Hydrogen peroxide modulate photosynthesis and antioxidant systems in tomato (Solanum lycopersicum L.) plants under copper stress

Chemosphere 230 (2019) 544e558

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Hydrogen peroxide modulate photosynthesis and antioxidant systems in tomato (Solanum lycopersicum L.) plants under copper stress Faroza Nazir a, Anjuman Hussain a, Qazi Fariduddin a, * a

Plant Physiology and Biochemistry Section, Department of Botany, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India

h i g h l i g h t s
H2O2 promote photosynthesis in Solanum lycopersicum at 40 DAT under Cu stress (10 or 100 mg kg-1 soil).
H2O2 enhance leaf gas exchange parameters, activity of carbonic anhydrase, and protein content.
H2O2 promoted photosynthetic capacity by reducing the hydrogen peroxide and superoxide content and by enhancing the antioxidant enzyme activity.
H2O2 modify root morphology, morphology and movement of stomata.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 August 2018 Received in revised form 9 April 2019 Accepted 1 May 2019 Available online 9 May 2019

Plant growth and development could be modulated by minute concentrations of hydrogen peroxide (H2O2) which serves as a signaling molecule for various processes. The present work was conducted with an aim that H2O2 could also modify root morphology, morphology and movement of stomata, photosynthetic responses, activity of carbonic anhydrase, and antioxidant systems in tomato (Solanum lycopersicum L.) plants under copper stress (Cu; 10 or 100 mg kg-1 soil). Roots of 20 d old plants were dipped in 0.1 or 0.5 mM of H2O2 solution for 4 h and then transplanted to the soil filled in earthen pots. High Cu stress (100 mg kg-1 soil) altered root morphology, reduced chlorophyll content and photosynthetic capacity and also affected movement of stomata and generation of antioxidant species at 40 d after transplantation. Further, root dipping treatment of H2O2 to plants under stress and stress-free conditions enhanced accumulation of proline and activity of catalase, peroxidase, and superoxide dismutase, whereas production of superoxide radical (O
2) and H2O2 were decreased. Overall, H2O2 treatment improved growth, photosynthesis, metabolic state of the plants which provided tolerance and helped the plants to cope well under Cu stress. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: T Cutright Keywords: Antioxidant Copper Hydrogen peroxide Photosynthesis Tomato

1. Introduction Due to changing environmental scenario, heavy metal stress has resulted in considerable loss in agribusiness worldwide (Lin and Aarts, 2012; Feng et al., 2017). Copper is an indispensable chemical element and an elemental part of various proteins and enzymes which is required by plants to perform wide variety of essential metabolic activities, including photosynthesis, respiration, protein synthesis, cell wall lignifications and protection to oxidative stress (Burkhead et al., 2009; Marschner, 2012; Yruela, 2013; Scheiber et al., 2013; Pichhode and Nikhil, 2015). Copper also acts as a cofactor for a number of oxygen-processing enzymes such as Cu/Zn-

* Corresponding author. E-mail address: [email protected] (Q. Fariduddin). https://doi.org/10.1016/j.chemosphere.2019.05.001 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

superoxide dismutase (Cu/ZnSOD), cytochrome c oxidase, ascorbate oxidase, amino oxidase, laccase, plastocyanin (PC), and polyphenol oxidase, and assist in the proper functioning of these enzymes (Yruela, 2009). However, high concentration of Cu induces excessive production of reactive oxygen species, (ROS) causing damage to photosystem II (PSII), decreases chlorophyll contents, hamper the development of chloroplast and altered the composition of thylakoid membrane (Quartacci et al., 2000; Huang et al.,
nchez-Pardo et al., 2014). However, 2012; Dey et al., 2014; Sa several feats have been made to countermine Cu-induced toxicity (Zhou et al., 2016; Shi et al., 2017; Zhang et al., 2018) along with proper hormonal status in various parts of the plants. H2O2 being a versatile molecule is involved in the regulation of plant growth and development at lower concentration under desirable and stressful environments, including the acquirement of

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resistance (Cheeseman, 2007), cell wall strengthening (Slesak et al., 2007), photosynthesis (Gao et al., 2010; Khan et al., 2016), senescence (Peng et al., 2005), stomatal movement (Deng et al., 2012), cell growth and development (Hossain et al., 2015). Various studies have established involvement of H2O2 in signaling cascades and metabolism (Wojtyla et al., 2016), stimulates the expression and activation of stress tolerant genes for physiological adjustment and their capacity to endure continued subjection to environmental changes (Bhattacharjee, 2013; Sathiyaraj et al., 2014; Fariduddin et al., 2014; Reczek and Chandel, 2015; Saxena et al., 2016). Earlier studies have been carried out which established that an exogenous H2O2 improved growth response of plants. In Brassica juncea, H2O2 supply has been found to increase growth and photosynthetic capacity under HM stress (Khan et al., 2016). In Cucumis sativus, exogenous application of H2O2 promoted photosynthesis and antioxidant metabolism leading to an efficient reduction of the harmful effects of drought stress (Sun et al., 2016). Importantly, H2O2 pretreatment improved growth, water status and mineral ion content in Zea mays and elevated proline levels and thereby regulated antioxidant defense system and increased Cu stress tolerance (Guzel and Terzi, 2013). Most of the work related to the role of H2O2 has been performed on the leaves, however, mitigative role as priming of roots with H2O2 under Cu stress has not been addressed yet. Therefore, the present study was performed to establish how minute concentration of H2O2 sourced through the roots of Cu stressed plants modifies root morphology, modulate movement of stomata and photosynthetic traits, and reduced generation of toxic superoxide radicals in Solanum lycopersicum.

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(shotgun approach). For each pot, three plants were managed after thinning and the experiment followed a completely randomized block design and number of replicates for each treatment were five (n ¼ 5). At 20 d after transplantation (DAT), the plants were exposed to the varying doses of Cu in the form of CuSO4$5H2O (10 or 100 mg kg-1) applied through the soil. Plants were given irrigation with tap water when required in order to ensure healthy growth of the seedlings. At 40 DAT, plants were sampled to determine various growth and physiological traits as well as biochemical characteristics. The rest of the plants were left to maturity and were harvested at 160 DAT to explore yield traits. 2.5. Growth biomarkers Plants were uprooted carefully from the pots, washed to remove adhered soil. The root and shoot length of a plant was measured with the help of meter scale and expressed in cm. Fresh mass of root and shoot of each plant was calculated with the help of an electronic weighing machine. The plants were then placed in a hot air oven run at 80  C till constant weight was achieved and then their dry mass was recorded. Leaf area was measured using leaf area meter (LA 211, Systronics, New Delhi, India). 2.6. Physiological traits

2. Materials and methods

2.6.1. Determination of leaf electrolyte leakage (EL) and SPAD (soil and plant analysis development) value of chlorophyll Estimation of EL was done by following the method of Sullivan and Ross (1979). The electrolyte leakage was computed using the formula:

2.1. Plant material

Electrolyte leakage ð%Þ ¼ ½ðECb  ECaÞ= ðECcÞ  100

The healthy and consistent seeds of tomato (Solanum lycopersicum L.) cv. S-22 were subsequently washed and surface sterilized with 1% sodium hypochlorite solution for 10 min. Seeds were repeatedly washed with double distilled water (DDW) before sowing.

The SPAD value of chlorophyll was assessed in the fully expanded leaves of the plant with the help of SPAD chlorophyll meter (SPAD- 502; Konica, Minolta sensing, Inc., Japan).

2.2. Preparation of hydrogen peroxide solutions Hydrogen peroxide was bought from SigmaeAldrich Chemicals Pvt. Ltd. India. 100 mM stock solution of H2O2 was maintained by dissolving the appropriate quantity of 30% pure H2O2 in 100 mL volumetric flask and the final volume was kept up to the point by DDW. Required concentrations (0.1 or 0.5 mM) of H2O2 were prepared by diluting the stock solution. Surface-active agent, tween-20 was added into the solution before the application.

2.6.2. Determination of leaf gas exchange parameters Gas-exchange parameters were analysed on the third fully expanded leaves between 11:00 and 12:00 h with the help of an infrared gas analyzer (IRGA) portable photosynthetic system (LICOR 6400, LI-COR, and Lincoln, NE, USA). For measuring the net photosynthetic rate (PN) and its related variables [stomatal conductance (gs), transpiration rate (E) and internal CO2 concentration (Ci)], the air temperature, relative humidity, CO2 concentration and photosynthetic photon flux density (PPFD) were upheld at 25  C, 85%, 600 mmol mol-1 and 800 mmol mol-2 s-2, respectively.

2.3. Source of copper stress

2.7. Biochemical analysis

Cu in the form of copper sulphate (CuSO4$5H2O) was used as the point of supply of Cu stress at different doses of 10 or 100 mg kg-1 of soil.

2.7.1. Estimation of H2O2 content H2O2 content in leaves was estimated as per the protocol of Patterson et al. (1984). Plant material (0.5 g) were homogenized with acetone using pre-chilled mortar and pestle and centrifuged at 5000  g for 15 min. After centrifugation, 20% titanium chloride (prepared in conc. HCl), 17 M ammonia solution and 1 mL supernatant were taken in the test tube. The supernatant was discarded and precipitate was subsequently washed with acetone and then dissolved in 10 mL 2 N H2SO4. In order to remove undissolved substance, the reaction mixture was again centrifuged. The intensity of the colour of supernatant was read at 410 nm. The content of H2O2 was estimated using standard curve plotted with known concentration of H2O2 and was expressed as n mol g-1 FM.

2.4. Treatment pattern and experimental design In earthen pots, the surface sterilized seeds were sown to set up the nursery. At 20 d after sowing (DAS) the seedlings were transplanted to the earthen pots (25  25 cm). Each pot carrying three plants, upheld under similar conditions to that of nursery pots. A consistent fundamental dose of N, P, and K was supplied to the soil at the time of transplant. The 20 d old plants were uprooted and dipped in 0, 0.1, or 0.5 mM H2O2 for 4 h before transplantation

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2.7.2. Estimation of reactive oxygen species The level of generation of O2
e and H2O2 was determined adopting the histochemical staining protocol of Kaur et al. (2016) with slight alteration using nitro blue tetrazolium chloride (NBT) and 3, 30 -diaminobenzidine (DAB) respectively, to stain the leaves. Pictures were taken with a NIKON digital camera (COOLPIX110). 2.7.3. Assay for carbonic anhydrase (CA) and nitrate reductase (NR) activity The activity of CA in the leaves was determined as per the protocol proposed by Dwivedi and Randhawa (1974). The leaf samples were cut into small pieces and then taken into the test tubes containing cysteine hydrochloride solution. Enzyme activity was expressed on per gram fresh mass (FM) basis. Nitrate reductase activity was assayed according to Jaworski (1971). The fresh leaf tissues were cut into small fragments and transferred it into plastic vials, containing phosphate buffer (pH 7.5), KNO3 and isopropanol. The intensity of the pink colour generated after some time was read spectrophotometrically at 540 nm. 2.7.4. Estimation of total protein content and antioxidant enzymes For the estimation of total protein content and the activities of antioxidant enzymes, leaf tissues were homogenized with an extraction buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethanesulfonylflouride (PMSF), 0.5% (v/v) Triton X-100 and 2% (w/v) polyvinyl pyrollidone (PVP). The homogenates were centrifuged at 12,000 g for 20 min at 4  C. The supernatant generated after centrifugation was utilized for the estimation of antioxidant enzymes and proteins. 2.7.4.1. Total protein content. Total protein content was estimated by the protocol as proposed by Bradford (1976). Four mL of Bradford reagent was taken in test tubes containing 200 mL of supernatant and were mixed lightly and tremendously. Sample was incubated at 25  C for 5e10 min and then recorded the absorbance at 595 nm using spectrophotometer. 2.7.4.2. Antioxidant enzyme activity. Catalase activity was determined using the method of Aebi (1984) to assess the disappearance of H2O2 during the beginning of the reaction. The reaction mixture containing 50 mM phosphate buffer (pH 7.0), 15 mM H2O2 and 100 mL enzyme extract was prepared for the samples. The optical density was measured at 240 nm for 2 min. Peroxidase activity was measured by the method of Sanchez et al. (1995) with slight changes. The reaction mixture contained 50 mM phosphate buffer (pH 7.0), 20 mM guaiacol, 1.5 mM H2O2 and 100 mL enzyme extract. The activity was measured by recording the absorbance at 436 nm for 1 min at 25  C. The activity of superoxide dismutase (SOD) was estimated according to the method of Beauchamp and Fridovich (1971). The reaction mixture consisted of 50 mM phosphate buffer (pH 7.8), 9.9 mM L-methionine, 55 mM nitrobluetetrazolium (NBT), 2 mM EDTA, 0.02% Triton X-100, 40 mL enzyme extract and 1 mM riboflavin which is added at last in the reaction mixture. The absorbance was calculated at 560 nm for 2 min at 25  C. 2.7.5. Proline content Proline content in fresh leaves was estimated by the protocol as described by Bates et al. (1973). Sulphosalicylic acid was used as an extraction reagent for each sample and in each test tube, glacial acetic acid and ninhydrin solutions were added. Optical density was measured at 528 nm.

2.7.6. Lycopene content Tomato fruits (3e4 in number) were smashed firmly in a blender. The colourless residue was obtained by repeated extraction of 5e10 g of pulp with acetone using a blender and then all the residues were pooled gently and transferred to a separating funnel containing about 200 mL petroleum ether and mixed smoothly. The solution was then left to separate into distinct phases and the lower aqueous phase was re-extracted with additional 20 mL petroleum ether till the aqueous phase become colourless. After adding 10 g anhydrous sodium sulphate solution in a brown bottle, the washed petroleum ether extract containing carotenoids was drained out into the brown bottle and was kept at room temperature for 30 min. The petroleum ether extract was poured into a 100 mL volumetric flask through a funnel containing cotton wool. Suspended particles of sodium sulphate were washed several times with petroleum ether until it become colourless. Volume was made up to mark and absorbance was read at 503 nm against petroleum ether as blank using spectrophotometer. 2.7.7. b-carotene content 5e10 g of sample was homogenized to a fine pulp using the food chopper. 2e5 g of homogenized samples was added with 40 mL acetone and then placed on the high speed blender for 5 min, adding 60 mL hexane and 0.1 g MgCO3. The residue was washed twice with 25 mL of hexane. The extract was washed with 100 mL of water in five portions and the topmost layer was transferred to 100 mL volumetric flask carrying 9 mL acetone and diluted with hexane to make the final volume 100 mL. The solution was shaken properly and then allowed to settle. The lower layer of water was retained. The hexane fraction containing carotene was washed with the help of separating funnel with distilled water for about three to four times. The hexane fraction was then transferred to conical flask and to this small quantity of anhydrous sodium sulphate was added to remove water completely in the final solution. The optical density of the solution was read spectrophotometrically at 448 nm. 2.7.8. Ascorbic acid 5 mL of working standard solution, prepared from the stock standard solution was transferred to a 100 mL conical flask. This was followed by the addition of 10 mL of 4% oxalic acid. The mixture was quantified by its titration against the 100 mL of dye (2, 6dichloro phenol indophenols). Pink colour developed which persisted for a few min, as terminating point and the amount of the dye used corresponded to the amount of ascorbic acid. 0.5e5 g of the sample was extracted in sufficient amount of 4% oxalic acid and the final volume was made to 100 mL with DDW. The supernatant was collected by centrifugation and then 5 mL of the supernatant was taken in a test tube followed by addition of 10 mL of 4% oxalic acid. It was titrated against the 100 mL of dye (V2). 2.8. Stomatal studies and root morphology The external structure of root and stomata was studied by envisioning freeze-dried segments using JEOL JSM-JSM 6510 scanning electron microscope. The fresh leaves and roots were dissected into 2  2 mm fragments and then were anchored with 2% paraformaldehyde, 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer (pH 7.3) for 2 h. The fragments were then post firmed with 1% osmium oxide. Ultimately, samples were dehydrated with ethanol graded series (50%, 70%, 80%, 90%, and 100%). The samples were glazed with gold-palladium in a splatter coater instrument (JEOL JFC-1600). The images were taken at 15 KV voltage and a magnification of 4000 .

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2.9. Estimation of Cu content

generated by higher concentration of Cu (100 mg kg-1).

For estimation of Cu content, root and shoot from uniformly grown plants were taken and washed with deionized water followed by drying in hot air oven at 80  C for 48 h. The dried tissue was weighed, ground to fine powder and then digested with concentrated HNO3/HClO4 (3:1, v: v). The content of Cu was determined in terms of mg g1 dry mass (DM) using atomic absorption spectrophotometer (GBC, 932 plus; GBC Scientific Instruments, Braeside, Australia).

3.4. Leaf gas exchange parameters

2.10. Statistical analysis Data were statistically analysed as per the accepted protocol of Gomez and Gomez (1984). Standard error was computed and analysis of variance (ANOVA) was operated on the data to assess the least significance difference (LSD) of the treatments at the 5% level of probability. All of the data are the mean of five replicates.

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The plants raised from root dipping treatment with H2O2 (0.1 or 0.5 mM) had improved net photosynthetic rate (PN) and related parameters (gs, Ci, and E) in comparison with the control. However, application of H2O2 (0.1 mM) was most effective in enhancing PN by 35.72%, gs by 32.21%, Ci by 33.14% and E by 31.64% respectively, compared to respective control plants. The combined application of H2O2 (0.1 mM) as root dipping and Cu (10 mg kg-1) supplementation generated more prominent increase in PN by 38.27%, gs by 34.34%, Ci by 35.24% and E by 34% compared to the unstressed control plants (Fig. 2CeF). Moreover, the damage caused by a higher concentration of Cu (100 mg kg-1) for the above parameters was partially neutralized by the follow-up treatment with H2O2 (Fig. 2CeF). 3.5. Carbonic anhydrase (CA) and nitrate reductase (NR) activity

3. Results 3.1. Growth biomarkers The plants exhibited distinctive response to the applied Cu concentrations. The plants exposed to lower Cu dose (10 mg Cu kg-1 soil) showed subtle increase in the growth biomarkers (i.e. length, fresh and dry mass of shoot and root and leaf area), whereas the plants exposed to Cu (100 mg kg-1 soil) had significant reduction of 27.52% (shoot length), 31.88% (root length), 28.91% (shoot fresh mass), 29.48% (root fresh mass), 28.63% (shoot dry mass), 29.32% (root dry mass) (Fig. 1AeF) and 30.92% (leaf area) (Fig. 2A), respectively, compared to control. Nevertheless, the plants exposed to H2O2 (0.1 or 0.5 mM) as root dipping treatment alleviated Cuinduced growth inhibition compared to control plants. Interestingly, maximum increase in these growth parameters was noted from combined application of H2O2 (0.1 mM) as dipping plus Cu (10 mg kg-1 soil) supplementation, in comparison to control plants. Moreover, the stressed plants treated with H2O2 partially restored the deleterious effect generated by 100 mg kg-1 soil of Cu.

The plants grown in Cu stress (100 mg kg-1) had decreased CA and NR activities, compared to control plants. (Fig. 4BeC). Treatment of H2O2 (0.1 or 0.5 mM) as root dipping to plants surpassed the negative impact of Cu on the above characteristics compared to the control. However, application of H2O2 (0.1 mM) was more pronounced and increased the CA and NR activity by 34.21%, and 34.02%, respectively compared with the control. However, the combined treatment of H2O2 (0.1 mM) as root dipping plus Cu (10 mg kg-1) generated maximum increase in the activities of CA and NR which were 37.72%, and 36.28% more than the control. Moreover, the follow-up treatment of H2O2 partially overcame the toxicity generated by Cu stress (100 mg kg-1). 3.6. H2O2 content Treatment with Cu (100 mg kg-1) enhanced the accumulation of H2O2 by 15.59% compared to the control plants. Application of H2O2 (0.1 or 0.5 mM) reduced H2O2 content, while 0.1 mM was most effective in reducing it, when compared to stress treatments only (Fig. 3E).

3.2. Electrolyte leakage 3.7. Production of hydrogen peroxide and superoxide Cu stress significantly increased the electrolyte leakage compared with the control. However, application of H2O2 (0.1 or 0.5 mM) to non-stressed plants reduced electrolyte leakage, but more prominently in the plants treated with (0.1 mM) H2O2 by about 20.7% in comparison to control. Moreover, H2O2 treatment to Cu-stressed plants reduced the electrolyte leakage in comparison with the Cu treated plants (Fig. 5F). 3.3. SPAD value of chlorophyll and chlorophyll fluorescence i.e. maximum quantum yield of PSII (Fv/Fm) The plants raised in the presence of Cu (100 mg kg-1) had significant reduction in chlorophyll content and Fv/Fm and the values were decreased by 27.11% (Figs. 2B) and 27.39% (Fig. 4A) respectively, compared with the control. However, root dipping treatment of non-stressed plants treated with H2O2 (0.1 or 0.5 mM) increased both chlorophyll content as well as Fv/Fm, respectively compared to their respective controls, but the increase was more obvious with lower concentration of H2O2 (0.1 mM). The combined dose of H2O2 (0.1 mM) as dipping plus Cu (10 mg kg-1) supplementation was most effectual in increasing chlorophyll content by 39.64% and Fv/ Fm by 30.23% compared to control plants. Moreover, application of 0.1 mM H2O2 to Cu-stressed plants partially neutralized the loss

The level of generation of O2
e (Fig. 3b) was shown by blue staining of leaves and H2O2 (Fig. 3a) by brownish colour. In Cutreated leaf discs, staining spots were more pronounced as compared to the control. Moreover, maximum decrease in the accumulation of O2
e and H2O2 in the leaves were observed by H2O2 (0.1 or 0.5 mM) application at 40 DAT. 3.8. Proline content As shown in Fig. 5B, Cu stress increased the level of proline in comparison to control. Treatments with H2O2 (0.1 or 0.5 mM) under stress free conditions also increased the proline content, whereas, H2O2 (0.1 mM) triggered more promising response, and increased proline content by 35.28% compared to the control. In addition, the plants subjected to combined stress of Cu (100 mg kg-1) and H2O2 (0.1 mM) showed maximal increase in proline content compared to control (Fig. 5B). 3.9. Total protein content Plants subjected to Cu (100 mg kg-1) showed decreased protein content (Fig. 4D). Moreover, the plants treated with H2O2 (0.1 or

Fig. 1. Effect of hydrogen peroxide (H2O2) on the copper induced changes in the (A) shoot length, (B) root length, (C) shoot fresh mass, (D) root fresh mass, (E) shoot dry mass, and (F) root dry mass of Solanum lycopersicum L. plants at 40 DAT.

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0.5 mM) as root dipping or Cu (10 mg kg-1) supplementation or their combined application possessed higher total protein content than their respective controls. Moreover, increase in protein content (32.72%) was noted in the plants by the combined treatment of H2O2 (0.1 mM) and Cu (10 mg kg-1). The loss brought about by Cu (100 mg kg-1) was partially restored by the subsequent treatment of H2O2.

shoot, and the greatest reduction was noted with the treatment of H2O2 (0.1 mM) compared to control plants (Fig. 7b). However, H2O2 (0.1 mM) together with Cu (100 mg kg-1) slightly reduced Cu content in the root and the shoot in comparison to Cu (100 mg kg-1) stressed plants.

3.10. Activity of antioxidant enzymes

It was established in our present study that Cu at 100 mg kg-1 soil significantly reduced SPAD level, gas exchange parameters, PN and their related imputes. However, a slight improvement in the aforesaid parameters was observed at 10 mg of Cu kg-1 soil. The increase in Cu accumulation and corresponding increase in oxidative stress resulted in reduction of photosynthetic and growth characteristics. Reduction in PN could be attributed to the Cu inhibition of stomatal conductance and photosynthetic pigment content as also observed in Sorghum bicolour under Cu stress (Avinash, 2017). Decrease in photosynthesis could either be through inhibition of electron transporters or accelerated degradation of photosystem II (PS II) (Swarna et al., 2012; Sirhindi et al., 2015; Feigl et al., 2015; Bhardwaj et al., 2015). Cu also inhibits chlorophyll biosynthesis by its reaction with thiol groups of the enzymes of d-amino levulinic dehydrogenase and protochlorophyllide reductase complex (Thounaojam et al., 2012). It was however observed that treatment of H2O2 improved photosynthetic characteristics. H2O2 treatment improved gas exchange parameters, chlorophyll content and PN along with improved scavenging of ROS and recovery of photosynthetic efficiency. These results are further corroborated by the others (Fariduddin et al., 2014; Sun et al., 2016). The role of H2O2 on photosynthesis in alleviating the Cu stress could be specified to the accumulation of proline and antioxidants, protection of structure of chloroplast and lowering the activity of ROS (Ashfaque et al., 2014). In addition to this, H2O2 improves stability and integrity of the mitochondrial electron transport complex II and also RuBisCO activity under Ni stress and thereby prevented Ni-induced leaf chlorosis in Brassica juncea (Khan et al., 2016). Moreover, H2O2 treatment along with BRs improved chlorophyll content in Vigna radiata (Fariduddin et al., 2014) and in tomato cultivars (Orabi et al., 2015) by modulating endogenous plant hormones. The plants treated with low concentration of Cu (10 mg of Cu kg1 ) improved all the growth traits. But higher concentration (100 mg of Cu kg-1) triggered reduction in all the growth parameters. The probable reason for this decrease could mainly be due to changes in ion toxicity and mineral interruptions in plants which led to inhibition of cell division and cell elongation (Kopittke and Menzies, 2006; Feigl et al., 2015). Our results are in agreement with Karmous et al. (2015) and Mwamba et al. (2016) who also pointed out inhibition in growth traits in Phaseolus vulgaris and Brassica napus under Cu stress. Growth reduction in Cu-treated plants has been ascribed due to the higher acquisition of Cu and reductions in the nutrients accessibility which causes disruption in the functions of Cu-containing proteins and the cell metabolism needed to sustain plant life (Ali et al., 2015). Application of H2O2 to the Cu-stressed plants improved growth of plants. Earlier reports have indicated that H2O2 conferred protection against Cu toxicity in Zea mays (Guzel and Terzi, 2013) and Vigna radiata (Fariduddin et al., 2014). Moreover, the suppression of inhibitory effect of Cu on growth parameters was mitigated by the application of H2O2 or salicylic acid which contributed in the regulation of endogenous growth regulators like GA3, IAA, and ABA in Lycopersicon esculentum, thereby minimizing the oxidative stress (Orabi et al., 2015). In the present study, a significant increase in EL was observed in tomato plants under Cu stress. The increase in EL by Cu could be

Treatment of H2O2 (0.1 or 0.5 mM) as root dipping to stressed as well as stress-free plants upregulated the activities of CAT, POX, and SOD. The activity of these enzymes increased under Cu stress as compared to control plants. Application of H2O2 (0.1 or 0.5 mM) to the stress free plants also enhanced the activity of antioxidant enzymes compared to the control. More pronounced increase in the activity was noted when H2O2 (0.1 mM) and Cu (100 mg Cu kg-1 soil) were given in combination than their individual effects leading to increase in CAT activity by 68.76%, POX activity by 78.71% (Fig. 4EeF), and SOD activity by 67.08% (Fig. 5A), respectively compared to control plants. 3.11. Lycopene, b-carotene and ascorbic acid content In the absence of Cu, H2O2 (0.1 or 0.5 mM) as root dipping treatment increased the lycopene and b-carotene content in the fruits of plants, but 0.1 mM of H2O2 generated more pronounced effect and increased substantially the content by 26.21%, and 23.72%, in comparison to control (Fig. 5CeD). In contrast, Cu stress (100 mg kg-1) decreased lycopene and b-carotene content by 32.47%, and 29.43% in comparison with the control. Moreover, application of H2O2 (0.1 mM) plus Cu (10 mg kg-1) supplementation triggered the most favourable response that increased the lycopene and b-carotene content in the fruits by 30.17% and 25.87% over the control. Subsequent treatment of H2O2 (0.1 or 0.5 mM) to the stressed plants incompletely restored the detrimental effect of Cu stress. Cu grown plants showed greater ascorbic acid content, but the marked increase of 27.85% was observed under Cu stress (100 mg kg-1) compared with control. Application of H2O2 (0.1 or 0.5 mM) decreased the ascorbic acid content in stressed as well as in stress-free plants in comparison to control (Fig. 5E). 3.12. Compound microscopy and SEM imaging The exogenous application of H2O2 (0.1 or 0.5 mM) in the absence of Cu increased stomatal width aperture compared to the control, but 0.1 mM of H2O2 was more spectacular in widening the stomatal width aperture in comparison to the 0.5 mM H2O2 treated plants. The compound microscopy observations were further consolidated by SEM observations. Stomata appeared normal with specialized guard cells in leaf samples of control and H2O2 treated. In Cu stressed plants, closed stomata with deformed guard cells were found partially open in presence of H2O2 (Fig. 6). Scanning electron micrographs of the roots of control plants and plants exposed to 100 mg kg-1 Cu are presented in (Fig. 7a). The plants grown in the presence of Cu (100 mg kg-1) had distorted morphology of the roots, whereas, root morphology of the control plants were not affected. 3.13. Cu accumulation in root and shoot The concentration of Cu was found higher in the root as compared to the shoot. However, root dipping treatment of plants with H2O2 (0.1 or 0.5 mM) reduced Cu accumulation in root and

4. Discussion

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Fig. 2. Effect of hydrogen peroxide (H2O2) on the copper induced changes in the (A) leaf area, (B) SPAD Chlorophyll content, (C) net photosynthetic rate (PN), (D) stomatal conductance (gs), (E) transpiration rate (E), and (F) internal CO2 concentration (Ci) of Solanum lycopersicum L. plants at 40 DAT.

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H2O2 (0.5 mM)

Control

H2O2 (0.1 mM)

Cu (100 mg kg-1)

(a)

Control

H2O2 (0.1 mM)

H2O2 (0.5 mM)

Cu (100 mg kg-1)

(b) 0 Fig. 3. Determination of level of generation of hydrogen peroxide (H2O2) and superoxide ion (O. 2 ) by 3,3 - diaminobenzidine (DAB) (a) and nitro blue tetrazolium (NBT) (b) staining in the leaves of Solanum lycopersicum L. after dehydration at 40 DAT under control (A), 0.5 mM H2O2 (B), 0.1 mM H2O2 (C), and Cu (100 mg kg1) (D). (E) shows the effect of hydrogen peroxide (H2O2) on the copper induced changes in the hydrogen peroxide content in leaves Solanum lycopersicum L. at 40 DAT.

attributed to decrease in water status by affecting integrity of the water transporters in the plasma membrane and thus directly impaired transpiration rates of plant tissues (Prasad, 2004). Our results provided conclusive evidence that how H2O2 decreased the values for EL in stressed and non-stressed plants (Fig. 5F). In the present study, excess Cu supplied to tomato plants resulted in decreased protein content. It has been revealed that Cu irreversibly breakdown the disulfide bonds present in the proteins, thereby facilitate the interruption of their structure and functions

(Mouratao et al., 2009). Moreover, H2O2 as root dipping treatment increased soluble protein content in stressed as well as in stressfree plants (Fig. 4D). This resulted in the maintenance of protein structure and regulation of enzymes responsible for protein synthesis (Guzel and Terzi, 2013). Our results indicated that Cu treatment increased levels of H2O2 content in the tomato plants, but exogenously applied H2O2 reduced levels of ROS as revealed by lower level of O2
e and H2O2 in leaf (Fig. 3). Similarly, Hossain et al. (2015) found that the

Fig. 4. Effect of hydrogen peroxide (H2O2) on the copper induced changes in the (A) maximum quantum yield of PSII (Fv/Fm), (B) carbonic anhydrase (CA) activity, (C) nitrate reductase (NR) activity, (D) protein content (PC), (E) catalase (CAT) activity, and (F) peroxidase (POX) activity of Solanum lycopersicum L. plants at 40 DAT.

Fig. 5. Effect of hydrogen peroxide (H2O2) on the copper induced changes in the (A) superoxide dismutase (SOD) activity, (B) proline content, and (F) electrolyte leakage (EL) of Solanum lycopersicum L. plants at 40 DAT and in the (C) lycopene content, (D) b-carotene content, and (E) ascorbic acid content (AsA) of Solanum lycopersicum L. plants at 160 DAT.

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exogenously applied H2O2 was effective in minimizing O2
e and H2O2 content. Furthermore, production of H2O2 is rigorously associated to O2
e generation (Slesak et al., 2007). High levels of O2
e could result in reduction in the photosynthetic capacity of PSII and PSI, thereby affecting development and economy of plants (Bondarava et al., 2010; Zulfugarov et al., 2014; Takagi et al., 2016). Excess Cu caused elevated level of H2O2 in Brassica juncea, Lemna minor, Vigna radiata and Citrus plants (Fariduddin et al., 2014; Panda, 2008; Hippler et al., 2017; Wang et al., 2004). The increased content of H2O2 under Cu stress may also be attributed to the upregulation of a wide variety of limited substrate oxidases, and

Control

H2O2 (0.1 mM)

NAD(P)H oxidases leading to the imbalances in the electron transport chains of chloroplasts and mitochondria via the Mehler reaction (Halliwell and Gutteridge, 1999). Treatment of plants with Cu resulted in the higher activity of APX, SOD, and CAT that could have reduced H2O2 content. Depending on the plant species used and the concentration applied, the application of H2O2 has been found to either increase or decrease the oxidative stress and antioxidant system in plants. In Zea mays, H2O2 also decreased the H2O2 content under Cu stress (Guzel and Terzi, 2013). Our results provide a conclusive evidence that H2O2 reduced H2O2 content in the stressed plants and are consistent with earlier findings about H2O2-

H2O2 (0.5 mM

Cu (100 mg kg-1)

Control

H2O2 (0.5 mM)

H2O2 (0.1 mM)

Cu (100 mg kg-1)

F

(E)

H2O2 (0.1 mM) + Cu (100 mg kg-1)

H2O2 (0.1 mM)+ Cu (100 mg kg-1)

(a)

(b)

Fig. 6. Stomatal response of Solanum lycopersicum L. at 40 DAT under control (A), 0.5 mM H2O2 (B), 0.1 mM H2O2 (C), Cu (100 mg kg1) (D), and 0.1 mM H2O2 þ Cu 100 mg kg1 (E) at 40  using compound microscope (a) and 4000  using scanning microscope (b).

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Control

Cu (10 mg kg-1)

555

Control

Cu (100 mg kg-1)

(a)

(b) Fig. 7. (a) Root response of Solanum lycopersicum L. at 40 DAT under control (A,B), Cu 10 mg kg1, (C) and Cu (100 mg kg1) (D) at 4000  using scanning microscope. (b) Effect of hydrogen peroxide (H2O2) on the copper induced changes in the (A) root content (RC) and (B) shoot content (SC) of Solanum lycopersicum L. plants at 40 DAT.

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induced mitigation of stress due to HMs (Guzel and Terzi, 2013; Yildiz et al., 2013; Hasanuzzaman et al., 2017). In the present study, plants treated with Cu and H2O2 exhibited higher accumulation of proline leading to greater water and osmotic potential that could have reduced oxidative stress induced by Cu stress. Proline has been reported to mitigate oxidative stress by serving as potential ROS scavenger and osmoprotectant in plants (Rejeb et al., 2014). The influence of H2O2 on proline accumulation under Cu stress substantially protected photosynthetic capacity to counteract Cu-caused oxidative stress. Recently, Fariduddin et al. (2014) also revealed that increased accumulation of proline enhanced Cu stress tolerance in Vigna radiata by limiting H2O2 production. Furthermore, increase in proline accumulation by H2O2 treatment of Cu-stressed plants might be due to the stimulation of activity of genes of proline biosynthetic pathway (Li et al., 2013). Therefore, increase in proline content with H2O2 in stressed as well as in stress-free plants was obvious and could have improved osmotic potential that resulted in the higher water uptake and influenced photosynthesis positively through its protective action on photosynthetic machinery leading to higher efficiency of PS II and enhanced chlorophyll content that cumulatively protected photosynthetic capacity under Cu stress. The present work demonstrated that antioxidant defense system (CAT, POX and SOD) in plants was induced when subjected to Cu toxicity as well as when H2O2 was applied through roots. The more prominent increase in the activities of CAT, POX and SOD were noted in the plants subjected to high Cu stress along with root dipping treatment of H2O2. Nanda and Agrawal (2016) also have observed activation of antioxidant defense system in the presence of Cu and Zn stress in Cassia angustifolia. Study of Yusuf et al. (2016) also suggested that improved tolerance to salt and Al toxicity could be induced by upregulation of antioxidant system in wheat. Other workers also firmly believe that improved expression of antioxidant enzymes provided tolerance to plants against HM stress (Abogadallah, 2010). Therefore, our results could help to establish that H2O2 prevent the increase of oxidative stress and endogenous hydrogen peroxide content by increasing the activity of antioxidant enzymes (SOD, POX and CAT) and accumulation of proline thereby quenching ROS which retained the cellular osmotic adjustment and protected photosynthetic machinery from Cu stress by acting as oxygen radical scrapper. Furthermore, addition of H2O2 also improved the activity of SOD, POX and CAT, when mung bean plants were exposed to Cu stress (Fariduddin et al., 2014) or salt stress (Sathiyaraj et al., 2014). Guzel and Terzi (2013) have also observed the protective role of H2O2 in relation to Cu toxicity by upregulating the expression of antioxidant genes. The present study revealed that the presence of excess Cu (100 mg kg-1) significantly decreased the activities of CA and NR (Fig. 4BeC). This could be associated with the metabolic disruptions and also loss of plasma membrane integrity (Hopkins and Hüner, 2004) which in turn reduced the uptake of nitrate to the plant, the inducer and the substrate of NR (Campbell, 1999). Moreover, Khudsar et al. (2008) revealed a decline in enzyme activity or synthesis as a result of its strong attraction with the sulfohydryl groups at the active site of the enzyme leading to a reduction in the flux of NO 3 to the site of enzyme action. It has also been reported that NR activity moderate various physiological functions, and activates specific photosynthates that could be essential for the maintenance of photosynthetic machinery. H2O2 treatment through roots significantly improved activity of NR in stressed and stress-free plants and this is in consistent with the results of Khripach et al. (1999). They suggested that H2O2 has the capability to increase activity of NR under stress conditions through expression of the specific genes related to enzyme biosynthesis. Other specific reason speculated to be the ability of H2O2 in increasing the

transcript level of NR, thereby upregulate the activity of substances including that of nitrate which has the capability of activating specific genes of NR (Campbell, 1999). Our findings further supported by the study of Fariduddin et al. (2014). It is believed that excess Cu leads to reduction in partial pressure of CO2 in the stroma and therefore, causes the stomatal closure resulting in gradual decline in CA activity (Bethke and Drew, 1992), which is in accordance with others (Azooz et al., 2012; Fariduddin et al., 2014). However, application of H2O2 to the Cu-stressed plants improved the activity of CA by promoting CO2 assimilation (Jannat et al., 2011; Gondim et al., 2013) which is also supported by increased level of Ci and gs in the present study. In addition to this, Borisova et al. (2012) suggested that enzyme CA plays a key role in maintaining the photosynthetic electron transport chain. Cu treatment also altered stomatal responses. In our study, Cu treated plants had comparatively closed stomata than the control plants (Fig. 6). However, application of H2O2 restored the normal stomata in Cu stressed plants. Moreover, the treatment of H2O2 conspicuously reduced the damage caused by Cu on stomatal width aperture. The results were further confirmed by the study executed by scanning electron microscopy shown in Fig. 6b. The treatment with H2O2 highly influenced GSH content by regulating the production of GSH that resulted in stomatal opening. Clark et al. (2011) revealed the involvement of H2O2 in the signal transduction mechanisms during the light-dark transition leading to stomatal opening. Moreover, the study of Xia et al. (2014) reported the involvement of H2O2 on stomatal response in Solanum lycopersicum, and suggested that a low concentration of H2O2 promotes stomatal opening, whereas a high concentration of H2O2 promotes stomatal closure. Similar response is also reported by Hao et al. (2012). High Cu-fed plants developed fruits having lower lycopene and b-carotene contents (Fig. 5CeD). In the present study, decreased lycopene and b-carotene contents was the consequence of Cu interference with the vital metabolic processes such as reduction in
ndez, 2010), chlorophyll and carotenoid contents (Astorga and Mele increasing the activity of chlorophyll degrading enzyme: cholorophyyllase or inhibiting the formation of 5-aminolaevulinic acid, a precursor of chlorophyll (Santos, 2004; Doganlar et al., 2010). However, root dipping treatment of H2O2 enhanced the b-carotene and lycopene contents in tomato, and this increase was related to differentiation of chloroplast into chromoplast, a developmental process accompanied by H2O2 and also the genes for transcription and translation have been found to be up-regulated (Mehta et al., 2002). In the present study treatment of H2O2 through roots to the stressed or non-stressed plants significantly reduced the ascorbic acid content in the fruits. Similar responses were reported by findings of Ali et al. (2006). The plants grown in the presence of Cu had significantly higher Cu accumulation (Fig. 7b). The acquisition of Cu was found markedly higher in root followed by shoot. It is considered that the restriction of HM translocation from root to shoot could be a possible mechanism that reinforced the plants to riposte damage caused by Cu toxicity (Adrees et al., 2015; Rizwan et al., 2016). These results are in close conformity with the results reported by Keller et al. (2014); Mostofa et al. (2015) and Ali et al. (2016). The accumulation of less Cu in shoot resulted in lesser the production of ROS and oxidative damage. Application H2O2 decreased Cu accumulation in root and shoot of plants under both in stress free and Cu stressed conditions. 5. Conclusion High concentration (100 mg kg-1) of copper supplied to Solanum

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lycopersicum through the soil reduced the growth, distorted the root morphology and altered the stomatal response and further reduced photosynthetic traits. H2O2 sourced through the roots ameliorated Cu-inflicted damage and restored normal morphology of roots and stomata of tomato plants. Moreover, root sourced H2O2 triggered up-regulation of antioxidant defense system and accumulation of proline which conferred tolerance to plants. H2O2mediated Cu tolerant mechanism and the signaling pathways could be further dissected in detail and how H2O2 modulate other phytohormones to activate defense systems to mitigate Cu stress. Conflicts of interest The authors declared that no competing interests exist. Acknowledgements We acknowledge support by UGC New Delhi India as Non Net fellowship at Aligarh Muslim University. References Abogadallah, G.M., 2010. Antioxidative defense under salt stress. Plant Signal. Behav. 5, 369e374. Adrees, M., Ali, S., Rizwan, M., Ibrahim, M., Abbas, F., Farid, M., Zia-ur-Rehman, M., Irshad, M.K., Bharwana, S.A., 2015. The effect of excess copper on growth and physiology of important food crops: a review. Environ. Sci. Pollut. Res. 22, 8148e8162. Aebi, H., 1984. Catalase in vitro. Methods in Enzymology. Academic Press, pp. 121e126. Ali, B., Hayat, S., Hasan, S.A., Ahmad, A., 2006. Effect of root applied 28homobrassinolide on the performance of Lycopersicon esculentum. Sci. Hortic. 110, 267e273. Ali, S., Shahbaz, M., Shahzad, A.N., Khan, H.A., Anees, M., Haider, M.S., Fatima, A., 2015. Impact of copper toxicity on stone-head cabbage (Brassica oleracea var. capitata) in hydroponics. Peer J. 3, 1119. Ali, S., Rizwan, M., Ullah, N., Bharwana, S.A., Waseem, M., Farooq, M.A., Abbasi, G.H., Farid, M., 2016. Physiological and biochemical mechanisms of silicon-induced copper stress tolerance in cotton (Gossypium hirsutum L.). Acta Physiol. Plant. 38, 262. Ashfaque, F., Khan, M.I.R., Khan, N.A., 2014. Exogenously applied H2O2 promotes proline accumulation, water relations, photosynthetic efficiency and growth of wheat (Triticum aestivum L.) under salt stress. Ann. Res. Rev. Biol. 4, 105e120.
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