Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Biochemical adaptations of four submerged macrophytes under combined exposure to hypoxia and hydrogen sulphide

  • Mahfuza Parveen,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama, Japan

  • Takashi Asaeda ,

    Roles Conceptualization, Data curation, Funding acquisition, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing

    Affiliations Department of Environmental Science and Technology, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama, Japan, Research Institute of Chuo University, Kasuga, Bunkyo, Tokyo, Japan

  • Md H. Rashid

    Roles Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Writing – original draft, Writing – review & editing

    Affiliations Department of Environmental Science and Technology, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama, Japan, Department of Agronomy, Bangladesh Agricultural University, Mymensingh, Bangladesh

Biochemical adaptations of four submerged macrophytes under combined exposure to hypoxia and hydrogen sulphide

  • Mahfuza Parveen, 
  • Takashi Asaeda, 
  • Md H. Rashid


A hydroponic experiment was performed to investigate the stress responses and biochemical adaptations of four submerged macrophytes, Potamogeton crispus, Myriophyllum spicatum, Egeria densa, and Potamogeton oxyphyllus, to the combined exposure of hypoxia and hydrogen sulfide (H2S, provided by NaHS). The investigated plants were subjected to a control, hypoxia, 0.1mM NaHS, 0.5 mM NaHS, 0.1 mM NaHS+hypoxia and 0.5 mM NaHS+hypoxia conditions. All experimental plants grew optimally under control, hypoxic and NaHS conditions in comparison to that grown in the combined exposure of hypoxia and hydrogen sulfide. For P. crispus and M. spicatum, significant decreases of total chlorophyll and increases in oxidative stress (measured by hydrogen peroxide, H2O2, and malondialdehyde, MDA) were observed with exposure to both sulfide concentrations. However, the decrease in catalase (CAT) and ascorbate peroxidase (APX) from exposure to 0.5 mM NaHS suggests that the function of the protective enzymes reached their limit under these conditions. In contrast, for E. densa and P. oxyphyllus, the higher activities of the three antioxidative enzymes and their anaerobic respiration abilities (ADH activity) resulted in higher tolerance and susceptibility under high sulfide concentrations.


Knowledge regarding submerged macrophytes and environmental factors is essential for understanding aquatic plant ecophysiology and ecosystem productivity. Submerged macrophytes are one of the key components in aquatic ecosystems and play an important role as primary producers. Any negative effects on them can hinder the viability of the aquatic ecosystem. The distribution of submerged macrophytes is dependent on several biotic and abiotic factors such as sediment anoxia [1], water column hypoxia [2,3], water movement [4,5], nutrient availability in both the sediment and water column [68], light availability [9], heavy metals, pH and temperatures. Among them, dissolved oxygen is one of the important environmental factors during the life cycle of submerged macrophytes. In fresh water and coastal marine ecosystems, dissolved oxygen (DO) can drastically change compared to other environmental factors such as flooding, stagnation and eutrophication.

Hypoxia may not act as a stressor alone, and it can co-occur in synergy with other stressors, such as hydrogen sulfide (a common toxic product of anoxic sediment). The pH of the water can have a strong influence on the chemical speciation of sulfide (H2S, HS- and S2-). Although all forms seem to be equally toxic [10], the gaseous H2S will normally prevail over both ionic forms in freshwater systems. Because the pH of most anaerobic soils is buffered at approximately 6–7 as a result of the HCO3- - CO2 buffering mechanism, relative H2S abundance is approximately 60–95% [11]. H2S is produced as a metabolic end product by microbially mediated organic matter decomposition and dissimilatory sulfate reduction in waterlogged soil. Regarding the aquatic plant responses to sulfide exposure, sulfide tolerance in sea grass species is relatively high (2000–6000 μM L-1) [11]. Moreover, larger halophytes show tolerance to high sulfide concentrations (500–1500 μM L-1) compare to other aquatic macrophytes (10–500 μM L-1). Toxicity effects were reported for E. nuttallii, P. compressus, and H. verticillata when they were exposed to 100–600 μM L-1 sulfide concentrations [12,13]. For the present study, two concentrations of H2S supplied by NaHS (0.1 and 0.5 mM) were selected based on previously cited literature and laboratory experiments. By observing differences in sulfide tolerance between species in the literature, it can be hypothesized that the enhancement of plant growth and tolerance to various H2S concentrations is species specific.

Hydrogen peroxide (H2O2) is a common reactive oxygen species (ROS) formed continuously as a by-product of the regular metabolism of oxygen. However, H2O2 levels can increase dramatically under stress conditions, which can cause damage in cells and tissues and seriously disrupt metabolism via the oxidation of membrane lipids, proteins, pigments and nucleic acids. [14]. To overcome this situation, cells are equipped with enzymatic and non-enzymatic mechanisms to eliminate or reduce their damaging effects [15]. Moreover, an effective antioxidant system is vital for keeping intracellular ROS pools at low levels and for processing ROS effectively [16]. The enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), glutathione peroxidase (GPx), and glutathione S-transferase (GST) and ascorbate peroxidase (APX). Submerged macrophytes have certain physiological adaptations to hypoxia and sulfide toxicity, such as antioxidative enzymes increment [17,18] and anaerobic respiration [19]. Alcohol dehydrogenase activity (ADH) is an active anaerobic fermentation enzyme that catalyses the terminal step in alcoholic fermentation [20] and is synthesized favourably under low O2 concentrations. Pyruvate is the end product of glycolysis that is converted to acetaldehyde, which is further converted to ethanol by ADH to generate NAD+ from NADH [21]. Therefore, pyruvate and ADH activity in plants subjected to sulfide and low oxygen stress appears to be an adaptation for anoxia tolerance.

The effects of hypoxia and sulfide on aquatic plants have been extensively studied [13,22,23], and only a few studies have determined the effects of hypoxia and dissolved H2S on submerged macrophytes. The effects of sediment anoxia on submerged macrophytes was evaluated by Zaman and Asaeda [1]. However, the study did not evaluate the effects of sulfide on submerged macrophytes under hypoxia. Therefore, the present study investigated the combined effects of water column hypoxia and exogenous H2S concentration on the biochemical adaptations of four submerged macrophyte species: Potamogeton crispus, Myriophyllum spicatum, Egeria densa, and Potamogeton oxyphyllus. These are cosmopolitan species and occur abundantly in Japan and the rest of the world.


Plant samples and experimental setup

We used data generated from laboratory experiments for the present study. Plants were collected from the rivers for planting and culturing in the tanks where no specific permission was required. We didn’t involve any endangered or protected species in any stage of the study. Plant samples of M. spicatum and P. crispus were collected from the Moto-Arakawa River, a tributary of the Arakawa River in southern Saitama, Japan (36° 7ʹ 30.1ʺ N, 139° 24ʹ 20ʺ E), and E. densa and P. oxyphyllus were collected from the Hofu River, Hiroshima (34° 11’ 390 ʺ N, 131.39’249ʺ E) and the Hii River, Shimane (35° 19´ 52.3ʺ N 132° 46´ 8.8ʺE), Japan, respectively. After collection, they were transported to the laboratory as early as possible and immediately cultured in a growth chamber at a controlled temperature of 23±3°C and a 12:12 (light:dark) photoperiod. The light intensity was maintained at approximately 100 μM m-2 s-1 by using fluorescent lamp tubes. Commercial river sand (DIY, Doite, Japan, 90% <1 mm particle) was used as the substrate. The experimental plants were obtained from these culture tanks. After one month 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’s nutrient solution (HNS) [24]. In total six treatments, control, hypoxia, 0.1mM NaHS, 0.5 mM NaHS, 0.1 mM NaHS+hypoxia and 0.5 mM NaHS+hypoxia were selected for each plant, with three replicates.For the hypoxic treatment, the beaker was placed in a 2.5 L AnaeroJar (Oxoid AG25, Oxoid Ltd., Basingstoke, England) [25] after deoxygenating the water by bubbling with anaerobic gas (a mixture of 9.38% CO2, 10.03% H2 with balanced N2). The oxygen concentration inside the jar was reduced with AnaeroPack (an atmospheric gas generating system, Mitsubishi Company, Japan), which can reduce the oxygen level by generating 10–15% CO2. Anaerobic indicators were used to check the low oxygen level (<0.1%) inside the jar. Such low oxygen and high CO2 under anoxia is a common phenomenon in natural conditions. For the H2S treatment, sodium hydrogen sulfide (NaHS) was used as a hydrogen sulfide (H2S) donor [2629]. For the combination of H2S and hypoxia, the beaker was placed in an AnaeroJar and the medium was deoxygenated with anaerobic gas (same as hypoxia), followed by an application of NaHS to achieve the desired H2S concentrations. The lid of the AnaeroJar closed immediately just after the application of NaHS. The culture medium of each treatment was renewed after 24 hours due to the relatively short half-life of H2S [30]. The experiment was conducted for 3 days as plants exposed to hypoxia+H2S showed brown discoloration. The pH of the solution was maintained at 5.0 to 5.5 using NaOH or HCl for every treatment.

Dissolved H2S and DO measurements in the water

Dissolved H2S was determined colorimetrically by the methylene blue method [31] using a diamine reagent. Four (4) ml of mixed diamine reagent was reacted with 50 ml water samples, and the amount of absorbance was measured spectrophotometrically at 670 nm after 20 minutes. NaHS was used as a calibration standard, and the results were expressed in mM. Dissolved oxygen (DO) was measured using a dissolved oxygen and temperature meter (HI 9146) and expressed as mg L-1.

Determination of chlorophyll, IAA, H2O2, POD, APX and CAT via assays

The chlorophyll (total chl.) content was determined spectrophotometrically by extracting fresh shoots in 5 ml of N,N-dimethylformamide for 24 h in the dark at 4°C [32] and calculated using the equations of Porra et al. [33]; chlorophyll content was expressed as mg g-1 FW.

The concentration of indole acetic acid (IAA), the most abundant form of auxin in plant tissues, was measured using the Salkowski reagent [34]. Approximately 100 mg of fresh weight (FW) plant tissue from the apical tip was ground in 2.5 ml of distilled water and centrifuged at 5,000 × g at 20°C for 15 min. After collecting the supernatant, 1 ml of the extract was added to 2 ml of the Salkowski reagent, and colour development was measured after 1 hr at 530 nm [5]. The results were presented as μg g-1 FW.

For H2O2, POD, APX and CAT assays, approximately 100 mg of fresh plant shoots were extracted in ice-cold phosphate buffer (50 mM, pH 6.0) that contained polyvinylpyrrolidone (PVP). The extractions were centrifuged at 5,000 × g for 20 min at 4°C. The supernatant was collected and immediately stored at -80°C for further analysis.

For the analysis of the endogenous H2O2 concentration, a 750 μl aliquot was mixed with 2.5 ml of 0.1% titanium sulfate in 20% (v/v) H2SO4 [35]. The mixture was centrifuged at 5,000 × g at 20°C for 15 min. The intensity of the yellow colour was measured spectrophotometrically at 410 nm. H2O2 concentrations were estimated using a standard curve prepared from known concentrations of H2O2. The results were presented as μmol g-1 FW. POD (EC was assayed according to the method of Goel et al. [36]. The change in absorbance was recorded at 470 nm in 15 s intervals for 3 min using an extinction coefficient of 26.6 mM-1 cm-1. APX (EC activity was assayed using the methods described by Nakano and Asada [37]. The decrease in absorbance at 290 nm was recorded at every 15 s, and APX activity was determined using the extinction coefficient of 2.8 mM-1 cm-1. CAT activity (EC was determined following the methods of Aebi [38] and was calculated using the extinction coefficient of 40 mM-1 cm-1. APX, POD and CAT activities were presented in μmol min-1 g-1 FW.

Determination of MDA

The level of lipid peroxidation was measured in terms of malondialdehyde (MDA), a product of lipid peroxidation, in plant samples using a thiobarbituric acid (TBA) reaction according to the formula developed by Heath and Packer [39]. Absorbance was measured at 532 and 600 nm where the molar extinction coefficient for MDA was 155 mM-1 cm-1. The results were presented as nmol g-1 FW.

ADH activity and pyruvate content

Alcohol dehydrogenase (ADH) activity (EC was extracted from shoot samples using the methodology described by John and Greenway [40]. Briefly, 50 mg of shoot tissue was ground in liquid nitrogen, and cold extraction buffer was added at 5 ml g− 1. The ADH extraction buffer was 50 mM HEPES (4-2-hydroxyethylpiperazine-1-ethanesulfonic acid) (pH 8.0) containing 5 mM MgCl2, 2 mM cysteine hydrochloride and 2% w/v PVP-40 (polyvinylpyrrolidone, MW ≈ 40,000). The samples were homogenized with a mortar and pestle and centrifuged at 10,000×g at 4°C for 10 min. From the collected supernatant, 0.1 ml enzyme extract was assayed in the presence of 80 μM NADH and 10 mM acetaldehyde in a buffer solution of 40 mM bicine and 5 mM MgCl2 (pH 8.0) [41]. The decrease in absorbance was monitored at 340 nm, and the enzyme activity was calculated using an extinction coefficient of 6.22 mM-1 cm-1 [42]. The ADH activities were presented as μmol min-1 g-1 FW.

The pyruvate content in the plant shoots was determined using the 2,4-dinitrophenylhydrazine method [43]. Approximately 50 mg of apical tip tissue was frozen with liquid N2, ground with 2.25 ml 8% trichloroacetic acid (TCA), and centrifuged at 4900×g at 4°C for 10 min. The supernatant was collected, and a 1 ml aliquot was mixed with 2 ml 8% TCA, 1 ml 0.1% 2,4 dinitrophenylhydrazine and 5 ml 1.5 M NaOH. The pyruvate concentration was calculated from the standard curve generated with known concentrations of sodium pyruvate [44] and expressed as μmol g-1 FW.

Statistical analyses

All experimental data were presented as the means ± SD (n = 3). The data were checked for normality before performing the statistical analysis. All data were subjected to a one-way analysis of variance (one-way ANOVA), followed by Tukey’s multiple comparison test to evaluate the mean differences at a 0.05 significance level (p<0.05). Pearson’s correlations were calculated among chlorophyll content, IAA, antioxidative enzymes, ADH activity, pyruvate content, MDA and H2O2. Statistical analyses were performed using SPSS for Windows (Release 17, SPSS INC., Chicago, IL).


In Fig 1(A), total chl. concentrations were varied among all four plants and treatments. For P. crispus and M. spicatum, total Chl. concentrations decreased significantly (P<0.05) when subjected to 0.1 and 0.5 mM NaHS+Hyp conditions. In contrast, the concentration was not significantly decreased for E. densa and P. oxyphyllus when plants were subjected to the first five treatments, although significant differences (P<0.05) were observed in 0.5 mM conditions. H2O2 and MDA content also increased significantly in the 0.1 and 0.5 mM NaHS+Hyp treatments, regardless of the species (Fig 1(B) and 1(C)). For P. crispus and M. spicatum, antioxidative enzymes (APX and CAT activity) significantly increased (P<0.05) in 0.1 mM NaHS+Hyp conditions, although they decreased in 0.5 mM NaHS+Hyp conditions (Fig 1(D) and 1(E)). Moreover, POD activity increased significantly (P<0.05) under both sulfide conditions (Fig 1(F)). For E. densa and P. oxyphyllus, antioxidative enzymes (APX, CAT and POD activity) significantly increased (P<0.05) in both the Hyp+ 0.1 and 0.5 mM NaHS treatments (Fig 1(D), 1(E) and 1(F)). No significant differences were observed for any of the studied parameters when plants exposed to hypoxic conditions were compared to the control. ADH activity increased significantly (P<0.05) under hypoxic conditions (Fig 1(G)). The increase was significantly different among the four studied plants (P. crispus, 17%; M. spicatum, 45%; E. densa, 70%; and P. oxyphyllus, 68%). In the 0.1 and 0.5 mM NaHS+Hyp treatments, ADH activity increased for E. densa (50% and 30%, respectively) and P. oxyphyllus (47% and 46%, respectively); however, ADH activity decreased for P. crispus (-9% and -30%, respectively) and M. spicatum (-8% and -16%, respectively). Likewise, pyruvate content for P. crispus and M. spicatum decreased significantly (P<0.05) in both treatments (Fig 1(H)); in contrast, the differences were not significant among the treatments for the remaining two species.

Fig 1.

Effects of hypoxia and NaHS on total chlorophyll (A), H2O2 (B), APX activity (C), MDA (D), CAT activity (E), POD activity (F), ADH activity (G) and pyruvate content (H) values in P. crispus, M. spicatum, E. densa and P. oxyphyllus. Values are the means of three replicates±SD. Bars with different letters are significantly different at P<0.05.

Fig 2 shows the correlations between H2O2 and total chlorophyll (Chl.) (Fig 2A to 2D), H2O2 and antioxidative enzymes (CAT+APX+POD) (Fig 2E to 2H), and H2O2 and ADH activity (Fig 2I to 2L) in four plants exposed to different treatments. Strong positive correlations were observed between the ADH activity and H2O2 concentrations, and antioxidative enzymes and H2O2 concentrations in P. oxyphyllus and E. densa compare to P. crispus and M. spicatum. Total Chl. contents were negatively correlated to H2O2 concentration for all plants (Fig 2A to 2D). Positive correlations were observed for antioxidative enzymes (CAT, APX, POD) and H2O2 concentration, irrespective of plant species (Fig 2E to 2H). However, the correlations were highly significant (P<0.01) for E. densa and P. oxyphyllus, significant for P. crispus (P<0.05) and not significant for M. spicatum (P<0.47). Significant negative correlations were observed between ADH activity and H2O2 concentration for P. crispus (R = -0.74) and M. spicatum (R = -0.63), and it was positive (not significant) for E. densa (R = 0.17) and P. oxyphyllus (R = 0.3).

Fig 2.

The correlations between H2O2 and total chlorophyll (A-D), H2O2 and antioxidative enzymes (CAT+APX+POD) (E-H), and H2O2 and ADH activity (I-L) in four plants exposed to different treatments.

The dissolved oxygen (DO) and H2S concentrations of experimental tanks were measured at the beginning and after 24 hours of the experiment (Table 1). For H2S+hypoxia experiment NaHS can produce desired amount of H2S at the beginning of the experiment, but decreased after 24 hours. To keep the desired H2S concentrations the media of every experiment changed after 24 hours. In Table 2 the ANOVA results (P and F values) of every treatment were listed.

Table 1. Dissolved oxygen (DO, mg L-1) and dissolved H2S concentrations (mM) in different treatments measured initially and after 24 hours.

Values are the means of three replicates (n = 3).

Table 2. ANOVA table for total chlorophyll (mg g-1 FW), H2O2 (μmol g-1 FW), APX (μmol min-1 g-1 FW), MDA (nmol min-1 g-1 FW), CAT (μmol min-1 g-1 FW), POD (μmol min-1 g-1 FW), ADH activity (μmol min-1 g-1 FW), pyruvate content (μmol min-1 g-1 FW) in P. crispus, M. spicatum, E. densa and P. oxyphyllus (n = 3).


The present study revealed that the four studied plants have the ability to survive in hypoxic conditions compared to the NaHS+Hyp conditions. Among them, P. oxyphyllus and E. densa have high tolerances to sulfide exposure compared to P. crispus and M. spicatum; additionally, the plants had different strategies in antioxidative responses and anaerobic respiration metabolism. Compared to P. crispus and M. spicatum, the higher antioxidant systems and anaerobic respiration abilities of P. oxyphyllus and E. densa stimulated their tolerances in high sulfide conditions.

The oxidative stress and the responses of antioxidative enzymes of the four submerged macrophytes were negatively affected by the presence of sulfide during water column hypoxia. Compared to control conditions, chlorophyll concentrations were reduced with increasing sulphide+hypoxia exposure for all study plants. The chlorophyll concentrations of E. densa and P. oxyphyllus were not significantly decreased at 0.1 mM NaHS+Hyp, which corroborates the findings of Dooley et al. [45] and Chen et al. [46] in an experiment with Zostera marina and Spinacia oleracea seedlings, respectively. Chloroplast biogenesis might be a partial reason for this phenomenon. Holmer and Bondgaard [47] also reported that chlorophyll a concentrations of eelgrass plant (Z. marina) decreased with increased sulfide concentrations under low oxygen exposure and different sulfide concentrations, thus demonstrating the consistency of the present study.

It was also visually observed that the investigated plants exposed to NaHS+Hyp conditions showed brown discoloration, which was quickly caused by chlorophyll degradation. This reduction can occur due to the accumulation of H2O2, given that chloroplast is one of the main organelles that produce ROS in plant cells. It is reported that H2O2 is a strong inhibitor of photosynthesis, which can inhibit CO2 fixation by 50% due to the oxidation of the thiol-modulated enzymes of the Calvin cycle [16].

Plants have evolved both enzymatic and non-enzymatic scavenging systems to mitigate the overproduction of ROS. In plants, catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD) are considered the most important H2O2 scavengers, and their increasing activities in plants indicate oxidative stress [4850]. The present results suggested that the overproduction of H2O2 under the different stresses was due to a concomitant increase in POD, APX and CAT activities (Fig 1). APX and CAT belong to two different classes of H2O2 scavenging enzymes; APX is responsible for the fine modulation of ROSs for signalling, whereas CAT is responsible for the removal of excess ROSs during stress [51]. Moreover, H2O2 is detoxified to H2O and O2 through CAT activity or through the ascorbate-glutathione cycle via the activity of APX [51]. Antioxidant systems have evolved not to completely remove ROS but to allow these signals to persist within the cellular environment. Hence, high or enhanced antioxidant capacity can be considered beneficial because it desensitizes photosynthesis, and in some cases, enhances the water-water cycle activity. In the present study, CAT and APX activity decreased for P. crispus and M. spicatum when they were exposed to the 0.5 mM NaHS+Hyp treatment, which suggested that these two enzymes were not able to scavenge the overproduction of ROS in a high sulfide environment.

POD is an essential component for plants for growth and senescence processes and is considered a stress marker enzyme with a high affinity for H2O2 [1,52]. It is activated as a short-term stress response [53], affects lignin and ethylene synthesis and the decomposition of IAA, and is involved in resistance against pathogens and promotes wound healing [54]. The results showed that the activities of POD increased to scavenge H2O2 during sulfide exposure in all studied plants. For M. spicatum and P. crispus, POD showed higher activity compared to APX and CAT during exposure to 0.5+hypoxia conditions but was not enough to survive under the reported conditions. During the exposure to high sulfide concentrations, these two plants lost their intrinsic balance due to the disturbance of the membrane system, which was measured as MDA content. MDA is a cytotoxic product of lipid peroxidation and has widely been used as an indicator of free radical production and consequent tissue damage [55]. The experimental results showed that with increasing sulfide concentration, the MDA concentration increased, which had a positive correlation with H2O2. Because there is a threshold of enzyme activity, the protective function of the three enzymes to the membrane system is limited [56]. For P. crispus and M. spicatum, the activities of the two antioxidative enzymes were low at the high sulfide concentration (0.5 mM), suggesting that the functions of the protective enzymes reached their limit under these conditions. The data are supported by several previous studies that evaluated submerged macrophytes subjected to heavy metal stress [5759]. In contrast, for E. densa and P. oxyphyllus, the higher activities of the three antioxidative enzymes resulted in higher tolerance and susceptibility in the high sulfide concentrations. In addition to the antioxidative enzyme pyruvate also have the ability to remove excess H2O2 from the cell [60].

Hypoxia and sulfide are two key environmental stresses found in a freshwater ecosystem. The anaerobic respiration of submerged macrophytes is an important survival mechanism in these stresses [21]. ADH activity catalyses the terminal step in anaerobic fermentation [41], which is necessary for a plant to survive in such conditions [42]. Maricle et al. [41] suggested that the ability to increase ADH activity is an adaptation of estuarine- and flooding-tolerant plants to tolerate their natural habitats, which also contain sulfide. In the present study, high ADH activity and pyruvate content were observed when plants were exposed to hypoxic conditions. This suggests a well-developed capacity of the studied plants to perform anaerobic respiration. A similar trend of ADH activity showed for two wetland macrophytes (S. alterniflora and P. hemitomon) under high sulfide exposure [20]. The ADH activity result is also consistent with several previous studies [21,42]. However, ADH activity is very sensitive to sulfide exposure. The increase in ADH activity of P. oxyphyllus and E. densa under sulfide exposure made them more tolerant than P. crispus and M. spicatum.

In plants, different sulfide tolerance mechanisms were discussed, which included mechanisms of avoiding sulfide exposure, oxidizing sulfide, or excluding sulfide from the body [61] and metabolic adaptations (cytochrome c oxidase and ADH activity) [62]. The present study suggests that the increase in antioxidative enzymes could be another possible mechanism for aquatic plants to become sulfide tolerant in high sulfide environments. This research provides an understanding of the distribution and habitat preferences of submerged macrophytes and can eventually be used as an ecosystem management tool.


  1. 1. Zaman T, Asaeda T(2013) Effects of NH4–N concentrations and gradient redox level on growth and allied biochemical parameters of Elodea nuttallii (Planch.). Flora-Morphology, Distribution, Functional Ecology of Plants 208: 211–219.
  2. 2. Summers JE, Ratcliffe RG, Jackson MB (2000) Anoxia tolerance in the aquatic monocot Potamogeton pectinatus: absence of oxygen stimulates elongation in association with an unusually large Pasteur effect. Journal of Experimental Botany 51: 1413–1422. pmid:10944155
  3. 3. Harada T, Satoh S, Yoshioka T, Ishizawa K (2005) Expression of sucrose synthase genes involved in enhanced elongation of pondweed (Potamogeton distinctus) turions under anoxia. Annals of Botany 96: 683–692. pmid:16033779
  4. 4. Atapaththu KSS, Asaeda T (2015) Growth and stress responses of Nuttall’s waterweed Elodea nuttallii (Planch) St. John to water movements. Hydrobiologia 747: 217–233.
  5. 5. Ellawala C, Asaeda T, Kawamura K (2011) Influence of flow turbulence on growth and indole acetic acid and H2O2 metabolism of three aquatic macrophyte species. Aquatic Ecology 45: 417–426.
  6. 6. Li H, Cao T, Ni L (2007) Effects of ammonium on growth, nitrogen and carbohydrate metabolism of Potamogeton maackianus A. Benn. Fundamental and Applied Limnology/Archiv für Hydrobiologie 170: 141–148.
  7. 7. Cao T, Xie P, Ni L, Wu A, Zhang M, et al. (2007) The role of NH4+ toxicity in the decline of the submersed macrophyte Vallisneria natans in lakes of the Yangtze River basin, China. Marine and freshwater research 58: 581–587.
  8. 8. Feijoó C, García ME, Momo F, Toja J (2002) Nutrient absorption by the submerged macrophyte Egeria densa Planch.: effect of ammonium and phosphorus availability in the water column on growth and nutrient uptake. Limnetica 21: 93–104.
  9. 9. Wu J, Cheng S, Liang W, He F, Wu Z (2009) Effects of sediment anoxia and light on turion germination and early growth of Potamogeton crispus. Hydrobiologia 628: 111–119.
  10. 10. Armstrong J, Armstrong W (2005) Rice: sulfide-induced barriers to root radial oxygen loss, Fe2+ and water uptake, and lateral root emergence. Ann Bot 96: 625–638. pmid:16093271
  11. 11. Lamers LP, Govers LL, Janssen IC, Geurts JJ, Van der Welle ME, et al. (2013) Sulfide as a soil phytotoxin—a review. pmid:23885259
  12. 12. Geurts JJ, Sarneel JM, Willers BJ, Roelofs JG, Verhoeven JT, et al. (2009) Interacting effects of sulphate pollution, sulphide toxicity and eutrophication on vegetation development in fens: a mesocosm experiment. Environmental pollution 157: 2072–2081. pmid:19285368
  13. 13. Wu J, Cheng S, Liang W, Wu Z (2009) Effects of organic-rich sediment and below-ground sulfide exposure on submerged macrophyte, Hydrilla verticillata. Bulletin of Environmental Contamination and Toxicology 83: 497–501. pmid:19565172
  14. 14. Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. The Plant Cell Online 17: 1866–1875.
  15. 15. Sinha S, Saxena R (2006) Effect of iron on lipid peroxidation, and enzymatic and non-enzymatic antioxidants and bacoside-A content in medicinal plant Bacopa monnieri L. Chemosphere 62: 1340–1350. pmid:16219336
  16. 16. Foyer CH, Shigeoka S (2011) Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant physiology 155: 93–100. pmid:21045124
  17. 17. Parveen M, Asaeda T, Rashid MH (2017) Hydrogen sulfide induced growth, photosynthesis and biochemical responses in three submerged macrophytes. Flora 230: 1–11.
  18. 18. KOK L, Bosma W, Maas F, Kuiper P (1985) The effect of short‐term H2S fumigation on water‐soluble sulphydryl and glutathione levels in spinach. Plant, Cell & Environment 8: 189–194.
  19. 19. Jackson M, Colmer T (2005) Response and adaptation by plants to flooding stress. Annals of Botany 96: 501–505. pmid:16217870
  20. 20. Koch MS, Mendelssohn IA, McKee KL (1990) Mechanism for the hydrogen sulfide-induced growth limitation in wetland macrophytes. Limnology and Oceanography 35: 399–408.
  21. 21. Wu J, Dai Y, Rui S, Cui N, Zhong F, et al. (2015) Acclimation of Hydrilla verticillata to sediment anoxia in vegetation restoration in eutrophic waters. Ecotoxicology: 1–9.
  22. 22. Pedersen MØ, Kristensen E (2015) Sensitivity of Ruppia maritima and Zostera marina to sulfide exposure around roots. Journal of Experimental Marine Biology and Ecology 468: 138–145.
  23. 23. DeBusk TA, Dierberg FE, DeBusk WF, Jackson SD, Potts JA, et al. (2015) Sulfide concentration effects on Typha domingensis Pers.(cattail) and Cladium jamaicense Crantz (sawgrass) growth in Everglades marshes. Aquatic Botany 124: 78–84.
  24. 24. Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Circular California Agricultural Experiment Station 347.
  25. 25. Blokhina OB, Chirkova TV, Fagerstedt KV (2001) Anoxic stress leads to hydrogen peroxide formation in plant cells. Journal of Experimental Botany 52: 1179–1190. pmid:11432936
  26. 26. Zhang H, Jiao H, Jiang C-X, Wang S-H, Wei Z-J, et al. (2010) Hydrogen sulfide protects soybean seedlings against drought-induced oxidative stress. Acta Physiologiae Plantarum 32: 849–857.
  27. 27. Hu L-Y, Hu S-L, Wu J, Li Y-H, Zheng J-L, et al. (2012) Hydrogen sulfide prolongs postharvest shelf life of strawberry and plays an antioxidative role in fruits. Journal of Agricultural and Food Chemistry 60: 8684–8693. pmid:22871304
  28. 28. Hou Z, Wang L, Liu J, Hou L, Liu X (2013) Hydrogen Sulfide Regulates Ethylene‐induced Stomatal Closure in Arabidopsis thaliana. Journal of Integrative Plant Biology 55: 277–289. pmid:23134300
  29. 29. Ali B, Qian P, Sun R, Farooq MA, Gill RA, et al. (2015) Hydrogen sulfide alleviates the aluminum-induced changes in Brassica napus as revealed by physiochemical and ultrastructural study of plant. Environmental Science and Pollution Research 22: 3068–3081. pmid:25231737
  30. 30. Napoli AM, Mason-Plunkett J, Valente J, Sucov A (2006) Full recovery of two simultaneous cases of hydrogen sulfide toxicity. Hospital Physician 42: 47.
  31. 31. Cline JD (1969) Spectrophotometric determination of hydrogen sulfide in natural waters. Limnology and Oceanography 14: 454–458.
  32. 32. Rashid MH, Asaeda T, Uddin MN (2010) Litter‐mediated allelopathic effects of kudzu (Pueraria montana) on Bidens pilosa and Lolium perenne and its persistence in soil. Weed Biology and Management 10: 48–56.
  33. 33. Porra R, Thompson W, Kriedemann P (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Acta (BBA)-Bioenergetics 975: 384–394.
  34. 34. Gordon SA, Weber RP (1951) Colorimetric estimation of indoleacetic acid. Plant Physiology 26: 192. pmid:16654351
  35. 35. Jana S, Choudhuri MA (1982) Glycolate metabolism of three submersed aquatic angiosperms during ageing. Aquatic Botany 12: 345–354.
  36. 36. Goel A, Goel AK, Sheoran IS (2003) Changes in oxidative stress enzymes during artificial ageing in cotton (Gossypium hirsutum L.) seeds. Journal of Plant Physiology 160: 1093–1100. pmid:14593811
  37. 37. Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and Cell Physiology 22: 867–880.
  38. 38. Aebi H (1984) Catalase in vitro. Methods Enzymol 105: 121–126. pmid:6727660
  39. 39. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics 125: 189–198. pmid:5655425
  40. 40. John C, Greenway H (1976) Alcoholic fermentation and activity of some enzymes in rice roots under anaerobiosis. Functional Plant Biology 3: 325–336.
  41. 41. Maricle BR, Crosier JJ, Bussiere BC, Lee RW (2006) Respiratory enzyme activities correlate with anoxia tolerance in salt marsh grasses. Journal of Experimental Marine Biology and Ecology 337: 30–37.
  42. 42. Pearson J, Havill D (1988) The effect of hypoxia and sulphide on culture-grown wetland and non-wetland plants II. Metabolic and physiological changes. Journal of experimental botany 39: 431–439.
  43. 43. Lou L, Gao N (2005) Determination of pyruvate by ultraviolet spectrophotometry. Anal Res 24: 11–13.
  44. 44. Lin M-W, Watson JF, Baggett JR (1995) Inheritance of soluble solids and pyruvic acid content of bulb onions. Journal of the American Society for Horticultural Science 120: 119–122.
  45. 45. Dooley FD, Wyllie-Echeverria S, Roth MB, Ward PD (2013) Tolerance and response of Zostera marina seedlings to hydrogen sulfide. Aquatic Botany 105: 7–10.
  46. 46. Chen J, Wu F-H, Wang W-H, Zheng C-J, Lin G-H, et al. (2011) Hydrogen sulphide enhances photosynthesis through promoting chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification in Spinacia oleracea seedlings. Journal of Experimental Botany 62: 4481–4493. pmid:21624977
  47. 47. Holmer M, Bondgaard EJ (2001) Photosynthetic and growth response of eelgrass to low oxygen and high sulfide concentrations during hypoxic events. Aquatic Botany 70: 29–38.
  48. 48. Noctor G, Foyer CH (1998) Ascorbate and Glutathione: Keeping Active Oxygen Under Control. Annual Review of Plant Physiology and Plant Molecular Biology 49: 249–279. pmid:15012235
  49. 49. Zhang F-Q, Wang Y-S, Lou Z-P, Dong J-D (2007) Effect of heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza). Chemosphere 67: 44–50. pmid:17123580
  50. 50. Zhang M, Cao T, Ni L, Xie P, Li Z (2010) Carbon, nitrogen and antioxidant enzyme responses of Potamogeton crispus to both low light and high nutrient stresses. Environmental and experimental botany 68: 44–50.
  51. 51. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends in plant science 7: 405–410. pmid:12234732
  52. 52. Andrews J, Adams S, Burton K, Edmondson R (2002) Partial purification of tomato fruit peroxidase and its effect on the mechanical properties of tomato fruit skin. Journal of experimental botany 53: 2393–2399. pmid:12432031
  53. 53. Domínguez D, García F, Raya A, Santiago R, (2010) Cadmium-induced oxidative stress and the response of the antioxidative defense system in Spartina densiflora. Physiol Plant 139: 289–302. pmid:20210872
  54. 54. Asada K (1992) Ascorbate peroxidase–a hydrogen peroxide-scavenging enzyme in plants. Physiologia Plantarum 85: 235–241.
  55. 55. Møller IM, Jensen PE, Hansson A (2007) Oxidative modifications to cellular components in plants. Annu Rev Plant Biol 58: 459–481. pmid:17288534
  56. 56. Xing W, Huang W, Liu G (2010) Effect of excess iron and copper on physiology of aquatic plant Spirodela polyrrhiza (L.) Schleid. Environmental toxicology 25: 103–112. pmid:19260045
  57. 57. Xing W, Li D, Liu G (2010) Antioxidative responses of Elodea nuttallii (Planch.) H. St. John to short-term iron exposure. Plant Physiology and Biochemistry 48: 873–878. pmid:20829054
  58. 58. Prasad M, Malec P, Waloszek A, Bojko M, Strzałka K (2001) Physiological responses of Lemna trisulca L.(duckweed) to cadmium and copper bioaccumulation. Plant Science 161: 881–889.
  59. 59. Ding B, Shi G, Xu Y, Hu J, Xu Q (2007) Physiological responses of Alternanthera philoxeroides (Mart.) Griseb leaves to cadmium stress. Environmental pollution 147: 800–803. pmid:17175077
  60. 60. Long LH, Halliwell B (2009) Artefacts in cell culture: pyruvate as a scavenger of hydrogen peroxide generated by ascorbate or epigallocatechin gallate in cell culture media. Biochemical and biophysical research communications 388: 700–704. pmid:19695227
  61. 61. Bagarinao T (1992) Sulfide as an environmental factor and toxicant: tolerance and adaptations in aquatic organisms. Aquatic Toxicology 24: 21–62.
  62. 62. Martin NM, Maricle BR (2015) Species-specific enzymatic tolerance of sulfide toxicity in plant roots. Plant Physiology and Biochemistry 88: 36–41. pmid:25635761