Effects of nitric oxide on zinc tolerance of the submerged macrophyte Hydrilla verticillata

Heavy metal pollution has become an increasingly recognized threat to the aquatic environment. In response to heavy metal stresses, various terrestrial plants produce the hormone nitric oxide (NO), but minimal information is available about the role of NO in aquatic macrophytes under high zinc (Zn) stress. In this study, we measured physiochemical parameters in the leaves of Hydrilla verticillata exposed to Zn2+ (10 mg l−1) alone or combined with NO (supplied as sodium nitroprusside, SNP) for 1 d and 4 d. Excess Zn disturbed nutrient uptake and altered the redox status of the aquatic plants. NO application partially reversed negative impacts of Zn stress on the contents of photosynthetic pigments, malondialdehyde, nutrient concentration, ascorbic acid, and the activity of antioxidant enzymes. However, the metabolites of SNP did not have similar effects. After treatments for 4 d, plants were further cultured in a solution containing Zn2+ (10 mg l−1) for 10 d. NO application partially reversed Zn-induced negative effects in parameters such as nutrient uptake, antioxidant enzymatic activities, and biomass, as compared to treatments with Zn alone. These results indicate that NO supply could mitigate Zn stress in H. verticillata, as a defense mechanism of the plant against Zn toxicity.


INTRODUCTION
Zinc (Zn) is an essential nutrient element for plants, as it is the component of many proteins such as Zn finger-containing transcription factors, Cu/Zn superoxide dismutase, carbonic anhydrase and Zn-metalloproteases (Broadley et al. 2007).Many of these proteins have critical roles in the uptake and transport of Zn, transcriptional regulation, RNA binding, regulation of apoptosis and protein−protein interactions (Beyersmann & Haase 2001, Ciftci-Yilmaz & Mittler 2008).However, excess Zn can cause damage to plants.High Zn affects mitotic activity of cells, photosynthesis, membrane integrity and permeability, and can even kill cells (Stoyanova & Doncheva 2002, Rout & Das 2003).Zn toxicity can result in nutrient imbalance and induce the generation of excess reactive oxygen species (ROS, including superoxide radical and hydrogen peroxide), which cause oxidative stress and lipid peroxidation (Wang et al. 2009).These negative effects may contribute to the inhibition of seed germination, plant growth and root development (Wang et al. 2009).
Plants have developed innate complex resistance/ adaptation mechanisms such as antioxidant systems to help them survive in the altered environment.Antioxidant enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and guaiacol peroxidase (POD), as well as antioxidant concentrations (for example ascorbic acid) are activated by plants to alleviate oxidative stress (Wójcik et al. 2006, Tewari et al. 2008, Wang et al. 2009).Zn stress can also induce the generation of nitric oxide (NO), an important stress-response hormone (Bartha et al. 2005, J. Xu et al. 2010).A recent report showed that NO was associated with longterm Zn tolerance in Solanum nigrum (J.Xu et al. 2010).
NO is a short-lived second messenger and acts as a diffusible free radical to readily react with a variety of intracellular and extracellular targets (Neill et al. 2003).It can be produced from NO 2 − nonenzymatically or catalyzed by nitrate reductase in plants, and is involved in regulating plant growth and development, such as photo morpho genesis, seed germination, de-etiolation, hy po cotyl elongation, organ maturation and senescence (Leshem et al. 1998).In addition, NO also participates in many processes as an antioxidant or anti-stress agent in response to abiotic stresses and has roles in the regulation of nutrient metabolism (Singh et al. 2008, Xiong et al. 2010).NO can also be released into waters by nitrifiers and denitrifiers in the aquatic environment, particularly at low O 2 concentrations (Neill et al. 2003).Submerged macrophytes play an important role in maintaining the health of aquatic ecosystems.The submerged sections of macrophytes growing under the water surface have adaptive mechanisms that differ from those of terrestrial plants.For example, their leaves can directly take up nutrients from the water, especially in water polluted with heavy metals.Consequently, submerged macrophytes are better adapted to absorbing heavy metals from water if they can thrive in those conditions (Wang et al. 2009).Although abundant data have shown that NO can improve the stress resistance in many terrestrial plants (Xiong et al. 2010), the role of NO in submerged macrophytes remains unclear.
Here we conducted a 2-stage experiment to study the role of NO in regulating the adaptation of submerged macrophytes to Zn stress using Hydrilla verticillata as a plant model.Sodium nitroprusside (SNP; a widely used NO donor) was used as the NO source, and the metabolic products of SNP were used as comparisons to confirm the effects of NO (Singh et al. 2008, 2009, Mostofa et al. 2014).The resultant changes in the levels of Zn and several other nutrient elements, the concentration of photosynthetic pigments and malondialdehyde, and the antioxidant response were assayed in leaves of H. verticillata treated with high levels of Zn alone or together with NO.

Materials and treatments
Hydrilla verticillata plants were purchased from an aquaculture company in Hangzhou, China.The tips (8 cm) of plant offspring were cultured in 10% Hoagland solution in 70 l plastic containers for 2 wk under laboratory conditions.SNP (Sigma Chemical) solution, used as an exogenous NO donor, was freshly prepared.SNP metabolite solution consisted of 50 µM sodium ferrocyanide, 50 µM sodium nitrite and 50 µM sodium nitrate.Fifty plantlets of similar growth stage were planted in 1 replicate for each treatment, and 4 replicates were used for each treatment (60 containers were used in total).
Throughout the acclimation and treatment periods, plants were cultured with a photoperiod of 12:12 h (light:dark) at a temperature of 24 ± 0.1°C and an irradiance of 120 µE m 2 s −1 under laboratory conditions.The culture solutions were renewed every 2 d.All of the plants (5 cm of the tip portion) harvested from 1 container were considered as 1 sample.Harvested material was rinsed with distilled water, blotted, and immediately frozen in liquid nitrogen.

Relative growth rate and mineral nutrient element content
The fresh weight of the plants was recorded before (W1, Day 0) and after treatment (W2, Day 14).The relative growth rate (RGR) was calculated based on the formula: 100% × (W2 − W1)/W2.
The harvested leaf samples were immediately washed with 50 µM EDTA solution and doubledistilled water (ddH 2 O) in sequence.The washed material was incubated in a forced-draught oven at 105°C for 20 min and then dried at 80°C for 24 h.A 0.1 mg aliquot of the dried sample was powdered and digested in 10:1 HNO 3 :HClO 4 solution and heated at 150−200°C until near dryness.The cooled residue was dissolved in 5 ml of 5% HNO 3 , and ddH 2 O was added to make 15 ml of total volume.The concentrations of Zn, Cu, Fe, Mn and Na were determined using inductively-coupled plasma atomic emission (Prodigy, Leeman-LABS).

Determination of photosynthetic pigments and metabolite
Concentrations of chlorophyll (chl) (a + b) and carotenoids were measured as described by Lichtenthaler & Wellburn (1983) after extraction with 80% acetone.Content of malondialdehyde (MDA), NO and non-protein thiols (NP-SH) was determined based on the methods described previously (Wang et al. 2011).

Assay of antioxidant enzymes
To assay the catalytic activities of various enzymes, 1 g of powdered sample was homogenized in 5 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 2 mM MgCl 2 , and 1% poly vinylpyrrolidone.For the APX assay, 1 mM ascorbic acid (AsA) was included in the same reaction as above.The homogenate was centrifuged at 12 000 × g (20 min at 4°C), and the supernatant was used for the enzyme assays.The protein content was determined by the method of Bradford (1976) using bovine serum albumin as a standard.
SOD activity was assayed according to the method described by Beauchamp & Fridovich (1971).One unit of SOD activity was defined as the amount of enzyme required to cause a 50% inhibition of the rate of nitroblue tetrazolium (NBT) as monitored spectrophotometrically at 560 nm.CAT activity was determined by measuring the change of absorbance at 240 nm that accompanied the consumption of H 2 O 2 (Aebi 1974).One unit of CAT activity (ε = 0.0394 cm 2 µmol −1 ) was defined as the amount required to decompose 1 nmol H 2 O 2 min −1 mg −1 protein.
POD activity was determined according to the method of Upadhyaya et al. (1985).One unit of POD (ε = 26.6 mM −1 cm −1 ) activity was defined as the amount required to convert 1 µmol guaiacol min −1 g −1 fresh weight.Glutathione reductase (GR) activity was assayed by following the reduction of NADPH reflected as a change of the absorbance at 340 nm as described by Carlberg & Mannervik (1985), and activity was calculated using the extinction coefficient ε = 6.22 mM −1 cm −1 .One unit of GR activity was defined as the amount required to decompose 1 nmol NADPH min −1 mg −1 protein.APX activity was determined essentially as described by Nakano & Asada (1981).One unit of APX activity (ε = 2.8 mM −1 cm −1 ) was defined as the amount required to decompose 1 µmol AsA oxidized min −1 mg −1 protein.

Statistical analyses
Each value shown in the figures and tables is mean ± SE of 4 replicates (n = 4).All data were analyzed by 1-way ANOVA and Tukey's test, and significance was considered at p < 0.05.All statistical analyses were performed with SPSS software (version 19.0).

RESULTS
Compared to the control, plant Zn concentrations increased significantly in all treatments on Days 1 and 4 (Table 1).However, the addition of SNP significantly inhibited the uptake of Zn on Day 4 (Table 1).A significant increase in plant Cu concentration was detected in ZS50 on Day 4 (Table 1).Plant Fe concentration increased significantly when exposed to ZS50 on Day 1; and by Day 4, it increased by 71, 135, 210 and 98%, respectively, in treatments Z10, ZS25, ZS50 and ZS50M, as compared to the control (Table 1).A significant increase in plant Mn was recorded in Z10, ZS25, ZS50 and ZS50M on Days 1 and 4, while plant Mn concentration was obviously lower in ZS25 and ZS50 than ZS50M (Table 1).In ZS25 and ZS50, plant Na concentration evidently decreased on Day 1, this was significant only on Day 4 (Table 1).
At the second stage, no significant alteration was observed among the treatments Z10, ZS25, ZS50, and ZS50M on Day 14. Plant Cu concentration was higher in ZS25 and ZS50 than in the other treatments, while the highest value in plant Fe was detected in ZS25.Plant Mn concentration was higher by 43.46%, 13.12%, 35.25%, and 35.48%, respectively, in ZS25, ZS50 and ZS50M, compared to the control.However, plant Na concentration was lower in ZS25 and ZS50.The RGR was higher in the control than all other treatments containing high Zn on Day 14, but the RGR was higher in ZS25 and ZS50 than Z10 and ZS50M.
NO increased significantly in plants of ZS25, ZS50 and ZS50M on Day 1 and in Z10, ZS25, ZS50 and ZS50M on Day 4, as compared to the control (Fig. 1A).The maximum NO concentration was detected in ZS50 on Days 1 and 4. Compared to the control, MDA concentration increased significantly in plants exposed to ZS50M on Day 1 and on Day 4, and was higher in Z10, ZS25 and ZS50M than in the control and ZS50 (Fig. 1B).The concentrations of NO and MDA were lower in the control than other treatments on Day 14.
Chl (a + b) content decreased significantly in ZS25 or ZS50M on Day 1 and in all treatments on Day 4, as compared to the control (Fig. 2A).In addition, chl (a + b) content was higher in ZS50 than ZS10 or ZS50M.No significant alteration was observed in plant carotenoid concentrations in all the treatments on Day 1, while carotenoid concentrations decreased in ZS10 and ZS25 on Day 4 (Fig. 2B).NP-SH concentration changed normally in Z10, ZS25 and ZS50M but significantly increased in ZS50M on Day 1; by Day 4, NP-SH increased in Z10 (21.7%),ZS25 (46.5%),ZS50 (32.7%) and ZS50M (29.1%;Fig. 2C).SOD activity increased significantly in ZS25 and ZS50 on Day 1 and in Z10, ZS25, ZS50 and ZS50M on Day 4 (Fig. 2D).From Days 5 to 14, the culture solution consisted of 10 mg l −1 Zn only in all 4 treatment groups (except for the control).Different superscripts indicate significant differences at p < 0.05 (n = 4; Tukey's test).The relative growth rate (RGR) was the increase in fresh weight after the treatment In the second stage (Day 14), values of chl (a + b), carotenoid, and NP-SH contents, and SOD activity were higher in ZS25 and ZS50 than in Z10 and ZS50M.Chl (a + b) and carotenoid contents decreased, but NP-SH content and SOD activity increased in Z10, ZS25, ZS50 and ZS50M, as compared to the control.Meanwhile, chlorosis was observed in plant leaves of Z10 and ZS50M, while no significant symptoms were observed in plant leaves of ZS25 and ZS50.
Plant AsA content increased by 9.6, 33.2, 17.9 and 4.8% of control values, respectively, in Z10, ZS25, ZS50 and ZS50M on Day 1; and by 5.9, 23.9, 32.1 and 9.8% on Day 4, respectively (Fig. 4A).As compared to controls, plant docosahexaenoic acid (DHA) content changed normally in all the treatments on Day 1 but significantly changed on Day 4. SNP application decreased the DHA concentration in plants ex posed to ZS25 and ZS50, as compared with ZS50M (Fig. 4B).In the second stage, AsA con- tent was obviously lower in controls than in ZS25 and ZS50 but was higher than in Z10 and ZS50M; however, DHA content was higher in controls than in other treatments.

DISCUSSION
In aquatic environments, heavy metals can accumulate in aquatic macrophytes, inducing a variety of cellular changes which may directly contribute to the metal tolerance capacity of plants ( (Singh et al. 2008, Q. Xu et al. 2010).NO, a type of hormone, is important for plants to cope with various stresses.However, Hydrilla verticillata can be damaged when exposed to high levels of NO (e.g. a long time of exposure to NO; Wang et al. 2010).In addition, in our experiment, NO was initially released quickly because SNP degrades quickly in the medium.There-fore, we replaced the solution every 2 d in the first stage of this experiment to reduce the potential toxic effects of NO.The increase of NO in ZS25 and ZS50 should be ascribed to the breakdown of SNP.However, an increase in NO was detected in H. verticillata plants exposed to Zn on Day 4, suggesting that excess Zn induced the generation of NO.High Zninduced NO was also found in the terrestrial plants Brassica juncea, Pisum sativum, and Solanum nigrum (Bartha et al. 2005, J. Xu et al. 2010).Increased NO concentration may be produced by peroxisomes and mitochondria and by the activity of nitrate reductase in plant cells (Xiong et al. 2010).
It is interesting to note that NO stimulated the uptake of Zn in leaves of H. verticillata on Day 1, but on Day 4, an inhibition effect on Zn accumulation was detected (Table 1).Zn uptake is associated with metallothionein proteins, which are the critical targets of NO (Desbrosses-Fonrouge et al. 2005, Arri - Metals like Cu, Fe, Mn and Na are important for plants.Plant Cu varied normally among the treatments on Day 1 (Table 1).Exogenous NO supply did not affect Cu uptake in roots of Panax ginseng on Day 1 (Tewari et al. 2008).However, plant Cu increased in ZS50 as compared with ZS50M and Z10 on Day 4 (Table 1).The increase of plant Fe content induced by NO (Table 1) might be attributed to the expression of Fe uptake-related genes, which could be induced by NO (Besson-Bard et al. 2009).A recent report by J. Xu et al. (2010) showed that NO was required for metal (especially Fe) uptake and homeostasis in plants exposed to excess Zn.Kumar et al. (2010) suggested that by means of modulating the redox, NO application could increase the intracellular availability of Fe.
Mn participates in both oxygen radical production in PSII and in the oxygen radical detoxification in mitochondria (Shenker et al. 2004).NO application inhibited Mn uptake in H. verticillata plants under Zn stress (Table 1).Na always causes osmotic stress for plants, thought it can substitute for K in some circumstances.NO application decreased Na content (Table 1).A decrease in Na content by NO in maize seedlings under NaCl stress was also re ported by Zhang et al. (2006), who suggested that NO might regulate the activities of vacuolar H + -ATPase and H + -PPase.
SNP application inhibited Zn-induced alteration in MDA (Fig. 1B) and chl (a + b) content (Fig. 2A) in leaves of H. verticillata.These results might be attributed to the enhancement of antioxidant responses (Neill et al. 2003, Shi et al. 2007, Xiong et al. 2010) and the improvement of nutrient composition (Wang et al. 2009).Meanwhile, SNP application induced a slight increase in carotenoid content (Fig. 2B) in H. verticillata, as compared to ZS50M.Kumar et al. (2010) showed that SNP application significantly increased the carotenoid content in maize.An increase in carotenoids may alleviate the damage from oxidative stress in chloroplasts, since carotenoids play a vital role in the photosynthetic reaction center of photosynthetic organisms.
The 25 µM SNP application increased the NP-SH content in leaves of H. verticillata on Day 4 (Fig. 2C).Similar results were found in the roots of P. ginseng (Tewari et al. 2008), while Kumar et al. (2010) showed a reduction in NP-SH content in maize plants exposed to SNP.NP-SH is a thiol pool containing monothiols (e.g.cysteine and reduced glutathione) and phytochelatins and has important roles in reducing heavy metal toxicity (Powell 2000).Thus, the effect of NO was likely a result of the adjustment of total or free SH levels (Abdel-kader 2007).
Efficient antioxidant mechanisms are crucial for cells to cope with excess ROS.In this study, NO application stimulated the activities of antioxidant enzymes including SOD, CAT and POD (Figs. 2B & 3A,B), suggesting that NO application enhanced the antioxidant response in H. verticillata to resist oxidative stress.Shi et al. (2007) found that SOD activity was regulated by NO in plants under NaCl stress on Day 4. The NO-induced increase in POD activity might contribute to the polymerization of phenoxy compounds to produce components such as lignins that stiffen cell walls (Reddy et al. 2005).APX and GR are 2 main enzymes of the ascorbate−glutathione cycle, which are localized mainly in chloroplasts and play an important role in combating oxidative stress (Shigeoka et al. 2002).APX and GR activity was induced by NO in H. verticillata plants under Zn stress (Fig. 3C,D).The increased antioxidant enzyme activity might be ascribed to the role of NO in stimulating the expression of their genes (Besson-Bard et al. 2009, Xiong et al. 2010).Recently, many NOinduced stress resistance genes were screened in different plants by a genomic microarray (Besson-Bard et al. 2009).AsA is the major redox regulatory antioxidant and is able to detoxify ROS by direct scavenging or by acting as a substrate in the ascorbate− glutathione cycle (Apel & Hirt 2004).AsA content increased in treatments with NO (Fig. 4A), suggesting that the exogenous NO supply enhanced the tolerance against Zn-induced oxidative stress in H. verticillata.
To study the effects of NO application on the adaptation of H. verticillata under Zn stress, after 4 d of treatments, plants of Z10, ZS25, ZS50 and ZS50M were further treated with 10 mg l −1 Zn 2+ for 10 d (until Day 14).Zn, Cu, Fe, Mn and Na contents were altered by NO in different patterns in leaves of H. verticillata plants under Zn stress, providing the evidence that NO is involved in the regulation of nutrient element uptake in plants under Zn stress.The disturbed nutrient uptake may be ascribed to the roles of NO in regulating metal uptake/transporterrelated gene expression and/or protein activity (Besson-Bard et al. 2009).NO obviously decreased in the treatments of ZS25 and ZS50 on Day 4, as compared to Day 14, demonstrating that exogenous NO decreased after the removal of SNP.Meanwhile, Cu, Fe and Mn contents in creased in treatments ZS25 and ZS50 on Day 14 as compared to Day 4. The improved growth traits in H. verticillata by NO application in this study might be associated with the altered nutrient element content.
No significant alterations in Zn content were observed among the treatments on Day 14.However, the RGR was higher in ZS25 and Z50 than in Z10 and ZS50M.These results indicate that pretreatment with NO could increase biomass production under Zn stress.The increased biomass production might be associated with the increase in photosynthetic pigment content and the alleviated damage, which can be revealed by the decreased MDA content and increased antioxidant response on Day 14.
In conclusion, exogenous NO partially reversed Zn-induced reduction of photosynthetic pigments and the increase of MDA content.NO application improved the nutrient balance and antioxidant ability in response to Zn stress.The effects of NO on these parameters may contribute to the accumulation of biomass.Consequently, pretreatment with NO may be a useful way to improve the effects of ecological remediation and phytoremediation of Zn-polluted waters.

Fig. 1 .
Fig. 1.Effect of SNP on levels of (A) nitric oxide (NO) and (B) malondialdehyde (MDA) contents in leaves of Hydrilla verticillata under Zn stress.Different lowercase letters indicate significant differences at p < 0.05 (Tukey's test).FW = fresh weight

Fig. 4 .
Fig. 4. Effect of SNP on the contents of (A) ascorbic acid (AsA) and (B) docosahexaenoic acid (DHA) of Hydrilla verticillata exposed to Zn. Different lowercase letters indicate significant differences at p < 0.05 (Tukey's test)

Table 1 .
Effects of sodium nitroprusside (SNP) on nutrient concentration (mg kg −1 dry weight) in leaves of Hydrilla verticillata.