Hydrogen sulfide induced growth, photosynthesis and biochemical responses in three submerged macrophytes

Flora 230 (2017) 1–11

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Hydrogen sulfide induced growth, photosynthesis and biochemical responses in three submerged macrophytes Mahfuza Parveen a , Takashi Asaeda b,∗ , Md H. Rashid b,c a b c

Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan Department of Environmental Science and Technology, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan Department of Agronomy, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

a r t i c l e

i n f o

Article history: Received 14 October 2016 Received in revised form 1 March 2017 Accepted 6 March 2017 Edited by Hermann Heilmeier Available online 9 March 2017 Keywords: Leaf pigments Chlorophyll fluorescence Indole acetic acid Oxidative stress Antioxidative response Lipid peroxidation

a b s t r a c t Hydrogen sulfide (H2 S) is a known phytotoxin for submerged macrophytes, which can affect plant species differentially and modify species composition in aquatic environments. To investigate the effects of H2 S on three submerged macrophytes, Elodea nuttallii, Myriophyllum spicatum and Potamogeton crispus were exposed to five treatments containing varying concentrations (0–1.0 mM, depending on species) of sodium hydrosulfide (NaHS) as the H2 S donor. NaHS can produce the desired levels of H2 S for the experiment. All the plants exposed to low concentrations of NaHS exhibited increased plant growth without showing oxidative stress. However, a decrease in growth rate, chlorophyll content, and an increase in hydrogen peroxide (H2 O2 ) and malondialdehyde content (MDA) were observed after exposure to high sulfide concentrations, which indicated the presence of increased oxidative stress in the three plant species of interest. For E. nuttallii and M. spicatum, the activity of guaiacol peroxidase (POD) and ascorbate peroxidase (APX) levels decreased in the presence of 0.5 and 1.0 mM NaHS concentrations, suggesting that the antioxidative enzymes were not able to scavenge the reactive oxygen species responsible for oxidative stress, furthering plant senescence. Compared with E. nuttallii, higher antioxidative responses in M. spicatum and P. crispus exhibited a higher tolerance to NaHS exposure. © 2017 Elsevier GmbH. All rights reserved.

1. Introduction Submerged macrophytes provide many important ecosystem services. They serve aquatic ecosystems by providing food and refuges for fishes, aquatic invertebrates and other aquatic organisms (Costanza et al., 1997; Harborne et al., 2006). They are the source of primary production, and other important services they offer are the control of transport, settling and resuspension of sediment (Madsen et al., 2001; Asaeda et al., 2010). Aquatic macrophytes also play an important role in bio-geochemical cycles and thus purify water in natural water bodies and constructed wetlands (Asaeda and Rashid, 2015). Due to their important services, they have been termed as ‘ecosystem engineers’ (Asaeda et al., 2010). However, aquatic macrophytes often encounter an array of biotic and abiotic stress factors. Most of these factors, individually or collectively, govern the growth of macrophytes (Ben Rejeb et al., 2014)

∗ Corresponding author. E-mail addresses: mahfuza [email protected] (M. Parveen), [email protected] (T. Asaeda), [email protected] (M.H. Rashid). http://dx.doi.org/10.1016/j.flora.2017.03.005 0367-2530/© 2017 Elsevier GmbH. All rights reserved.

and determine their distribution (Chambers et al., 2008). Among many other factors, eutrophication and submergence are considered the major drivers for plant growth and species distribution (Zhang et al., 2016). Hydrogen sulfide (H2 S) is a known phytotoxin in aquatic environments (Lamers et al., 2013). It can be produced either in the course of microbial organic matter decomposition or dissimilatory sulfate reduction in waterlogged soil (Reddy and DeLaune, 2008). In eutrophic waters, it can also be formed by anaerobic decomposition of organic wastes. Sulfides can be trapped in the sediment by precipitation with metal ions. However, some portion of it remains undissociated in the form of H2 S, dissolved in sediment pore water, especially at pH < 7 (Thode-Andersen and Jørgensen, 1989). Despite being present in low concentration, H2 S plays a very important role in the biological, physical and chemical processes in aquatic ecosystems. Among several forms of sulfides, being dissolved in water, it is the only form that can freely penetrate through the cell membrane affecting growth of submerged macrophytes (Koren et al., 2015). In addition to direct interference with plant physiological processes, the production of H2 S can deplete dissolved oxygen (DO) in waters by increasing the sediment oxygen demand rate and thus creates toxicity to aquatic plants (Dunnette et al., 1985). Anoxic

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production of H2 S exerts strong toxicity in submerged plants and reduces growth (DeLaune et al., 1983) by interfering with nutrient uptake (King et al., 1982), photosynthesis and metabolism (Holmer et al., 2005). In addition, high H2 S can be also responsible for the formation of reactive oxygen species (ROS), which can lead to protein degradation and peroxidation of membrane lipids (lipid peroxidation), resulting in production of malondialdehyde (MDA) (Brodersen et al., 2015). However, recent studies reported that H2 S at very low concentrations acts as a gaseous signal molecule and alleviates oxidative damage in plants by antioxidant enzymes (Shi et al., 2015; Li et al., 2016). Despite the potent toxicity of sulfide, some aquatic macrophytes, such as Spartina alterniflora (Lee et al., 1999) and S. anglica (Lee, 2003) have adapted to sulfide-rich sediments. Sulfide concentration has been linked to the distribution of emergent macrophytes, Phragmites australis, S. alterniflora (Chambers et al., 1998) and other species (Ingold and Havill, 1984) in salt marshes. Li et al. (2009) suggested that this kind of macrophyte distribution is due to the differential responses of plants to sediment sulfides. Though we do not have any report that sulfide determines the composition and distribution of submerged macrophytes, its effect on the growth of the same have been studied elsewhere (DeBusk et al., 2015; Pedersen and Kristensen, 2015). Toxicity symptoms appeared in the tropical seagrass Thalassia testudinum at 2 mM sulfides (Erskine and Koch, 2000), and 100 ␮M sulfides in Potamogeton compressus (Geurts et al., 2009). Currently, eutrophication in different water bodies is increasing due to effluent wastewater from factories or agricultural leakage, and submerged macrophyte populations are declining in eutrophic lakes globally (Kemp et al., 1983; Chai et al., 2006). Sulphate enrichment can also lead to eutrophication and cause serious problems in freshwater wetlands (Lamers et al., 1998). On the other hand, anaerobic decomposition in eutrophic waters produces H2 S (Castel et al., 1996). Therefore it is necessary to investigate the effects of dissolved H2 S on the growth and biochemical responses of submerged macrophytes. Though the occurrence of H2 S in aquatic environments is temporary, it can either exert oxidative stress (at higher concentration) by depleting oxygen in the water column (Dunnette et al., 1985) or promote growth of submerged macrophytes at lower concentration (Shi et al., 2015; Li et al., 2016). Although works cited above studied the effects of sulfides in sediment on the growth response of aquatic macrophytes, none of these studies has investigated dissolved H2 S as a stressor for the growth of submerged macrophytes. Though the tolerance of a plant to abiotic stress factors is a physiological trait, it can also be determined by the morphology of plants (Cedergreen et al., 2004). Likewise, aquatic macrophytes have been reported to adopt phenotypic plasticity to cope with certain abiotic stress (Gratani, 2014). The level of tolerance to sediment sulfide among several species varied greatly as reported by some researchers (Geurts et al., 2009; Wu et al., 2009). Therefore it can be assumed that growth promotion or stress tolerance of aquatic macrophytes to H2 S exposure is species specific, and it depends on the morphology of the plants. To test this hypothesis, we observed growth rate, chlorophyll content, maximum photochemical efficiency (Fv/Fm), concentrations of indole acetic acid (IAA), contents of hydrogen peroxide (H2 O2 ) and malondialdehyde (MDA) as oxidative stress indicators, and activities of antioxidant enzymes, viz. ascorbate peroxidase (APX) and guaiacol peroxidase (POD) of three submerged macrophytes grown under varying concentrations of NaHS (as the H2 S donor). The investigated macrophytes are Elodea nuttallii St. John, Myriophyllum spicatum L. and Potamogeton crispus L. They are very common and abundant species in Japan. These macrophytes have conspicuous differences in the foliar structures (Fig. 1). M. spicatum has feather like leaves, composed of more than 10 thin and

short (≈1.0 cm) leaflets. Both P. crispus and E. nuttallii have lamina leaves. However, P. crispus leaves are curly and much larger (4–10 cm) than those of E. nuttallii (0.5–1.5 cm). E. nuttallii and M. spicatum leaves are whorled around the stem, whereas P .crispus leaves have alternate arrangement.

2. Materials and methods 2.1. Plant materials, growth conditions, and treatment Plant samples of E. nuttallii, M. spicatum and P. crispus were collected from Moto-Arakawa River, a tributary of the Arakawa River in southern Saitama, Japan (36◦ 7 30.1 N, 139◦ 24 20 E) (Asaeda and Rashid, 2015). Plant materials were cultured in tanks (50 cm × 35 cm × 35 cm) in a growth chamber at a controlled tem◦ perature of 23 ± 3 C and a photoperiod of light: dark of 12 h: 12 h. The photosynthetic photon flux density was maintained at approximately 100–120 ␮mol photons m−2 s−1 by using fluorescent lamp tubes. Commercial river sand (90% <1 mm) was used as the substrate, which was purchased from the local market (DIY, DoIt, Japan). Experimental plants were obtained from the culture tanks. After 21 days of acclimation, two apical tips (≈6 cm) were clipped and plugged into silicone sponge clumps and placed in a 500 ml glass beaker. The culture medium was 5% Hoagland nutrient solution (HNS) (Hoagland and Arnon, 1950). The pH of the solution was maintained at 6.0–6.5 using 1 M NaOH or HCl. A preliminary experiment was set up with different concentrations of NaHS applied to E. nuttallii, M. spicatum and P. crispus and shoot length and vigor (naked eye observation) were observed. It appeared that E. nuttallii was weaker in comparison to the other two and could not survive at 1.0 mM NaHS concentration after 15 days. Therefore, on the basis of the observations of this preliminary experiment and previous literature (Geurts et al., 2009; Wu et al., 2009), different sets of treatments were established for E. nuttallii, and M. spicatum and P. crispus. Five treatments were selected for each plant, with three replicates (30 similar sized plants from each species) for each of the following treatments: NaHS concentrations of 0, 0.01, 0.05, 0.1 and 0.5 mM for E. nuttallii; and 0, 0.01, 0.1, 0.5 and 1.0 mM for M. spicatum and P. crispus. To achieve the desired H2 S concentrations, sodium hydrogen sulfide (NaHS, Sigma) was used as a hydrogen sulfide (H2 S) donor (Ali et al., 2015). Previous studies reported that NaHS can generate the highest amount of H2 S contents compared to other chemicals. Chen et al. (2011) used a series of sulphur- and sodiumcontaining chemicals, including NaHS, Na2 S, Na2 SO4 , Na2 SO3 , NaHSO4 , NaHSO3 and NaAC, and exposing Spinacea oleracea to them, and suggested that H2 S rather than the other sulphurcontaining compounds or sodium was responsible for the increase in chlorophyll content in S. oleracea after NaHS treatment. The targeted H2 S concentrations were achieved by adding 10 mM NaHS via syringe, after deoxygenating the media with inert gas. The beakers were sealed with Parafilm M and disturbed as few as possible to prevent the escape of H2 S gas. The culture medium of each treatment was renewed everyday due to the relatively short half-life of H2 S (Napoli et al., 2006). To maintain the target concentrations of the treatments, a 5 ml portion of the medium from each flask was extracted daily by syringe and H2 S was measured colorimetrically (Cline, 1969). The experiment was conducted for 7 days, as plants exposed to high H2 S concentrations showed brown discoloration and increased mortality after 7 days. At the end of the experiment, plant samples were collected for physical and biochemical analyses, washed with distilled water, and dried by blotting with laboratory tissue. Final shoot length (length of main stem) was measured with the help of a ruler to calculate the growth rate of plants. The same

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Fig 1. A comparative view of the shoot sections of investigated plants.

plant samples were used to analyze chlorophyll concentrations and fluorescence measurements. Other plant samples were collected and frozen immediately in liquid nitrogen and stored at −80 ◦ C for further analysis. 2.2. Morphological and photosynthesis related parameters Shoot growth rate (SGR) was obtained from the difference between the final and initial length divided by the number of days and was calculated as cm/day. Chlorophyll contents were determined spectrophotometrically (UV–vis spectrophotometer, Shimadzu, Japan) by extracting fresh leaves and stems in 5 ml of N, N-dimethylformamide for 24 h in the dark at 4 ◦ C (Rashid et al., 2010). Chlorophyll contents were calculated using the equations described by Porra et al. (1989), expressing chlorophyll in mg/g FW. The chlorophyll a fluorescence of fresh leaves was determined using a Handy Fluorocam (FC1000-H, Photon Systems Instruments, Czech Republic) via auto image segmentation and 15 min of dark adaptation. The maximum photochemical efficiency of PSII (Fv/Fm) was automatically calculated using the following equation by DeEll and Toivonen (2003): Fv/Fm = (Fm-F0)/Fm where Fm and F0 are the maximum and minimum fluorescence in dark adapted leaves, respectively. 2.3. IAA, H2 O2 , POD and APX assays The concentration of indole acetic acid (IAA), the most abundant form of auxins in plant tissues, was measured using the Salowski reagent (Gordon and Weber, 1951). Approximately 100 mg of fresh weight (FW) from the apical tip was ground in 2.5 ml of distilled water and centrifuged at 5000 × g at 20 ◦ C for 15 min. After collecting the supernatant, 1 ml of the extract was added to 2 ml of the Salowski reagent, and the color development was measured after 1 h at 530 nm (Ellawala et al., 2011). The results are presented as ␮mol g−1 FW. For H2 O2 , POD and APX assays, approximately 100 mg of fresh plant shoots were extracted in an ice-cold phosphate buffer (50 mM, pH 6.0) which contained polyvinylpyrrolidone (PVP). The extractions were centrifuged at 5000 × g for 20 min at 4 ◦ C. The supernatant was collected and immediately stored at −80 ◦ C for further analysis. For the analysis of endogenous H2 O2 concentration, 750 ␮l aliquot was mixed with 2.5 ml of 0.1% titanium sulfate in 20% (v/v) H2 SO4 (Jana and Choudhuri, 1982). The mixture was centrifuged

at 5000 × g at 20 ◦ C for 15 min. The intensity of the yellow color was measured spectrophotometrically at 410 nm. H2 O2 concentrations were estimated using a standard curve prepared from known concentrations of H2 O2 . The results are presented as ␮mol g−1 FW. POD (EC 1.11.1.7) was assayed according to the method of Goel et al. (2003). The reaction mixture containing 1.2 ␮l of 30% H2 O2 , 1.68 ␮l of 100% guaiacol, and 2.89 ml of 0.1 M potassium phosphate buffer (pH 6.0) was freshly mixed. The reaction was initiated with the addition of 0.1 ml of enzyme extract. The change in absorbance was recorded at 470 nm at an interval of 15 s for 3 min using an extinction coefficient of 26.6 mM−1 cm−1 . APX (EC 1.11.1.11) activity was assayed using the methods described by Nakano and Asada (1981). The reaction mixture contained 100 ␮l of enzyme extract, 200 ␮l of 0.5 mM ascorbic acid in 50 mM potassium phosphate buffer (pH 7.0) and 2.0 ml of 50 mM potassium phosphate buffer (pH 7.0). The reaction began after adding 60 ␮l of 1 mM H2 O2 . The decrease in absorbance at 290 nm was recorded every 15 s, and the APX activity was determined using the extinction coefficient of 2.8 mM−1 cm−1 . Ascorbate peroxidase and POD activities are presented as ␮mol/min g−1 FW. 2.4. Determination of MDA The level of lipid peroxidation was measured in terms of malondialdehyde (MDA), a product of lipid peroxidation in the plant samples that was estimated using a thiobarbituric acid (TBA) reaction according to the formula developed by Heath and Packer (1968): MDAequivalents(nmol ml−1 ) = [(A532 -A600 )/155000] where A532 is the absorbance at 532 nm and A600 the absorbance at 600 nm. Approximately 100 mg of fresh plant samples were homogenized in 0.5 ml of 0.1% (w/v) TCA before centrifuging at 15,000 × g for 10 min at 4 ◦ C. Then, 0.5 ml of the collected supernatant was mixed with 1.5 ml 0.5% TBA diluted in 20% TCA. The mixture was incubated in a 95 ◦ C water bath for 25 min and the reaction stopped by placing in an ice bath. The results are presented as nmol MDA g−1 FW. 2.5. Dissolved oxygen and dissolved H2 S measurements in water Dissolved oxygen (DO) was measured using a Dissolved Oxygen and Temperature meter (HI 9146). Dissolved H2 S was determined colorimetrically using the methyleneblue method (Cline, 1969).

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Table 1 Average applied NaHS concentrations (mM) and achieved H2 S concentrations (mM) in different treatments. Values are the means of three replicates ± SE. Elodea nuttallii

Myriophyllum spicatum

Potamogeton crispus

Applied NaHS (mM)

Measured H2 S (mM)

Applied NaHS (mM)

Measured H2 S (mM)

Applied NaHS (mM)

Measured H2 S (mM)

0 0.01 0.05 0.1 0.5

0±0 0.008 ± 0.0 0.044 ± 0.0 0.093 ± 0.0 0.365 ± 0.0

0 0.01 0.1 0.5 1.0

0±0 0.008 ± 0.0 0.088 ± 0.0 0.453 ± 0.0 0.957 ± 0.0

0 0.01 0.1 0.5 1.0

0±0 0.008 ± 0.0 0.085 ± 0.0 0.423 ± 0.0 0.897 ± 0.0

Fig. 2. NaHS induced changes in shoot growth rate (SGR) and Indole acetic acid (IAA) concentrations in Elodea nuttallii, Myriophyllum spicatum and Potamogeton crispus. Values are the means of three replicates ± SE. Bars with different letters are significantly different at P < 0.05.

Water samples were carefully collected to avoid the inclusion of air bubbles by using a 50 ml glass syringe and transferred to a 50 ml serum bottle that was sealed with a rubber cap. Then, 4 ml of mixed diamine reagent (2.4 g Ferric chloride (FeCl3 ·6H2 O) and 1.6 g N, Ndimethyl-p-phenylenediamine sulfate dissolved in 100 ml 6 M HCl solution) were injected into the sample using a glass syringe. After 20 min, the absorbance was spectrophotometrically measured at 670 nm. For each set of samples, NaHS was used as a calibration standard, and the results are expressed in mmol l−1 (mM). 2.6. Statistical analyses Data analyses were carried out using R (R Development Core Team, 2010). Raw data of all variables were checked for normal distribution with the one-sample Kolmogorov-Smirnov test as well as for homogeneity of variances with the Levene’s test and when necessary, arcsin transformation was performed. Data were subjected to a one-way analysis of variance (one-way ANOVA) followed by Tukey’s multiple comparison test to detect significant differences at p < 0.05. To explore correlations among treatments, Pearson’s correlation analysis was used. The ‘rcorr.adjust’ function of ‘RcmdrMisc’ package was used to compute matrices of Pearson correlations along with the pairwise p-values among the correlations. The p-values were corrected for multiple inferences using

Holm’s method. The nonmetric multidimensional scaling (NMDS) of plant growth and stress components data were conducted using the function ‘metaMDS’, which is incorporated in the statistical package ‘vegan’ (Oksanen et al., 2013). Bray-Curtis similarity was used as the pair-wise distance among samples. 3. Results Hydrogen sulfide concentrations in the media were measured every day after the application of NaHS because the concentration of the medium changed every day. The average applied NaHS and measured H2 S concentrations in the media are given in Table 1. There were no significant differences observed among the applied and achieved H2 S concentrations in the media (p < 0.05). A significant gradual decrease in shoot growth rate (SGR) was observed when all three plant species were exposed to high H2 S concentrations (p < 0.01) (Fig. 2A, C, E). Overall, there was a significant SGR decrease by 95% for E. nuttallii, 90% for M. spicatum and 62% for P. crispus observed under highest stress concentrations of 0.5, 1.0, and 1.0 mM NaHS, respectively (Fig. 2). Alternatively, no significant decrease of SGR was observed in the three plant species when they were exposed to low NaHS concentrations up to 0.05, 0.1 and 0.1 mM. The IAA concentrations of the plants also decreased under high and very high concentrations of NaHS exposure (Fig. 2B,

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Fig. 3. NaHS induced changes in total chlorophyll and Fv/Fm values in Elodea nuttallii, Myriophyllum spicatum and Potamogeton crispus. Values are the means of three replicates ± SE. Bars with different letters are significantly different at P < 0.05.

D, F) for all three plant species. The highest IAA concentrations were observed for P. crispus compared to E. nuttallii and M. spicatum. The total chlorophyll concentrations and Fv/Fm values of exposed plants also decreased significantly under high and very high H2 S concentrations (Fig. 3). For all species, higher total chlorophyll concentrations and Fv/Fm values were observed when plants were exposed to 0.01, 0.1, 0.1 mM H2 S and control conditions (Fig. 3). The oxidative stress of the three submerged macrophyte species, measured as the MDA and H2 O2 concentrations, increased gradually in the three plant species with exposure to high and very high H2 S concentrations (Fig. 4). Activity of the antioxidative enzymes POD and APX also increased significantly under high H2 S exposure (p < 0.01). However, for E. nuttallii and M. spicatum, POD and APX activities were decreased under the exposure of 0.5 and 1.0 mM H2 S concentrations (Fig. 5). Table 2 shows the correlations among total chlorophyll, Fv/Fm, IAA, APX, POD, H2 O2 , MDA, and SGR in the three species of submerged macrophytes. Significant positive correlations were observed among SGR and total chlorophyll (TotChl), and SGR and Fv/Fm for all investigated species. Significant negative correlations were observed for all three species among SGR and MDA, TotChl and H2 O2 , and TotChl and MDA, and for E. nuttallii and P. crispus among IAA and H2 O2 , and IAA and MDA concentrations. In all studied plants, the concentration of H2 O2 was found in significant positive correlation with the MDA concentration. For E. nuttallii and M. spicatum, no significant correlations were observed between H2 O2 and APX or between H2 O2 and POD, when they exposed to 0.5 and 1.0 mM H2 S concentrations (not shown in Table 2). Shoot growth rate (SGR) increased with increasing IAA and total chlorophyll concentrations for all three species (Fig. 6). In contrast, SGR decreased with increasing H2 O2 concentrations for all three plant species. Fig. 7 depicts the antioxidant activities (POD and APX) and MDA content as a function of H2 O2 . MDA concentration

increased linearly with the increment of tissue H2 O2 concentration in all three species. APX and POD activities increased in P. crispus tissues with the increment of H2 O2 , whereas these correlations were either negative or neutral in E. nuttallii and M. spicatum.

4. Discussion In the present study exposure to H2 S clearly reduced the growth and photosynthetic pigment contents in all the investigated plant species in a concentration dependent manner. However, species differed in their response to different concentrations of H2 S, with P. crispus performing (in terms of growth and photosynthetic pigments) the best, followed by M. spicatum, and E. nuttallii showed the least tolerance to H2 S exposure, since there was very little biomass growth at 0.5 mM H2 S exposure. Van Der Welle et al. (2007) also observed absence of biomass increment for E. nuttallii, when this species was exposed to 949 ␮M sulfide concentrations in field experiments, which supports our present results. One possible reason for the decreased growth rate of plants under H2 S toxicity can be attributed to the inability of the plants to take up nutrients (Van Der Welle et al., 2007). The decrease in photosynthetic pigments at high H2 S concentrations was visually observed by the brown discoloration of plants. The damage of the photosynthetic apparatus was also confirmed by the Fv/Fm value which is used as one of the indices of stress in photosystem II (Calatayud and Barreno, 2004). Plants subjected to high NaHS concentrations showed low Fv/Fm values (<0.8). Commonly, the Fv/Fm value of stress-free plants is approximately 0.8 (Maxwell and Johnson, 2000). In the present study, MDA and total chlorophyll concentration were found in significant negative correlation for E. nuttallii, M. spicatum and P. crispus (Table 2). Increased levels of MDA might have degraded the photosynthetic apparatus and chlorophyll pigments were lost as suggested by some researchers (Abdel Latef, 2013; Ahmad et al., 2015).

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Fig. 4. Hydrogen peroxide (H2 O2 ) and malondialdehyde (MDA) levels in Elodea nuttallii, Myriophyllum spicatum and Potamogeton crispus grown under different NaHS (H2 S donor) concentrations. Values are the means of three replicates ± SE. Bars with different lowercase letters are significantly different at P < 0.05.

Fig. 5. Guaiacol peroxidase (POD) and ascorbate peroxidase (APX) activities of Elodea nuttallii, Myriophyllum spicatum and Potamogeton crispus grown in different NaHS (H2 S donor) concentrations. Values are the means of three replicates ± SE. Bars with different lowercase letters are significantly different at P < 0.05.

The oxidative stress imposed on E. nuttallii, M. spicatum and P. crispus after H2 S exposure was shown by the significant increase in H2 O2 and MDA levels (Fig. 4). Malondialdehyde is a major

toxic product of lipid peroxidation, the end product of peroxidized linoleic acid (Møller et al., 2007), and has widely been used as an indicator of free radical production (Zhang et al., 2014; Atapaththu

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Table 2 Pearson correlation coefficients among total chlorophyll (mg/g FW), Fv/Fm, IAA (␮mol/g FW), ascorbate peroxidase (APX, ␮mol/min/g FW), guaiacol peroxidase (POD, ␮mol/min/g FW), hydrogen peroxide (H2 O2 , ␮mol/g FW), malondialdehyde content (MDA, nmol/g FW) and SGR (cm/day) in Elodea nuttallii, Myriophyllum spicatum and Potamogeton crispus. E. nuttallii SGR TotChl Fv/Fm IAA APX POD H2 O2 MDA M. spicatum TotChl Fv/Fm IAA APX POD H2 O2 MDA P. crispus TotChl Fv/Fm IAA APX POD H2 O2 MDA a b

TotChl

Fv/Fm

IAA

APX

POD

H2 O2

0.85 0.80b 0.83b 0.38 0.43 −0.72a −0.71a

0.87b 0.90b 0.22 0.21 −0.81b −0.75a

0.90b 0.13 0.20 −0.96b −0.84b

0.03 0.07 −0.89b −0.85b

0.85b 0.02 0.19

0.001 0.09

0.86b

0.76a 0.77a 0.52 −0.10 0.20 −0.63 −0.79a

0.74a 0.87b −0.12 0.06 −0.83b −0.75a

0.59 −0.36 0.15 −0.75a −0.88b

0.01 0.09 −0.69 −0.60

0.55 0.005 0.22

−0.27 −0.34

0.81b

0.85b 0.80b 0.83b 0.38 0.43 −0.72a −0.71a

0.87b 0.89b 0.22 0.21 −0.80b −0.74a

0.90b 0.13 0.20 −0.97b −0.84b

0.03 0.06 −0.89b −0.85b

0.87b 0.02 0.19

0.001 0.08

0.96b

b

Correlation is statistically significant at the 0.05 level. Correlation is statistically significant at the 0.01 level.

Fig. 6. Relationship between shoot growth rate (SGR) and Total chlorophyll concentration (A–C), SGR and H2 O2 concentration (D–F), SGR and Indolic acetic acid (IAA) concentration (G–I) of Elodea nuttallii, Myriophyllum spicatum and Potamogeton crispus grown in different NaHS concentrations. Values are the means of three replicates ± SE.

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Fig. 7. Relationship between H2 O2 concentration and APX activity (A, D, G), H2 O2 concentration and POD activity (B, E, H), H2 O2 and MDA concentrations (C, F, I), of Elodea nuttallii, Myriophyllum spicatum and Potamogeton crispus grown in different NaHS concentrations. Values are the means of three replicates ± SE.

and Asaeda, 2015). ROS damage plant cells by reacting with proteins, lipids, and other biomolecules (Ahmad et al., 2008). However, the application of lower concentrations of NaHS as the H2 S donor decreases the production of H2 O2 and MDA (Fig. 4). H2 S may play a role in stomatal closure induced by abscisic acid (ABA) (García-Mata and Lamattina, 2010), and subsequently prevents the H2 O2 signaling in guard cells by countering H2 O2 concentrations, thus reducing oxidative stress in leaf and root tissues (Ali et al., 2015). A significant positive correlation in E. nuttallii, M. spicatum and P. crispus was observed between H2 O2 level and MDA content. In addition, a sharp increase in MDA and H2 O2 content in high H2 S-treated plants indicates that oxidative stress increased under higher H2 S concentrations compared to the control (Zhang et al., 2009; Zhang et al., 2011). Zaman and Asaeda (2013) also reported that H2 O2 level and MDA content in E. nuttallii tissue were significantly correlated when grown in anoxic condition. The oxidative stress resistance of a plant depends on the activation of the defense system (de Azevedo Neto et al., 2006), which includes the production of antioxidant compounds and several antioxidative enzymes, such as POD and APX. Among these antioxidative enzymes, POD is considered a stress-marker enzyme having a high affinity for H2 O2 (Andrews et al., 2002; Zaman and Asaeda, 2013). In addition to scavenge H2 O2 , peroxidases are also involved in growth and development of plants (Khan and Panda, 2007). Moreover, there is a threshold level of enzyme activity below which the antioxidative system of a plant is unable to function as ROS scavenger (Xing et al., 2010a). In our study, higher activities of POD and APX were found when E. nuttallii, M. spicatum and P. crispus were exposed to NaHS concentrations of 0–0.1, 0–0.5

and 0–1.0 mM, respectively. For E. nuttallii and M. spicatum the decreases of the POD and APX levels in the presence of 0.5 and 1.0 mM NaHS concentrations indicated that the production of protective antioxidative enzymes fell below the threshold levels and were not enough to neutralize the ROS, and thus plant growth was severely affected. These data were supported by several previous studies when submerged macrophytes were subjected to different heavy metal stress (Prasad et al., 2001; Ding et al., 2007; Xing et al., 2010b). Therefore, NaHS concentrations of 0.05, 0.1 and 0.5 mM in water may reflect the exposure tolerance limit (in terms of SGR and photosynthetic pigments) for E. nuttallii, M. spicatum and P. crispus, respectively. During anoxic events in the aquatic sediment environment, H2 S is produced by the reduction of sulfate and putrefaction (Dunnette et al., 1985). In a major sediment re-suspension event, H2 S production decreases the dissolved oxygen in overlying water, which potentially disrupts the physiological functions of plants and increases the mortality of aquatic plants (Koch et al., 1990; Koch and Erskine, 2001). However, increasing evidence has demonstrated that H2 S also acts as an important signaling molecule for plants, which involves the modulation of physiological traits such as heat tolerance, photosynthetic gene expression, etc. (Zhang et al., 2010; Chen et al., 2011; Li et al., 2013). These studies suggest that the role of H2 S in plants is multi-faceted. Favorable dose dependent exogenous treatments of the H2 S donor (NaHS) can play a protective role in abiotic stress tolerance in Arabidopsis (0.1 mM NaHS) (Shi et al., 2015), and, for Spinacia oleracea, growth, chlorophyll content and photosynthesis increment was observed in a dose-dependent manner, with 0.1 mM NaHS being the optimal

M. Parveen et al. / Flora 230 (2017) 1–11

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internal tissue aeration for leaf surface area, photosynthetic and dark respiration rates as suggested by Koren et al. (2015) and Elgetti Brodersen et al. (2016). Intra-plant O2 can be transported to belowground parts of submerged plants and in the course of transportation and radial loss of the same can oxidize the water column (Colmer, 2003). By adapting a similar process seagrass can detoxify the effect of H2 S in reduced sediment (Hasler-Sheetal and Holmer, 2015).

5. Conclusion

Fig. 8. Nonmetric multidimensional scaling (NMDS) based on the average values of plant response variables measured under NaHS treatments. Bray-Curtis similarity as the pair-wise distances among samples was used for grouping. The label at the centroid of each convex hull groups the macrophyte species of interest. Each species is connected to the cluster centroids by lines using functions ‘ordispider’ and ‘ordihull’ (statistical package ‘Vegan’).

concentration (Chen et al., 2011). Moreover, Ali et al. (2015) and Zhang et al. (2010) also observed that the application of 0.3 mM NaHS and 0.05–0.1 mM NaHS significantly improved plant growth, photosynthetic pigments, and alleviated oxidative stress of Brassica napus and Glycine max plants under Al and drought stress respectively. Fig. 8 represents the Nonmetric Multidimensional Scaling (NMDS) analysis of the response variables of the investigated species of submerged macrophytes under H2 S treatments; the NMDS analysis shows that there are distinct groupings of the plant species (stress = 0.58). This signifies that these species respond to different magnitudes of H2 S. However, though there was grouping among the species due to H2 S stress, the alignment of the convex hull and observed variables, with respect to coordination axes, implies that all species growth parameters (SGR, Chl, IAA, Fv/Fm) were inversely related to stress components (H2 O2 , MDA) and antioxidants (APX and POD) in plant tissue. H2 O2 functions as a stress signal and triggers the production of antioxidants (Ahmad et al., 2008). Therefore, a higher activity of antioxidants in tissue implies that the plants are in stress condition, and hence the growth parameters in our study were found in significant negative correlations with antioxidants. Internal O2 pressure of the aerenchyma tissues of submerged plants is highly correlated with the O2 content in the water column (Pedersen et al., 2004; Borum et al., 2007). Therefore, under anoxic condition, lack of tissue aeration creates a pressure gradient with the water column and H2 S permeates the cell membrane (Brodersen et al., 2015). Nielsen and Sand-Jensen (1989) reported the relative surface area (0.29 ± 0.02, 0.26 ± 0.01, 0.15 ± 0.01 m2 g−1 DW, respectively), photosynthetic rates (17.98 ± 0.34, 9.11 ± 1.04, 5.16 ± 0.45 mg O2 g−1 DW h−1 , respectively), and dark respiration rates (1.09 ± 0.12, 1.10 ± 0.07, 0.54 ± 0.01 mg O2 g−1 DW h−1 , respectively) of P. crispus, M. spicatum, and Elodea canadensis. In our study, P. crispus showed the highest tolerance to H2 S stress (in terms of growth, chlorophyll content, and antioxidative enzymes production) followed by M. spicatum and E. nuttallii. This tolerance sequence of these species might be attributed to the variation of

In the present study, species-specific differences in the response patterns of the plants to NaHS as the H2 S donor indicated that the submerged macrophyte Elodea nuttallii is more sensitive to H2 S than the other two species Myriophyllum spicatum and Potamogeton crispus. Elodea nuttallii, M. spicatum and P. crispus can grow well in the presence of 0.01, 0.1 and 0.1 mM NaHS and can tolerate 0.05, 0.1 and 0.5 mM NaHS, respectively. The above results indicate that the concentrations of H2 S in water can affect the growth of submerged macrophytes; the tolerance of a species to H2 S depends on the morphology and ability to scavenge ROS. Plants with larger relative leaf surface area are more tolerant than smaller-leaved plants.

Acknowledgements A Research Grant-in-Aid (15H04045) from the Japan Society for Promotion of Science, the River Fund by the River Foundation in Japan, and the Education, Culture, Sports, Science and Technology (MEXT) financially supported this research.

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