up on 50 lM CdCl2 treatment (Supplementary data 2). It was also noticed that the size of aperture was small in plants grown under red and blue lights compare with white light. Relative water content was highest in plants grown under red and blue lights (Table 1). Chlorophyll a content was higher in plants grown under white and red lights (Fig. 1A). Chlorophyll b content found to be least under blue light (Fig. 1B). Carotenoid and anthocyanin content were found maximum under blue and red lights (Fig. 1C and D).

3.2. Photosynthetic performance and transpiration Plants grown under blue and red light showed more photosynthetic efficiency compare to those grown under white light. It is noteworthy that increase of Cd treatment had negative effect on photosynthetic performance under all the light sources. Among various treatments, sharp decrease in photosynthesis was observed under white light at 50 lM CdCl2 (Fig. 1E). Quantum efficiency of photosystem II found to be highest in plants grown under red light at 25 lM CdCl2 (Fig. 2A). Lowest value of quantum efficiency was observed in plants grown under white light at 50 lM CdCl2 treatment. Electron transport rate also showed above trend (Fig. 2B). Non-photochemical quenching was observed highest in plants grown under blue or red light and least in plants grown under white light at 50 lM CdCl2 (Fig. 2C). Transpiration was highest under blue light followed by red and white lights respectively (Fig. 1F). 3.3. Components of oxidative stress and its detoxifying agents NADPH dependant superoxide production as well as NADPH oxidation, and chloroplast dependant superoxide production were higher in plants grown under white light (Table 1). It was also noticed that Cd treatment increased production of these free radicals. Hydrogen peroxide production in leaves was significantly increased at 50 lM CdCl2 under white light compare with blue and

Fig. 1. Plant pigments, photosynthesis and transpiration in response to Cd under white, red and blue light treatments. Since photosynthesis oxygen evolution measured with 1200 lmol photons m2s1 white light, Fig. 2E indicates efficiency of plants to perform photosynthesis. The difference in photosynthesis occurred under the different light source during period of treatments is appeared in terms of sugar accumulated; Table 1. (Abbreviations: Numerical 0, 25 and 50 represents CdCl2 treatment at micro-molar concentrations. Letters W, R and B represent white, red and blue lights respectively. This pattern is followed throughout all the figures and tables). Nb: Alphabets in each bar indicate result of Duncan’s multiple range test for statistical means separation. Figure not showing a common letter are significantly differ from one another at the 5% error level where letters a, b, c, d etc. represents first, second, third, fourth and so on level of significance.

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fect was absent in red and blue light grown plants (Supplementary data 3). Organic acid content of plants was highest under red and blue lights (Table 1). Cd treatment also had significant influence on production of organic acids especially at 25 lM CdCl2 treatment where production of organic acids found to increase. Among various functional group analyzed; amines showed maximum shift followed by alkanes, esters and amides (Supplementary data 4). 3.5. Cd accumulation and nutrient content It was found that leaves of plants grown under red and blue light accumulate lower Cd compare with white light (Fig. 6B). This effect was due to decrease in Cd translocation under these lights (Fig. 6C). It was found that higher amount of Cd held in roots of red and blue light grown plants compare with plants grown under white light (Fig. 6A). All the above results point decrease of Cd translocation was due to retaining Cd in root under red and blue lights. Red and blue light treatments also increased amount of essential nutrients in plant (Table 2). Potassium uptake was higher in plants grown under red and blue lights compare with white light. Potassium content found to increase with increase of Cd treatment. Sulfur content also increased under red and blue lights. In leaves; increase of sulfur content with increase of Cd conc. was prominent under blue light. Phosphorous content was found to increase under both blue and red lights reared plants during Cd stress. Similar effect was observed under white light at 25 lM CdCl2 treatment. Magnesium increased at 25 lM CdCl2 treatment under white and blue lights. Calcium in leaves found to higher in blue light compare with red and white lights. Fig. 2. Chlorophyll fluorescence analysis parameters such as quantum efficiency of PSII, electron transport rate and non-photochemical quenching under the influence of light and Cd stress (Nb: Post hoc. test results for YII, ETR, NPQ; follows in the same sequence: W0 – a, a, b; W25 – a, c, a; W50 – b, c, c; R0 – a, a, a; R25 – a, b, a; R50 – a, a, a; B0 – a, a, a; B25 – a, a, a; B50 – a, a, b).

red lights (Table 1). Malonyldialdehyde content was also found highest in plants grown under white light (Table 1). Activity of enzymes in Asada–Halliwell pathway such as monodehydroascobate reductase (Fig. 3A), dehydroascorbate reductase (Fig. 3B), glutathione reductase (Fig. 3C), and enzymes which are known to act as antioxidant enzymes such as superoxide dismutase (Fig 4A), catalase (Fig. 4B), ascorbate peroxidase (Fig. 4C) and guiacol peroxidase (Fig. 4D) were found to be up regulated by red and blue lights. Cd treatments showed stimulatory effect on activity of these enzymes particularly during exposure to red light. Total antioxidants (Fig. 3F) as well as GSH (Fig. 5A and B) were found to be highest during red and blue light treatments. Phenolics and flavanols also found to increase under these lights upon Cd treatment (Table 1). Total ascorbate and dehydroascorbic acid were formed more under red and blue lights. Cd treatment not only decreased total ascorbate but also increased dehydroascorbic acid content (Fig. 3D and E). GSSG content was higher in plants grown under white light (Fig. 5C and D). In roots; protein thiols and non-protein thiols were higher during red and blue light treatments (Fig. 5E and F). 3.4. Major cellular macromolecules and shift in functional groups Sugar content was highest in plants grown under red light and lowest in plants grown under white light (Table 1). Treatments with 50 lM CdCl2 under white light showed least sugar content. Protein content in leaves and roots decreased upon Cd treatments except in roots of red light reared plants at 25 lM CdCl2 treatment (Table 1). It was found that amount of rubisco large subunit decrease with increase in Cd concentration under white light. This ef-

4. Discussion Morphological appearance of plants in the current study indicates that light wavelength had significant role to cope with Cd stress. Difference in amount of biomass observed between treatments indicates that red and blue light grown plants were able to tolerate Cd stress more efficiently compare with plants grown under white light. Reason for such a difference was proven as stability of metabolic functions in the presence of Cd. This functional stability in turn maintained plant growth and biomass production. Blue and red light stand as the most effective light wavelength for photosynthesis. Increase in photosynthesis support biosynthetic pathways by providing essential intermediates for the metabolism [41,42]. Metabolic stability accelerated plant growth was reflected in the current study as increase of biomass. Stress tolerance of plants depends on photosynthetic efficiency which plays central role in biosynthesis of aminoacids, proteins, organic acids and secondary metabolites such as polyamines [43]. Steady photosynthesis efficiency observed under red and blue lights compare to white light with increase of conc. of Cd points that photosynthetic performance played crucial role in current study to reduce Cd translocation. Organic acids found in plants are known to bind and exclude Cd [44,45]. Carboxylic acid synthesis in plants depends on photosynthesis and keeping balance of these acids is crucial for rhizocomplexation of metals in plants [46]. Cd rhizocomplexation occurs through trafficking of Cd in to vacuoles of root with help of metal chelators such as organic acids and thiols derivatives. It is the abundance of high affinity metal binding groups such as – COOH and –SH that makes organic acids and thiol derivatives to form respective metal complexes. Organic acids that are reported to be involved in Cd chelation in root are malic acid, citric acid and oxalic acid. Thiol compounds that are involved in Cd trafficking in to vacuoles are glutathione and phytochelatins. Apart from these compounds, protein called metallothionein is also involved in Cd detoxification in plant roots. The pathway that direct cytoplasmic

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Fig. 3. Response of components in Asada–Halliwell pathway such as monodehydroascobate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) along with essential metabolites in the pathway during different light and Cd treatments.

Cd to vacuole is complex and yet to be studied in detail. Lack of specificity of uptake and distribution system observed in case of Cd indicate Cd trafficking occurs through micronutrient homeostasis network operate in plants [8]. But phytochelatin synthesis that occurs only during toxic heavy metal stress differentiate trafficking of mineral nutrients and toxic heavy metals such as Cd. Transport of phytochelatin–metal complex to vacuole is reported to be mediated by ABC transporter, Hmt 1 in fission yeast but corresponding proteins in plants have yet to be identified. Compare with organic acids, thiol compounds play key role in Cd rhizocomplexation. In the present study, organic acids as well as thiols such as protein thiol which is an indicator of phytochelatins or metallothioneins, glutathione, and non-protein thiols found to increase in root under red and blue lights treatments. Significant difference observed among amount of these metabolites between white light and blue or red lights indicate that Cd rhizocomplexation reduced Cd translocation in to leaves under blue or red lights. The reason for increase of above mentioned metal chelators relate to maintenance of photosynthesis under Cd stress which had been described in following sections. Carbon fixation during photosynthesis depends on amount of rubisco. Decrease in typical rubisco large subunit band (53 kDa) observed under white light at 50 lM CdCl2 compare with red and blue lights accounts for low sugar and protein contents among these plants (Supplementary data 4). This is because of the reason that carbon fixation is the key process which channels intermediates for the synthesis of these molecules. Low intensity rubisco band observed under white light is also a clear indication of higher level of Cd accumulation which is well known to cause oxidative

stress and inhibition of protein synthesis in plants. Since red and blue light treatments increased antioxidant activity, these plants were also able to overcome oxidative stress due to Cd which entered in leaves. Increase in antioxidant activity was reflected as increase in total antioxidant activity, ascorbic acid content, glutathione content, and activity of enzymes in antioxidant pathway such as Asada–Halliwell pathway under red and blue light treatments. This effect is due to effectiveness of red and blue light to cause light saturation of photoelectron transport compare with white light that end up in stimulation of antioxidant activity [50]. During normal plant growth condition, light saturation leads production of ROS such as superoxide from photosynthetic electron transfer chain which can be detoxified by antioxidant enzymes [51]. Thus red and blue light grown plants that were acclimatized with water–water cycle survived from Cd inducible oxidative stress and this process in turn protected photosynthesis under Cd stress. Decrease of quantum efficiency of PSII and electron transport rate across PSII of white light grown plants indicate that damage of PSII function due to loss of protein counterpart since there was no significant changes in chlorophyll content among all the light treatments. Higher non-photochemical quenching observed among red and blue light grown plants compare with white light grown plants indicate that proper functioning of energy quenching mechanism which protected reactive oxygen formation in these groups of plants. More amount of carotenoids observed among these plants also support above finding because carotenoids play major role in non-photochemical quenching. Increase in anthocyanin observed among all light treatments could be justified as a protecting mechanism operated against Cd inducible ROS irrespective

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Fig. 5. Redox regulators such as glutathione and metal chelators such as thiols in response to light and Cd treatments. Relatively higher amount of oxidized glutathione accumulated in plants grown under white light indicate inability of these plants to reduce glutathione which is essential for operation of antioxidant system as well as synthesis of thiol containing peptides such as phytochelatins.

Fig. 4. Antioxidant enzyme activity such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guiacol peroxidase (GPX) in response to Cd and Light treatments. It is noteworthy that light saturation dependant ROS production was nullified because of increase of antioxidant activity under red and blue lights which also protected these plants from Cd inducible ROS production (see Table 1).

of light wavelength [52]. Since chlorophyll a content not significantly affected by Cd treatments among all the light sources; the chance of reactive oxygen formation through excited chlorophyll a is more prominent under white light where photochemical quenching as well as non-photochemical quenching were least. It was also noticed that increase of Cd treatment leads more prominent increase in NADPH dependant reactive oxygen production under white light. This kind of reactive oxygen formation is common in presence of divalent metal ion such as Cd2+ and the rate of production of which further supports higher amount of Cd entered in leaves of white light exposed plants [53]. The deleterious effect of ROS under white light was further demonstrated by increase of lipid peroxidation product malonyldialdehyde with increase of conc. of Cd treatment in the present study. Photosynthesis helps to keep nitrogen and carbon balance where nitro compounds in leaves upon breakdown release organic acids which later translocate in to roots for nitrogen assimilation [47]. Thus increase of organic acid production in leaves through photosynthesis helps to translocate nitrogen from root to leaves along with increase of metal chelation ability in roots. This process enhanced during blue or red

lights exposure and reflected as more biomass which is an indicator of nitrogen status of the plant. Balance of sugar metabolism also assists various secondary metabolic pathways that are involved in synthesis of phenolics and flavanols. These compounds help plants to cope with stress [48]. Increase in flavanols and phenols observed in the study points involvement of these compounds on metal detoxification. Functional group shift observed among amines especially under blue light support that operation of secondary metabolism played significant role in the current study by providing components that act as antioxidant agents during Cd stress [49]. Oxidative stress observed in the study stands as a clear evidence of physiological challenge that arise because of damage of photosynthetic electron transport and carbon fixation during Cd stress. Blue and red lights control rhythm of physiological processes such as stomatal opening crucial for mineral nutrient uptake in plants [54]. Increase of stomata frequency observed stand as reason for high transpiration rate observed under red and blue light. Together with stomata frequency, influence of red and blue lights on stomatal opening also needs to be considered with regard to increase of transpiration during exposure of blue or red lights. Higher transpiration rate accounts for superior uptake and allocation of nutrients such as potassium; magnesium, sulfur, phosphorous, and calcium under red and blue lights except for magnesium at 50 lM CdCl2 treatment; phosphorous and calcium at 25 lM CdCl2 treatments respectively. It was noticed that increase of Cd treatment cause increase of phosphorous accumulation in roots. This defense strategy helps to precipitate toxic Cd2+ in root by forming phosphates of Cd. On the other hand; increase of sulfur accumula-

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A. Sebastian, M.N.V. Prasad / Journal of Photochemistry and Photobiology B: Biology xxx (2014) xxx–xxx

tion with increase of Cd concentration indicates operation of thiol mediated protective mechanism [55]. An increase of sulfur content in roots helped to increase glutathione level which is important to keep redox balance as well as metal chelation. The difference in sulfur content in roots and leaves also points relation between Cd detoxification and sulfur translocation. Decrease of Cd content along with increase in Mg and Ca under red and blue light supports competitive uptake of Cd in to the plant along with these elements. In summary, blue and red light treatments caused light saturation of photosynthesis. Light saturation caused up regulation of antioxidant mechanism to cope with photo oxidative stress which in turn protected plants from Cd inducible oxidative stress. Thus photosynthesis efficiency of plants was increased under red and blue lights during Cd stress. This enabled higher assimilate turn over for the synthesis of proteins including rubisco, organic acids, antioxidants, and secondary metabolites along with increase of assimilate accumulation in root. Buildup of assimilate in roots supported channeling of carbon skeleton for synthesis of metal chelators such as glutathione, protein thiols, and non-protein thiols along with organic acids all which are involved in Cd rhizocomplexation. Increase of Cd rhizocomplexation reduced Cd translocation. Even though transpiration is a passive process, regulation of this process by red and blue light influenced Cd accumulation in plant by increasing uptake and translocation of macronutrients that are known to reduce Cd uptake and transport. 5. Abbreviations

Fig. 6. Cd accumulation (A – root, B – Leaf) and Cd translocation factor (C) in response to light treatments under Cd stress. It is clear that reduction in Cd translocation factor caused reduction in Cd accumulation in leaf.

Treatments

Mg

P

K

S

Ca

Wo

0.9 ± 0.3c 1.0 ± 0.3b 0.1 ± 0.1d 2.6 ± 0.5a 2.1 ± 0.1a 0.7 ± 0.3c 0.5 ± 0.2c 1.3 ± 0.1b 1.4 ± 0.3b 1.0 ± 0.1b 0.2 ± 0.1d 0.7 ± 0.3c 1.8 ± 0.3b 1.6 ± 0.3b 2.1 ± 0.4a 2.3 ± 0.6a 1.4 ± 0.8b 2.7 ± 0.2a

1.3 ± 0.4d 1.9 ± 0.4c 1.9 ± 0.4d 7.3 ± 0.7a 0.7 ± 0.4e 0.9 ± 0.3e 1.3 ± 0.4d 1.4 ± 0.7d 3.2 ± 0.5b 2.8 ± 0.2c 2.7 ± 0.8c 3.0 ± 0.6c 3.3 ± 0.3c 4.6 ± 0.4b 5.0 ± 0.6b 8.8 ± 0.9a 6.1 ± 0.8a 8.4 ± 3.0a

7.20 ± 2.0b 9.50 ± 0.7c 8.00 ± 1.5b 9.70 ± 1.6c 1.20 ± 0.6c 6.90 ± 1.9d 2.80 ± 0.9c 8.20 ± 2.2c 8.70 ± 0.1b 10.3 ± 1.2c 16.6 ± 5.0a 14.3 ± 0.4b 7.10 ± 2.0b 16.2 ± 3.0b 11.3 ± 1.1a 25.8 ± 1.5a 25.7 ± 0.6a 25.7 ± 0.8a

0.2 ± 0.1d 1.5 ± 0.0c 0.9 ± 0.1b 0.9 ± 0.3d 0.4 ± 0.2c 1.1 ± 0.4c 0.6 ± 0.2c 1.4 ± 0.3c 0.6 ± 0.2b 1.8 ± 0.1c 1.2 ± 0.3a 2.4 ± 0.4b 0.8 ± 0.3b 2.3 ± 0.4b 1.1 ± 0.1a 3.7 ± 0.1a 2.3 ± 0.4a 3.6 ± 0.6a

0.4 ± 0.2c 1.1 ± 0.1c 0.3 ± 0.1c 4.3 ± 0.4a 0.2 ± 0.1c 0.8 ± 0.1d 0.4 ± 0.2c 0.3 ± 0.2d 1.9 ± 0.5b 1.0 ± 0.3c 0.3 ± 0.2c 0.8 ± 0.3d 4.2 ± 0.7a 1.5 ± 0.0c 0.6 ± 0.3c 2.1 ± 0.5b 2.9 ± 0.7b 5.2 ± 0.5a

W25 W50 R0 R25 R50 B0 B25 B50

nicotinamide adenine dinucleotide phosphate glutathione reduced glutathione oxidized Fourier transform infrared spectroscopy sodium dodecyl sulfate–polyacrylamide gel electrophoresis matrix assisted laser desorption/ionization time of flight.

Acknowledgements

Table 2 Atomic percent of elements in response to Cd and light treatments.

Leaf Root Leaf Root Leaf Root Leaf Root Leaf Root Leaf Root Leaf Root Leaf Root Leaf Root

NADPH GSH GSSG FTIR SDS– PAGE MALDI TOF

Nb: W, R and B indicate white, red and blue lights and numerical associated with these letters indicate CdCl2 treatment. Alphabets along with quantity of elements indicate result of Duncan’s multiple range test for statistical means separation. Figure not showing a common letter are significantly differ from one another at the 5% error level where letters a, b, c and d represents first, second, third and fourth level of statistical significance respectively.

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