S (13) Reference: HAZMAT 15178

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1 Title: Alleviation of cadmium toxicity in Medicago sativa by hydrogen-rich water Author: Weiti Cui Cunyi Gao Peng Fang Guoqing Lin Wenbiao Shen PII: S (13) DOI: Reference: HAZMAT To appear in: Journal of Hazardous Materials Received date: Revised date: Accepted date: Please cite this article as: W. Cui, C. Gao, P. Fang, G. Lin, W. Shen, Alleviation of cadmium toxicity in Medicago sativa by hydrogen-rich water, Journal of Hazardous Materials (2013), This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 1 Running title: H 2 -induced alleviation of Cd toxicity Title: Alleviation of cadmium toxicity in Medicago sativa by hydrogen-rich water Weiti Cui a, Cunyi Gao a, Peng Fang a, Guoqing Lin b, Wenbiao Shen a,* a College of Life Sciences, Nanjing Agricultural University, Nanjing , China b Laboratory Center of Life Sciences, Co. Laboratory of Nanjing Agricultural University and Carl Zeiss Far East, Nanjing Agricultural University, Nanjing , China * Corresponding author. Tel.: ; fax: address: wbshenh@njau.edu.cn (W.B. Shen). Word count: 5000 words. Abstract (199 words); Introduction (524 words); Materials and methods (980 words); Results (1316 words); Discussion (1000 words); Conclusion (93 words); Acknowledgment (24 words);table legend (75 words); Figure legends (789 words). Number of table and figures: table and 7 figures, including four color figures in Fig. 2, 3, 4 and 6 (If accepted, we prefer color only on the web, but not in the print form); 1 supplementary data, including 1 table. 1 Page 1 of 49

3 23 AB S T R A C T Hydrogen gas (H 2 ) induces plant tolerance to several abiotic stresses, including salinity and paraquat exposure. However, the role of H 2 in cadmium (Cd)-induced stress amelioration is largely unknown. Here, pretreatment with hydrogen-rich water (HRW) was used to characterize physiological roles and molecular mechanisms of H 2 in the alleviation of Cd toxicity in alfalfa plants. Our results showed that the addition of HRW at 10% saturation significantly decreased contents of thiobarbituric acid reactive substances (TBARS) caused by Cd, and inhibited the appearance of Cd toxicity symptoms, including the improvement of root elongation and seedling growth. These responses were related to a significant increase in the total or isozymatic activities of representative antioxidant enzymes, or their corresponding transcripts. In vivo imaging of reactive oxygen species (ROS), and the detection of lipid peroxidation and the loss of plasma membrane integrity provided further evidence for the ability of HRW to improve Cd tolerance significantly, which was consistent with a significant enhancement of the ratio of reduced/oxidized (homo)glutathione ((h)gsh). Additionally, plants pretreated with HRW accumulated less amounts of Cd. Together, this study suggested that the usage of HRW could be an effective approach for Cd detoxification and could be explored in agricultural production systems. 2 Page 2 of 49

4 42 43 Keywords: Alfalfa (Medicago sativa L.) roots Cadmium toxicity Glutathione homeostasis Hydrogen gas Oxidative stress 3 Page 3 of 49

5 48 1. Introduction Cadmium (Cd), one of the toxic heavy metals, has become a major pollutant due mainly to natural and diverse anthropogenic activities. Cd is readily accumulated by higher plants, thus leading to severe inhibition of plant photosynthetic, respiratory and nitrogen metabolisms. Subsequently, visible symptoms of Cd toxicity, including plant growth inhibition, chlorosis, necrosis or programmed cell death (PCD), and even cell death are observed [1]. If Cd is allowed to accumulate in crop plants, toxic Cd poses a severe threat to human health through food chains [2,3]. A close link between Cd toxicity and oxidative stress in plants reveals that Cd toxicity is, at least partially, caused by stimulated generation of reactive oxygen species (ROS), which is able to modify the antioxidant defence and elicit oxidative stress [4 6]. The antioxidant network consists of enzymatic system and non-enzymatic components. Enzymatic system including superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX), etc, are predominantly associated with the maintenance of cellular redox steady state in plant cells upon Cd stress [7]. Similarly, of the various detoxification processes activated in plant cells upon Cd stress, complexing of Cd by phytochelatins and compartmentalization in vacuoles, etc, plays very important roles [8,9]. Most importantly, maintenance of the glutathione (GSH) pool plays a central role in above processes and therefore is important for plant tolerance to heavy metals [10,11]. For example, cell imaging by using fluorescent probes of Cd-treated alfalfa 4 Page 4 of 49

6 70 71 plants confirmed that accumulation of peroxides and depletion of GSH and homoglutathione (hgsh; the glycine of GSH is substituted by alanine), could cause redox imbalance, and even cell necrosis [12]. Recent studies revealed that hydrogen gas (H 2 ) is a vital physiological regulatory molecule with antioxidant, anti-inflammatory and antiapoptotic protective effects on animal cells and organs [13]. Although the primary molecular target(s) of H 2 is not fully understood, the main mechanism of H 2 s action might be related to the preferential scavenging of hydroxyl and peroxynitrite radicals, thereby reducing the oxidative damage to both membrane lipids and DNA [14]. The metabolism of H 2 by bacteria, green algae and higher plants has also been reported for many years [15,16]. Subsequent literatures show that this gas can affect seed germination, and improve the growth performance of crops [17 19]. More recently, there has been a renewed interest in the effect that H 2 has on plant physiology, especially in the stressed conditions. For example, our results revealed that H 2 could act as an important gaseous molecule with multiple biological functions in plant responses against salinity and paraquat exposure in rice, Arabidopsis and alfalfa plants [20 22]. However, little information is known about whether and how H 2 modulate the Cd-induced toxicity in the roots of alfalfa. To investigate physiological roles of H 2, in this context, we investigated some physiological and biochemical events induced by the pretreatment of hydrogen-rich water (HRW), followed by Cd stress in alfalfa seedlings. Therefore, our results support the idea that H 2 reduces Cd uptake, suppresses ROS production and alleviates 5 Page 5 of 49

7 92 93 Cd-induced oxidative stress in plants by re-establishing redox homeostasis. These results advocate a positive role for HRW in reducing pollutant residues for food safety in the fields. 6 Page 6 of 49

8 95 2. Materials and methods Plant material and growth conditions Alfalfa (Medicago sativa L. cv. Victoria) seeds were surface-sterilized with 5% NaClO for 10 min, and rinsed extensively in distilled water then germinated for 1 d at 25 C in the darkness. Uniform seedlings were selected and transferred to the plastic chambers and cultured in quarter-strength Hoagland s solution. Seedlings were grown in the illuminating incubator (14 h light with a light intensity of 200 μmol m -2 s -1, 25±1 C, and 10 h dark, 23±1 C). 5-day-old seedlings were incubated in different pretreatment and treatment solutions as described in the corresponding figure or table legends. The sample without chemicals was the control (Con). The ph for both nutrient medium and treatment solutions was adjusted to 6.0. After various treatments, plants were photographed and root tissues were sampled for used immediately or flash-frozen in liquid nitrogen, and stored at 80 C for further analysis Preparation of hydrogen-rich water (HRW) Purified hydrogen gas (99.99%, v/v) generated from a hydrogen gas generator (SHC-300; Saikesaisi Hydrogen Energy Co., Ltd., Shandong, China) was bubbled into 1000 ml quarter-strength Hoagland s solution (ph 6.0, 25 C) at a rate of 150 ml min -1 for 60 min [20]. Then, the corresponding HRW was immediately diluted to the required concentrations [1, 10 and 50% concentration, (v/v)]. The H 2 concentration in freshly prepared HRW analysed by gas chromatography (GC) [23] was 0.22 mm, and 7 Page 7 of 49

9 117 maintained at a relative constant level in 25 C for at least 12 h Determination of root growth and thiobarbituric acid reactive substances (TBARS) contents Alfalfa seedlings were photographed after the beginning of pretreatment, 24, 48 and 72 h of various treatments, then the root length was measured with IMAGE J (available at [24]. Three replicated experiments were analyzed (n=12). Lipid peroxidation was estimated by measuring the amount of TBARS [25] DNA extraction and gel electrophoresis DNA extraction by the cetyltrimethylammonium bromide (CTAB) method was used [24]. For analysis of DNA laddering, after incubation with RNase at 37 C for 30 min, equal amounts of DNA samples were used for electrophoresis. After run on 2% agarose gel and stained with ethidium bromide, the gel was photographed under a UV light box Fluorescence microscopy and laser scanning confocal microscopy assays The cell viability was determined by fluorescein diacetate-propidium iodide (FDA-PI, FDA from ICN Biomedicals, Inc., and PI from Sigma) double fluorescent assay [26]. Root samples were loaded with FDA-PI double assay (20 μg ml -1 FDA and 1 μg ml -1 PI) for 10 min and washed 3 times with distilled water for 5 min. Red 8 Page 8 of 49

10 and green fluorescence and concurrent differential interference contrast images were obtained with a fluorescent microscope (Axio Imager A1, Carl Zeiss, Germany) The 4,6-diamidino-2-phenylindole (DAPI, Roche) assay was based on the method described previously [27,28]. In brief, root samples were stained using DAPI at 0.5 μg ml -1 for 10 min after fixed with 4% paraformaldehyde (ph 7.4) for 3 h, then washed twice with distilled water. Fluorescence was imaged with a Zeiss LSM 710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany) Histochemical analyses Reactive oxygen species (ROS) production was detected by 3,3 -diaminobenzidine tetrahydrochloride (DAB) staining [29]. Histochemical detection of lipid peroxidation and loss of plasma membrane integrity in root apexes were performed with Schiff's reagent [30] and Evans blue [31], respectively. Afterwards, all the roots were washed extensively, then observed under a light microscope (model Stemi 2000-C; Carl Zeiss, Germany) and photographed on colour film (Powershot A620, Canon Photo Film, Japan) Assay of enzyme activity Lipoxygenase (LOX) activity was analyzed as described previously [32]. Activities of superoxide dismutase (SOD) and guaiacol peroxidase (POD) were analyzed by the former methods [33,34]. Ascorbate peroxidase (APX) activity was measured as described by Nakano and Asada [35]. Glutathione peroxidase (GPX) activity was 9 Page 9 of 49

11 measured as described previously [36]. Protein content was determined by the method of Bradford [37] Gel electrophoresis The isozymes of SOD, POD and APX were separated on discontinuous polyacrylamide gels (stacking gel 5% and separating gel 12%) under non-denaturing conditions. For each lane, 30 μg of protein extract was applied. SOD, POD and APX isozymatic activities on the gel were visualized by activity staining [20,38]. For the determination of the relative activity of different isozymes, gels were scanned and the intensity of bands was calculated by using the Quantity One v4.4.0 software (Bio-Rad, Hercules, California, USA), and the band intensities of the individual isozymes were expressed as % of the control values Real-time quantitative RT-PCR analysis Real-time quantitative RT-PCR reactions were performed using a Mastercycler ep realplex real-time PCR system (Eppendorf, Hamburg, Germany) with SYBR Premix Ex Taq TM (TaKaRa Bio Inc., Dalian, China) according to the user manual. The cdna was amplified using specific primers (Supplementary Table 1). The expression levels of corresponding genes are presented as values relative to the corresponding control samples under the indicated conditions, with normalization of data to the geometric average of two internal control genes MSC27 and Actin2 [39] Page 10 of 49

12 Determination of (h)gsh and (h)gssg(h) contents (h)gsh (GSH+hGSH) and (h)gssg(h) (GSSG+hGSSGh) contents were measured by the 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB)-glutathione reductase (GR) recycling assay [25,40]. Frozen root tissues were homogenized in cold 5% 5-sulfosalicylic acid. The homogenate was centrifuged at 12,000 g for 20 min at 4 C and the supernatant was collected. Total glutathione ((h)gsh plus (h)gssg(h)) was determined in the homogenates spectrophotometrically at 412 nm, using GR, DTNB, and NADPH. (h)gssg(h) was determined by the same method in the presence of 2-vinylpyridine and (h)gsh content was calculated from the difference between total glutathione and (h)gssg(h) Determination of Cd content in plant tissues The concentration of Cd element was measured by an Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES, Perkin Elmer Optima 2100DV) Statistical analysis Values are means ± SE of three independent experiments with at least three replicates for each. Differences among treatments were analysed by one-way ANOVA, taking P < 0.05 as significant according to Duncan s multiple range test Page 11 of 49

13 Results Cd toxicity is dose-dependently influenced by HRW pretreatment Exposure to Cd was observed to decrease the biomass accumulation in terms of fresh weight of ten seedling roots significantly when compared with the untreated control (Fig. 1A). Further experiments showed that pretreatments with different concentrations of HRW (1, 10, 50 and 100%) brought about the improvement of the fresh weight by 4.4, 20.1, 16.8, and 11.1% in root tissues, with respect to stressed alone plants. A maximal inducible response was observed in 10% HRW-pretreated plants. However, no significant difference was found between 10% HRW applied alone sample with respect to the Cd-free control. To further assess the Cd toxicity to the roots of alfalfa, the oxidative damage to membranes was investigated by measuring the contents of thiobarbituric acid reactive substances (TBARS), an important indicator of lipid peroxidation and free radical generation. Cd stress alone caused 81.2% increase in TBARS content compared with the control sample. However, compared with the Cd treatment alone, the addition of HRW produced a similar dose-dependent reduction in the amount of TBARS as was found for seedling root growth (Fig. 1A and B). Similarly, the optimal reduction was observed at 10% HRW pretreatment. A slight but no significant reduction in TBARS content was observed in 10% HRW-pretreated alone sample, with respect to the Cd-free control sample. To confirm the regulatory roles of H 2 at a molecular level, we further detected DNA 12 Page 12 of 49

14 laddering, a hallmark of cellular PCD in plant cells when exposed to heavy metal, which is frequently used to monitor molecular injury caused by Cd stress Interestingly, Cd-induced DNA degradation could be partially attenuated by the pretreatment with 10% HRW. Comparatively, use of other concentrations of HRW did not exhibit such visible effective in ameliorating DNA laddering. Similarly, by using fluorescein diacetate-propidium iodide (FDA-PI) double fluorescent assay and 4,6-diamidino-2-phenylindole (DAPI) staining, we observed that, pretreatment with 10% concentration of HRW could partially block the Cd-induced increases of dead cells (red fluorescence; Fig. 2A). Meanwhile, nuclei of Cd-treated roots showed increases in the degree of disorganization, and displayed more chromatin condensation. However, further investigation showed that the above phenomena were differentially alleviated by HRW pretreatment (Fig. 2B). Therefore, we used 10% concentration of HRW to investigate the role of H 2 in the regulation of Cd tolerance HRW improves root growth and suppresses Cd uptake Similar ameliorating responses of H 2 against the Cd-induced growth inhibition were also observed, as evaluated by morphology and root elongation (Fig. 3A and B). For example, the Cd-induced inhibition of root elongation became more distinct with time, such that root elongation was reduced from 1.24±0.12 cm to 0.52±0.05 cm after d incubation in CdCl 2. The reduction in root elongation was significantly recovered when roots were preincubated in 10% HRW, especially in the 3 d of Cd treatment. These results clearly revealed that HRW attenuated the Cd-induced inhibition of root 13 Page 13 of 49

15 growth. As expected, Cd treatment alone brought about a rapid uptake of Cd in the root tissues during the initial 3 d. However, the pretreatment of 10% HRW significantly slowed down the accumulation rate of Cd. For instance, at 1 and 3 d of treatments, the Cd content in HRW-pretreated root tissues was 29.4 and 22.9% lower than that of Cd-stressed alone sample (Fig. 3C) Cd-induced ROS production and oxidative stress are decreased by HRW Results of DAB staining showed that root tissues exhibited marked brown coloration after Cd treatment, suggesting more ROS accumulation compared to Cd-free control samples (Fig. 4A). Comparatively, those pretreated with HRW followed by Cd treatment had only slight staining. Evaluation of lipid peroxidation and the loss of plasma membrane integrity in alfalfa seedling roots were performed by histochemical staining with Schiff s reagent and Evans blue. Similarly, the roots treated with Cd alone were stained extensively, but those pretreated with HRW had only slight staining (Fig. 4B and C), both of which were consistent with the changes in activities of lipoxygenase (LOX) (Fig. 4D), which is partially contributed to the increase of oxidation products formation in plant cells. These results further indicated that H 2 prevents the root cells against Cd-induced 265 oxidative stress HRW activates antioxidant enzyme activities in roots exposed to Cd 14 Page 14 of 49

16 Cd-induced oxidative stress in roots was associated with ROS production. Therefore, it was necessary to investigate the antioxidant enzymes responsible for ROS scavenging. Further results revealed that there was a significant decrease in total activities of antioxidant enzymes (SOD, APX and GPX) in alfalfa seedling roots exposed to Cd treatment, being 36.1, 24.3 and 28.3% lower than the Cd-free control samples, respectively (Fig. 5A, C and D). By contrast, when HRW was pretreated, it significantly alleviated the effects of Cd on SOD, APX and GPX activities, being 60.2, 25.1 and 25.5% higher, respectively, than those with Cd treatment alone. In contrast to SOD, APX and GPX, total activity of POD was increased in the presence of Cd (Fig. 5B). However, pretreatment with HRW could significantly aggravate the increasing tendency, being 12.0% higher than Cd-stressed alone samples. To confirm total activities of above antioxidant enzymes, a native PAGE analysis for SOD, POD and APX isozymatic activities was performed. At least four SOD isozymes were detected in alfalfa roots (Fig. 6A and B). Among these, only the SOD-I isozymes was Mn-SOD (as confirmed by the inhibitor test, data not shown; located in the mitochondrial and peroxisome); and the rest of the isozymes belonged to the Cu,Zn-SODs (located in the cytosol). In response to Cd exposure, isozymes I-IV showed obvious decreased activities; whereas, those with HRW pretreatment showed increased activities, especially isozymes I, III, and IV. At least six isozymes of POD were detected (Fig. 6C and D). All isozymes of POD in root tissues with Cd showed increasing activities. Pretreatment with HRW increased all isozymes except isozyme IV. Testing another H 2 O 2 -scavenger enzyme, APX, seven bands of isozymes could be 15 Page 15 of 49

17 detected, and the APX-VI isozyme contributed the most activity (Fig. 6E and F). Treatment with Cd generally resulted in decreases in all band size of APX isozymes However, the decreases in the amount of APX isozymes could be differentially reversed by the pretreatment with HRW. Thus, we also observed that the patterns of different isozymes (Fig. 6) were similar to those of in-tube assay (Fig. 5) Transcript levels of antioxidant enzyme genes Cd treatment resulted in the significant decreases in the transcripts levels of corresponding antioxidant genes, such as Cu,Zn-SOD, Mn-SOD, APX1/2, and GPX in root tissues of alfalfa plants (Fig. 7A, C and D). However, pretreatment with HRW significantly blocked the decreases in the transcripts levels. Compared with the Cd-free control samples, Cd caused increases in the transcription level of POD, and this increase was substantially strengthened by pretreatment with HRW (Fig. 7B). We also noticed that HRW applied alone differentially increased antioxidant gene transcripts, in comparison with those in the Cd-free control samples Re-establishing glutathione homeostasis As expected, treatment with Cd induced an obvious decrease in content of (h)gsh and an increase of (h)gssg(h) in roots (Table 1). By contrast, the HRW pretreatment significantly eliminated the effects of Cd treatment alone on (h)gsh and (h)gssg(h) contents. Meanwhile, a higher ratio of (h)gsh/(h)gssg(h), an important parameter for the intracellular redox status, was observed in the HRW pretreatment followed by 16 Page 16 of 49

18 Cd exposure, with respect to the solely Cd-treated sample. On the other hand, HRW applied alone did not significantly affect (h)gssg(h) content, but increased (h)gsh concentration in root tissues, with respect to the Cd-free control samples. Furthermore, the expression of genes involved in the synthesis and metabolism of (h)gsh and (h)gssg(h), were investigated. Results of Fig. 7E-H revealed that pretreatment with HRW significantly enhanced the Cd-induced γ-glutamylcysteine synthetase (ECS), glutathione synthetase (GS) and glutathione reductase1/2 (GR1/2) gene expression, and significantly blocked the Cd-induced decrease of homoglutathione synthetase (hgs) transcription. We also noticed that, a pretreatment with HRW alone brought about a slight but not significant increase in above genes expression. 17 Page 17 of 49

19 Discussion H 2 is a colourless, odourless, tasteless and flammable gas. However, recent results in animals revealed that H 2 is a potent anti-oxidative, anti-apoptotic and anti-inflammatory agent and therefore has potential medical application [13]. Although several reports discovered H 2 evolution and uptake in illuminated leaves and germinating seeds in fifty years ago [15 17], little information was known about the exact mechanism of its biosynthesis and even physiological roles of endogenous or exogenous H 2 in plants, and hydrogenase and nitrogenase were merely thought of as potential sources of H 2 releasing in plant cells [15,18,19]. Despite these unsolved problems, the methodology using exogenous HRW provides a useful research tool to investigate the specific physiological role(s) of H 2 in plants. Ample evidence has confirmed that the use of HRW in clinical trials is a clearly more convenient method for the delivery of molecular hydrogen since HRW can be made relatively easily and safely [13,14,41]. In this study, we analyzed the potential ameliorative role of HRW, against Cd stress in alfalfa seedlings by evaluating its performance on the major symptoms of Cd toxicity, including the inhibition of seedling growth, enhancement of oxidative stress and Cd uptake. Our results clearly demonstrated that pretreatment of alfalfa seedlings with 10% HRW for 12 h attenuated Cd-induced seedling growth inhibition (Figs. 1A, 3A and B) and Cd accumulation (Fig. 3C) in root tissues. Meanwhile, re-establishment of redox homeostasis is involved in HRW-mediated cytoprotective roles (Figs. 4-7, Table 1). 18 Page 18 of 49

20 These findings are consistent with H 2 -indcued plant tolerance to various abiotic stresses, including salinity and paraquat exposure [20 22]. At least several explanations may account for the positive effect of HRW on Cd-challenged alfalfa seedlings and are discussed in the following. First, overproduction of ROS and subsequent oxidative stress is the mechanisms of phytotoxicity of Cd, although production of ROS is tightly controlled at a very low level under the normal growth conditions [7]. Recently, several endogenous gaseous molecules (e.g. nitric oxide, carbon monoxide, hydrogen sulfide) are showed to modulate Cd-induced oxidative stress [25,42]. In this context, our results illustrated that, besides providing a significant promotion of plant growth as compared with Cd-treated samples (Figs. 1A, 3A and B), pretreated alfalfa seedlings with 10% HRW exhibited obvious decreases of Cd-induced ROS overproduction and alleviation of oxidative stress. These conclusions were supported by up-regulation and/or activation of representative antioxidant enzymes, including SOD, POD, APX, and GPX total and/or isozymatic activities (Figs. 5 and 6), or corresponding transcripts (Fig. 7A-D), all of which are correlated with plant heavy metal tolerance [7]. Therefore, increased antioxidant enzyme activities led to partially preventing oxidative damage to membranes, evaluated as TBARS formation (Fig. 1B) and LOX activity (Fig. 4D) in alfalfa seedling root tissues upon Cd exposure. Such effects were confirmed by histochemical staining of ROS (Fig. 4A), lipid peroxidation (Fig. 4B) and the loss of plasma membrane integrity (Fig. 4C) in alfalfa root tips. We speculate that one of the mechanisms of this decrease in ROS content is the ability of H 2 to directly reduce 19 Page 19 of 49

21 ROS in vivo which has been confirmed in animals, showing that H 2 acts as a therapeutic anti-oxidant by selectively reducing hydroxyl radicals in rat pheochromocytoma12 cultured cells [14]. The scavenging of ROS by electrolyzed-reduced water, which dissolved large amounts of H 2, was also reported [43]. Previously, our in vitro analysis further showed that HRW was able to directly quench hydrogen peroxide (H 2 O 2 ), but not singlet oxygen radical [21]. Another explanation is that H 2 can readily permeate the cell membrane thereby influencing gene expression of antioxidant genes, including SOD, CAT, POD, stromal and cytoplasmic APX, etc, all of which have confirmed both in animals and recently in plant kingdoms [20-22,44]. Notably, the overall cytoprotective roles of HRW pretreatment is, at least partially, attributed to the decreased Cd uptake in root tissues (Fig. 3C). However, the detailed mechanism behind HRW-induced decrease in Cd accumulation is unclear. Third, molecular evidence revealed that Cd treatment could trigger either necrosis or PCD in onion roots, Arabidopsis and tobacco suspension cultures, at least partial, via an ROS and/or nitric oxide-dependent manner [1,45,46]. Previous results revealed that electrolyzed-reduced water was able to protect DNA from oxidative damage [43]. In agreement with these results, we noticed that Cd toxicity is closely linked with the results of DNA laddering (Fig. 1A and C). This observation was also confirmed by using FDA-PI dual fluorescent assay and DAPI staining (Fig. 2), all of which could be explained by the observed increases in ROS production, the cellular lipid peroxidation or plasma membrane injury (Fig. 4A-C). Pretreatment with HRW, however, was able 20 Page 20 of 49

22 to partially prevent DNA fragmentation, cell vigor, nuclear phenotypes, etc (Figs. 1C and 2), which was consistent with the antioxidant behaviors triggered by HRW (Figs ). This result, along with the decreased Cd uptake and the alleviation of seedling growth inhibition (Fig. 3), clearly indicates that H 2 can confer tolerance of the plant cells to Cd stress. It was suggested that many benefic roles of GSH are also performed by hgsh in alfalfa and soybean plants, particularly in the control of the cellular redox homeostasis and ROS scavenger [9]. Our former experiments demonstrated that high GSH/GSSG ratios might provide some useful explanation for the cytoprotective roles of carbon monoxide (CO) in alfalfa plants upon Cd exposure [25,47]. Heme oxygenase-1-mediated salicylic acid-induced alleviation of oxidative stress due to Cd stress is confirmed to be attributed to the significant enhancement of the ratio of reduced/oxidized homoglutathione [48]. In the subsequent test, we observed that HRW pretreatment could result in a higher (h)gsh/(h)gssg(h) ratio in root tissues of alfalfa seedlings 1 d after Cd exposure (Table 1), which might be related to the regulation of ECS, GS, hgs, and GR1/2 transcripts (Fig. 7E-H). Meanwhile, HRW-up-regulated (h)gsh contents was closely associated with ROS-scavenging capacity (Fig. 4A-C). Similarly, GSH content in γ-irradiated mice, was also induced by H 2 pretreatment [49]. These results clearly demonstrated that HRW may help maintain the glutathione homeostasis in alfalfa seedlings upon Cd stress. Therefore, redox balance is reestablished as to adapt to Cd exposure Page 21 of 49

23 Conclusions In summary, it was observed that HRW pretreatment resulted in the alleviation of Cd-induced plant growth inhibition and oxidative stress. Notably, re-established glutathione homeostasis and less quantity of accumulated Cd were observed in HRW-pretreated plants than those treated with Cd alone. All the parameters clearly indicate an anti-stress property of HRW against Cd contamination. Thus, these findings suggest the potential feasibility of HRW application to reduce Cd residues for food safety. Further genetic and molecular investigations will be required to better understand the detailed molecular mechanisms of HRW-induced Cd stress tolerance in plants. 22 Page 22 of 49

24 Acknowledgment This research was supported by the National Natural Science Foundation of China ( ) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. 23 Page 23 of 49

25 Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at 24 Page 24 of 49

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29 carbon monoxide in tuna by gas chromatography with micro-thermal conductivity detector, J. Chromatogr. Sci. 46 (2008) [24] T. Ling, B. Zhang, W. Cui, M. Wu, J. Lin, W. Zhou, J. Huang, W. Shen, Carbon monoxide mitigates salt-induced inhibition of root growth and suppresses programmed cell death in wheat primary roots by inhibiting superoxide anion overproduction, Plant Sci. 177 (2009) [25] Y. Han, J. Zhang, X. Chen, Z. Gao, W. Xuan, S. Xu, X. Ding, W. Shen, Carbon monoxide alleviates cadmium-induced oxidative damage by modulating glutathione metabolism in the roots of Mdicago sativa, New Phytol. 177 (2008) [26] L. Lin, W. Zhou, H. Dai, F. Cao, G. Zhang, F. Wu, Selenium reduces cadmium uptake and mitigates cadmium toxicity in rice, J. Hazard. Mater (2012) [27] N. Watanabe, E. Lam, BAX inhibitor-1 modulates endoplasmic reticulum stress-mediated programmed cell death in Arabidopsis, J. Biol. Chem. 283 (2008) [28] M. Katsuhara, T. Kawasaki, Salt stress induced nuclear and DNA degradation in meristematic cells of barley roots. Plant Cell Physiol. 37 (1996) [29] H. Thordal-Christensen, Z. Zhang, Y. Wei, D.B. Collinge, Subcellular localization of H 2 O 2 in plants. H 2 O 2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction, Plant J. 11 (1997) Page 28 of 49

30 [30] A. Pompella, E. Maellaro, A.F. Casini, M. Comporti, Histochemical detection of lipid peroxidation in the liver of bromobenzene-poisoned mice, Am. J. Pathol (1987) [31] Y. Yamamoto, Y. Kobayashi, H. Matsumoto, Lipid peroxidation is an early symptom triggered by aluminum, but not the primary cause of elongation inhibition in pea roots, Plant Physiol. 125 (2001) [32] H. Zhang, W.B. Shen, L.L. Xu, Effects of nitric oxide on the germination of wheat seeds and its reactive oxygen species metabolisms under osmotic stress, Acta Bot. Sin. 45 (2003) [33] K. Liu, S. Xu, W. Xuan, T. Ling, Z. Cao, B. Huang, Y. Sun, L. Fang, Z. Liu, N. Zhao, W. Shen, Carbon monoxide counteracts the inhibition of seed germination and alleviates oxidative damage caused by salt stress in Oryza sativa, Plant Sci. 172 (2007) [34] S. Xu, L. Wang, B. Zhang, B. Han, Y. Xie, J. Yang, W. Zhong, H. Chen, R. Wang, N. Wang, W. Cui, W. Shen, RNAi knockdown of rice SE5 gene is sensitive to the herbicide methyl viologen by the down-regulation of antioxidant defense, Plant Mol. Biol. 80 (2012) [35] Y. Nakano, K. Asada, Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts, Plant Cell Physiol. 22 (1981) [36] A. Drotar, P. Phelps, R. Fall, Evidence for glutathione peroxidase activities in cultured plant cells, Plant Sci. 42 (1985) [37] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram 29 Page 29 of 49

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32 apoptotic changes in tobacco cells, Plant Cell Environ. 23 (2000) [46] B.S. Behboodi, L. Samadi, Detection of apoptotic bodies and oligonucleosomal DNA fragments in cadmium-treated root apical cells of Allium cepa Linnaeus, Plant Sci. 167 (2004) [47] G. Fu, L. Zhang, W. Cui, Y. Wang, W. Shen, Y. Ren, T. Zheng, Induction of heme oxygenase-1 with β-cd-hemin complex mitigates cadmium-induced oxidative damage in the roots of Medicago sativa, Plant Soil 345 (2011) [48] W. Cui, L. Li, Z. Gao, H. Wu, Y. Xie, W. Shen, Haem oxygenase-1 is involved in salicylic acid-induced alleviation of oxidative stress due to cadmium stress in Medicago sativa, J. Exp. Bot. 63 (2012) [49] L. Qian, F. Cao, J. Cui, Y. Huang, X. Zhou, S. Liu, J. Cai, Radioprotective effect of hydrogen in cultured cells and mice, Free Radic. Res. 44 (2010) Page 31 of 49

33 Table 1 (Homo)glutathione ((h)gsh and (h)gssg(h)) and the ratio of (h)gsh/(h)gssg(h) in alfalfa seedling roots. 5-day-old seedlings were treated with 0 or 75 μm CdCl 2 for 24 h with or without 12 h pretreatment with 10% HRW. The sample without chemicals was the control (Con). Values are means SE of three independent experiments with at least three replicates for each. Different letters within columns indicate significant differences (P < 0.05) according to Duncan s multiple range test. Treatment (h)gsh (nmol g -1 FW) (h)gssg(h) (nmol g -1 FW) (h)gsh/(h)gssg(h) Con Con b b 9.25 Con Cd d a 4.55 HRW Cd c ab 6.45 HRW Con a b Page 32 of 49

34 580 Figure legends Fig.1. Effects of hydrogen-rich water (HRW) pretreatment on fresh weight (A), TBARS accumulation (B) and DNA laddering (C) in the roots of Medicago sativa under Cd stress. 5-day-old seedlings were pretreated with 0-100% HRW for 12 h and then exposed to 0 or 75 µm CdCl 2 for 72 h (A), 24 h (B) or 48 h (C). After that, root fresh weight (A) and TBARS content (B) were measured. DNA isolated from root tissues was analyzed by agarose gel electrophoresis (C). M, DNA size marker. The sample without chemicals was the control (Con). Values are means ± SE of three independent experiments with at least three replicates for each. Bars with different letters are significantly different at P < 0.05 according to Duncan s multiple range test. Fig.2. Effects of hydrogen-rich water (HRW) pretreatment on the cell vigor (A) and nuclei (B) of root tips from Medicago sativa seedlings subjected to Cd stress. 5-day-old seedlings were pretreated with 0 or 10% HRW for 12 h and then exposed to 0 or 75 µm CdCl 2 for 48 h. Afterwards, the cells were counterstained in situ by FDA-PI dual (A) and DAPI (B) fluorescent assays. The sample without chemicals was the control (Con). 1, Con Con; 2, Con Cd; 3, HRW Cd; 4, HRW Con. The right panels of Fig. 2B are the amplifications of corresponding square frames in the left panels, which showed the region of about 0.5 cm from root tip. The arrow shows shrunken nuclear morphology. Figure is representative from at least three independent 33 Page 33 of 49

35 602 experiments. Bar = 50 μm Fig.3. Effects of HRW pretreatment on seedling growth (A), root length (B) and Cd content (C) in the roots of Medicago sativa under Cd stress. 5-day-old seedlings were pretreated with 0 or 10% HRW for 12 h and then exposed to 0 or 75 µm CdCl 2 for 72 h. The sample without chemicals was the control (Con). Bar=2 cm (A). ND, none detected. Values are means ± SE of three independent experiments with at least three replicates for each. Bars with different letters are significantly different at P < 0.05 according to Duncan s multiple range test. Fig.4. Effects of HRW pretreatment on reactive oxygen species (ROS) localization (A), lipid peroxidation (B), loss of plasma membrane integrity (C) and lipoxygenase (LOX) activity (D) in the roots of Medicago sativa under Cd stress. 5-day-old seedlings were pretreated with 0 or 10% HRW for 12 h and then exposed to 0 or 75 µm CdCl 2 for 24 h. Bar=1 mm. The sample without chemicals was the control (Con). Values are means ± SE of three independent experiments with at least three replicates for each. Bars with different letters are significantly different at P < 0.05 according to Duncan s multiple range test (D) Fig.5. Effects of HRW pretreatment on SOD (A), POD (B), APX (C) and GPX (D) activities in the roots of Medicago sativa under Cd stress. 5-day-old seedlings were pretreated with 0 or 10% HRW for 12 h and then exposed to 0 or 75 µm CdCl 2 for Page 34 of 49

36 h. The sample without chemicals was the control (Con). Values are means ± SE of three independent experiments with at least three replicates for each. Bars with different letters are significantly different at P<0.05 according to Duncan s multiple range test. Fig.6. Effects of HRW pretreatment on isozymatic activities of SOD, POD and APX in the roots of Medicago sativa under Cd stress. 5-day-old seedlings were pretreated with 0 or 10% HRW for 12 h and then exposed to 0 or 75 µm CdCl 2 for 24 h. The sample without chemicals was the control (Con). After that, total protein from roots was extracted and loaded onto the native PAGE. Following the electrophoresis, the gels were stained and photographed for SOD (A), POD (C) and APX (E) in-gel activities, respectively. Corresponding band intensities of the individual isozymes (B, D and F) are expressed as a percentage of the control values of isozyme I. The arrows indicate the bands corresponding to various isozymes. Fig.7. Effects of HRW pretreatment on gene expression of Cu,Zn-SOD and Mn-SOD (A), POD (B), APX1/2 (C), GPX (D), ECS (E), GS (F), hgs (G), and GR1/2 (H) in the roots of Medicago sativa under Cd stress. 5-day-old seedlings were pretreated with 0 or 10% HRW for 12 h and then exposed to 0 or 75 µm CdCl 2 for 12 h. The sample without chemicals was the control (Con). Expression levels of corresponding genes are presented relative to the control samples, with normalized against expression of two internal reference genes in each sample. Date are the means ± SE of at least three 35 Page 35 of 49

37 independent experiments. Within each set of experiments, bars with different letters are significantly different at P < 0.05 according to Duncan s multiple range test Page 36 of 49

38 Highlights: HRW can alleviate Cd-induced alfalfa seedling growth inhibition and DNA laddering. HRW alleviates Cd-induced oxidative stress by activating antioxidant enzymes. Cd uptake in alfalfa seedling roots was decreased by HRW. HRW can re-establish glutathione homeostasis under Cd stress. 37 Page 37 of 49

39 Graphical Abstract (for review) Accepted Manuscrip Page 38 of 49

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