1 INTRODUCTION

Aquatic macrophytes are fundamental components in littoral zone and play key roles in ecological functions by means of being highly productive primary producer, providing food (Laguna et al., 2016), shelter and nursery habitats (Wetzel, 2001) for aquatic animals, competing with phytoplankton on nutrients (Ibáñez et al., 2012; Dai et al., 2017) and altering nutrients cycle due to growth and death, etc. They occupy a wide range of different habitats from irregular to permanent waterlogged, and develop mainly three life forms including emergent, floating-leaved and submersed. The different life forms are results due to adaptive evolution in order to cope with various inorganic and biotic environments. For instance, all of the leaves of emergent macrophytes (EM) are exposed in air, the whole plant of submersed macrophyte (SM) is surrounded by water, while the upper leaf surface of floating-leaved macrophytes (FM) open to air and the other side down in water. The characters of leaves make difference in gas (CO₂ and O₂) exchanges and light availability. Furthermore, EM and FM species generally experience mechanical damages from predation of large animals due to their higher caloric contents (Boyd, 1968), while SM species are damaged by high nutrients and strong wave force (Zhang et al., 2010; Su et al., 2016; Zhu et al., 2018) and suffer from physiological stress induced by toxins of ammonia, sulfide and microcystins, etc. (Cao et al., 2007; Zhang et al., 2011).

Littoral zone is a buffer between land and open water. It is characteristic of high biodiversity and complex environmental conditions. Intensive interactions were found not only among the organisms but also the organisms and their habitat environments, such as high nutrients input and high intensity of human disturbance, etc. Like other plants, macrophytes have to deal with different stresses from external environment and also metabolism in vivo, and some productions of metabolism are harmful to organisms. One of them is the reactive oxygen species (ROS), includes superoxide anion radical (·O^2-), hydrogen peroxide (H₂O₂), and hydroxyl radical (·OH) (Metcalfe and Alonso-Alvarez, 2010). The ROS producing reaction happens rapidly and strongly, and is harmful to macrophytes, for example, ROS usually cause death of biomolecules and cells, lipid membrane overoxidation and protein denaturation (Bowler et al., 1992; Mittler, 2002).

Except for environmental factors, eutrophication in lakes is another strong stress on the survival of macrophytes. During the process of eutrophication, physical and chemical properties of water change dramatically, such as decreasing transparency, dropping dissolved oxygen level, increasing nutrient content (Anderson et al., 2002; Conley et al., 2009; Qin et al., 2015; Watson et al., 2016). These variations may also change some aspects of physiological process of macrophytes, for example the antioxidant system. It has been found that activities of superoxide dismutase (SOD) and ascorbate oxidase (APX) of Potamogeton crispus L. fitted a lognormal distribution with increasing ammonium levels in an aquarium experiment (Cao et al., 2004), and potentially significant biochemical damage occurred when NH₄-N≥5 mg/L. A field experiment (Zhang et al., 2011) in an eutrophic lake (Taihu Lake, China) showed that synthetic action of ammonium, microcystins and hypoxia arising from cyanobacteria caused oxidative stress in terms of high SOD activity in FM and SM, and FM were more resistant to the stresses of eutrophication.As a result of eutrophication, submerged macrophytes would finally disappear (Ozimek and Kowalczewski, 1984; Phillips et al., 2016), and the aquatic plant community will succeed to the stage with emerged and floating-leaved macrophytes dominated. Such succession could be related with the antioxidant system, as it is vital to the survival of plants.

Plants develop antioxidant system to remove ROS and lead to a dynamic balance between producing and removing of ROS in the normal growth process (Almeselmani et al., 2006). Antioxidant system in plants contains antioxidant enzymes and antioxygen (Shao et al., 2008), it is generally recognized that they are synergistically working in the process of plants' coping with external adversities (Larson, 1988). However, the mechanisms of oxidation resistance in plants are very distinct in different taxa. Antioxidant enzymes are constructional components in all plant cells, in which SOD is the first defensive line of them changing ·O^2- into H₂O₂ and then the other enzymes, such as catalase (CAT) and APX, further clearing the H₂O₂ jointly. While phenolics, a class of secondary metabolic compounds in plants, its antioxidant mechanisms are more complex, mainly being summarized for three pathways (Leopoldini et al., 2004; Leopoldini et al., 2006): 1) transformation of phenolic hydroxyl hydrogen atom; 2) get electronics from highly reactive free radicals; 3) complexation with metal ions stopping the Feton reaction (Fe(Ⅱ)+H₂O₂——Fe(Ⅲ)-OH^-+·OH).

As for the two parts of antioxidant system in plants, former studies have demonstrated that phenolics production concerns with UV radiation (Song et al., 2015), herbivores' predation and pathogens (Lodge, 1991; Dudt and Shure, 1994; Vergeer and Van Der Velde, 1997; Bixenmann et al., 2016; Frew et al., 2016) and are affected by C and N metabolism (Cao et al., 2008), while antioxidant enzymes relate to stresses of nutrient (Cao et al., 2004, 2009; Zhang et al., 2010, 2011), light (Zhang et al., 2010) and heavy metal stresses (Monferrán et al., 2009), etc. But the potential relationship between phenolics and antioxidant enzymes and how the plants evolved different antioxidant system according to different life-forms are still not clear.

In this study, we examined the antioxidant systems of phenolics and enzymes among emergent, floating-leaved and submersed macrophyte, and discuss the relationships between the two antioxidative systems with regard to the plant habitats. Before the study, We supposed that: 1) total phenolic contents in EM and FM should be higher than SM, while antioxidant enzymes activities could be lower on the contrary; 2) As effects of taxonomy on physiological and chemical characteristics of macrophytes are nonnegligible (Smolders et al., 2000; Su et al., 2016), some interspecific differences within each lifeform would be observed; 3) the EM and FM should represent higher adaptability than SM based on the characters of antioxidant system and natural succession of aquatic macrophytes.

2 MATERIAL AND METHOD 2.1 Study area

The study was carried out in two adjacent bays (Haichao Bay and Shaping Bay) in the northern part of Lake Erhai (25°52'N, 100°06'E) in subtropic Yungui Plateau, China (Fig. 1). The lake has a total area of 249.8 km², with moderate water depth (max 20.5 m, mean 10.5 m) and a mesotrophic status. These two bays are rich in similar aquatic macrophyte species and share parallel external environments (He et al., 2015).

2.2 Plant material

Mature leaves in good conditions of 26 aquatic macrophyte species (7 EM, 6 FM and 13 SM) were randomly collected with a long-handled scythe-type sampler (0.2 m² in sampling area) in the two bays (Haichao Bay and Shaping Bay) in zones with water depth lower than 3 m in September 2013. The distance of each two sampling sites is over 30 m. Leaves were cleaned, sorted by species and put into ziplocked bags with waterproof labels. Then all the samples were put into icebox and taken back to laboratory for further analysis. All species belong to 14 families, and the biggest family is Potamogetonaceae (Table 2).

2.3 Measurement

Each plant material in every sampling site was harvested into two replicate samples. One for antioxidant enzymes analysis was ground into fine powder in liquid nitrogen with a mortar and a pestle. 0.5 g fine powder was extracted at 4℃ for 20 min in 5 mL ice-cold buffer (50 mmol/L potassium phosphate, pH 7.8) containing 0.1 mmol/L EDTA, 1 mmol/L PMSF, 1 % (w/v) PVP, then centrifuged at 15 000 r/min for 20 min, at 4℃, and the supernatant was used for antioxidant enzymes examine. Soluble protein content was assayed by the Coomassie blue dyeing method (Bradford, 1976) using bovine serum albumin as a standard. SOD (EC 1.15.1.1) activity was assayed based on a method of T-SOD reagent box from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), one unit of SOD activity is defined as the amount of SOD when its inhibition ratio is 50% of per mg protein in 1 mL reaction liquid. CAT (EC 1.11.1.6) activities was measured according to the modified method (Chance and Maehly, 1955), and one enzyme activity units of CAT is defined as the reduction of A240 by 0.1, respectively. The activity of APX (EC 1.11.1.11) was measured according to (Rout and Shaw, 2001). One enzyme activity unit is defined as oxidation of 1 μmol ascorbate acid of per mg protein per min. The other one for total phenolics (TP) was oven-dried at 80℃ for 48 h to constant weight, and then ground into fine powder with a pestle and mortar. About 50 mg of the powder was extracted with 5 mL of 80 % ethanol at 80℃ for 20 min, then centrifugated for 10 min at 5 000 r/min with supernatant dissociated. The residue was reextracted and recentrifuged, then merged the supernatant for determination of TP which was determined mainly following the method described by Mole and Waterman (1987). Tannic acid (Sigma Chemical Company) was used as a standard. All measurements were carried out in duplicate using a Shimadzu spectrophotometer (UV-2550).

2.4 Data analysis

Mean values and standard errors of all parameters within each macrophyte were calculated from replicates and one-way ANOVA followed by Tukey test (EM 52 replicates, FM 49 replicates and SM 115 replicates) was used to test differences between individual means. Pearson's correlation analysis (EM 52 replicates, FM 49 replicates and SM 115 replicates) was performed for pairs of the signatures using SPSS 22.0 software (SPSS, Chicago, IL), with significance set at P < 0.05. Before analysis, all data were test for normality and homogeneity, and non-normal variables were log10-transformed to get normality. Principal component analysis (PCA, 26 samples) was conducted by CANOCO for windows (version 5) to elucidate the relationship between macrophyte life forms and antioxidants. Map location of sampling sites was made by AutoCAD 2015 (Autodesk, Inc).

3 RESULT 3.1 Background environmental factors

Data showed that water nutrient conditions and macrophytes structures (unpublished data) were similar (Table 1), so were the whole external environments, and the dominant macrophytes were Ceratophyllum demersum and Potamogeton maackianus (He et al., 2015).

3.2 Antioxidant systems

Overall, antioxidants in these three life forms were obviously different and systematic differences were found in them, also prodigious differentiation within each type (Table 2). The average activities of SOD, CAT and APX were 219.4 U/mg protein, 11.4 U/mg protein and 1.0 U/mg protein for EM, and 250.4 U/mg protein, 3.2 U/mg protein and 0.5 U/mg protein for FM, and 411.0 U/mg protein, 2.6 U/mg protein and 1.95 U/mg protein for SM, respectively. The activities of SOD and APX in leaves of SM were much higher than those of EM and FM (P < 0.05), CAT activity in leaves of FM and SM was much lower than those of EM (P < 0.05), and the contents of TP in SM and FM were much higher than that of SM (P < 0.05) (Table 2 and Fig. 2). TP contents among all macrophytes were from 2.7 mg/g dry weight (DW) to 69.4 mg/g DW with mean value of 18.2 mg/g DW. EM and FM have significantly higher TP contents than that with submerged leaves (P < 0.05), which was in accord with previous studies (Smolders et al., 2000; Cao et al., 2008).

In EM, TP were negatively correlated with CAT (R=-0.37, P < 0.05). SOD was negatively correlated with CAT (R=-0.71, P < 0.001), and CAT was positively correlated with APX (R=0.42, P < 0.01) (Table 3). In SM, apparent inhibition among TP and all three enzymes was observed: TP-SOD (R=-0.41, P < 0.001), -CAT (R=-0.47, P < 0.001), -APX (r=-0.22, P < 0.05). While mutual positive correlations between three enzymes were significant: SOD-CAT (R=0.36, P < 0.001), SOD-APX (R=0.36, P < 0.001), and CAT- APX (R=0.26, P < 0.01) (Table 5). However, in FM, there were no obvious correlations among these four antioxidants, except SOD and CAT (R=-0.32, P < 0.05) (Table 4).

In PCA analysis, most EM and FM species were closed to TP which means high TP contents, while SM ones gathered around SOD indicating that these species were characterized by high antioxidant enzymes (Fig. 3).

4 DISCUSSION

In the present study, the activities of antioxidant enzymes (SOD, CAT and APX) and total soluble phenolic content differed greatly among three life forms. The results demonstrated that the aquatic macrophytes developed different antioxidant systems in response to the magnitude of water logging, with the increasing water logging the plants tended to depend more on antioxidant enzymes than phenolics to cope with oxidative stress. Considering submersed macrophytes are difficult in gaining carbon dioxide and experience low light in water, which make it having low photosynthetic carbohydrate production, and thus depend more on antioxidant enzymes——a protein instead of phenolics that consumes much carbohydrates, particularly in eutrophic water where nitrogen and phosphorus are relatively cheap for protein synthesis.

Submersed macrophytes had an obviously higher SOD activity than emergent and floating-leaved macrophytes, this suggested that SM was subjected to more strongly oxidative stress of •O^2- than EM and FM on average. EM and FM living in habitats on or above the water surface with high oxygen concentration have relatively more complex organization structures (Wetzel, 2001), and higher photosynthesis rate (Bowes, 1985) combined with strong photoelectron transfer strength, which likely enable them generating more ROS and consequently showing high SOD activity. While SM under the water surface suffered the lower light intensity due to dense floating macrophyte mats and/or phytoplankton (Barko and Smart, 1981; Klančnik et al., 2018), and excessive absorption of nutrients which is an energy consumption behavior (Cao et al., 2007, 2009). In addition, some studies do have shown that in algal blooming period, FM and SM have a rapid rise in SOD activity, and SM are more vulnerable to stresses of eutrophication than FM (Zhang et al., 2011). Therefore, we speculate that these eutrophic factors might have stronger combined stress on SM than those on EM and FM.

EM and FM have significantly higher caloric contents than SM (Boyd, 1968; Lodge, 1991), thus they might be more attractive to herbivores. They also have high carbon (C) and nutrient contents which effects the synthesis of total phenolics (Cao et al., 2008), therefore they are potential to hold chemical defense against pathogens and herbivore. In addition, stronger intensity of photosynthesis in EM and FM brings higher photoelectronic transfer frequency than in SM, this would consume more energy and generate more ROS. However, in this study, SM produced more ROS which brought more intense oxidative stress reaction under eutrophic water than emergent and floating leaves. This would be a plausible explain for the above results. CAT and APX both take H₂O₂ as catalytic substrate (Mittler, 2002). The reason why CAT activity of SM was lower than those of EM and FM might be linked to the production of H₂O₂.

Phenolics production of plants is affected by environmental factors. For example, high light and low nutrient availability generally increase the production of phenolics by plants (Dudt and Shure, 1994; Vergeer and Van Der Velde, 1997; Cronin and Lodge, 2003). Environments above or on the water and underwater are so distinct that EM and FM are more vulnerable to herbivores and UV radiation than SM. According to Fryer et al. (1970), the number of phytophagous insects was highest for helophytes (semi-submerged) species and much lower for submerged macrophytes, with floating-leaved species having intermediate numbers of phytophagous species. Furthermore, most of EM and FM have a more conservative growth strategy with low turnover rate and underground storage organs such as stolon and rhizomes, which are consistent with a greater investment in chemical defense such as phenolics in these species (Smolders et al., 2000).

Compared to EM and FM, SM are under low predated pressure duo to the low nutrients contents (Yuan et al., 2016) and poor palatability, and low UV radiation which is inhibited by dissolved organic carbon (DOC) in water (Schindler et al., 1996). Hutchinson (1957) found that SM are also less well equipped with alkaloids and other secondary plant compounds than EM and FM. Therefore, we might infer that high content of phenolics is not necessary for SM, otherwise most SM are not well-equipped conditions for synthesis of phenolics. Moreover, some submerged species have low phenolics contents, such as Chara spp. (mean total soluble phenolics content 2.70±0.2 mg/g DW), producing allelopathic substances (Wium-Andersen et al., 1982; Ostrofsky and Zettler, 1986; Pakdel et al., 2013), and ones equipped with spines, which protect them from adverse environment.

In addition, high UV radiation in Lake Erhai might be another reason for the variations of TP in this study. Some scholars also believed that the content of phenolics is mainly related to the degree of photodamage (Close and McArthur, 2002), and the light intensity has positive effects on the synthesis of phenolics (Dudt and Shure, 1994). Herbivores and infections by pathogens, for instance, can induce denovo synthesis of phenolics (Hall, 1999), while differences in nutrients status and light intensity also lead to intraspecific variation in TP levels (Dudt and Shure, 1994; Vergeer and Van Der Velde, 1997). Previous study showed that content of TP was positively correlated with leave C content (R=0.600, P < 0.001) (Yuan et al., 2016), therefore we conjectured that high TP contents of EM and FM might partly be related to the strong light intensity and high carbon dioxide content in the air.

Significant negative relationships between TP and antioxidant enzymes were found in EM and SM in this study, representing that the two antioxidant pathways are reciprocally inhibited to a certain extant among them. Carbon-nutrient balance hypothesis (Bryant et al., 1983) suggests that resources in vivo only be used for synthesis of chemical defensive substances after meeting the normal growth need of plants, and their concentrations are limited by environmental carbon and nutrients availability, which indicate that secondary metabolic compounds like phenolics are depending on the internal carbon and nutrients levels. Related study (Cao et al., 2007) showed that a submerged macrophyte, Vallisneria natans L., absorbed excessive nutrients to produce more carbohydrates for detoxification of excess NH₄⁺ into non-toxic free amino acids. Yuan et al. (2016) found that content of TP was positively correlated with those of carbon (C) and nitrogen (N) in leaves of all these macrophytes in Erhai, and unpublished data showed that activities of antioxidant enzymes also had some correlations with C and N contents. Therefore, these two pathways (antioxidant enzymes and phenolics) might have some competition on resources in vivo, and the low interdependence of them in FM might imply that the resources for antioxidant enzymes and phenolics were sufficient, while those of EM and SM were shortage.

5 CONCLUSION

Antioxidant strategies are diverse among macrophytes with different life forms, emergent and floating-leaved macrophytes both have a strategy with high phenolics might due to their exposure to air where there are high UV radiation and strong predation pressure from herbivores in their habitats. Photosynthetic carbon is relatively cheap in the air compared to the water column, so they might synthesize more phenolics to fight against herbivores, and it might be related to synthesis of lignin which is abundant in emergent macrophytes. While submerged macrophytes were more relying on antioxidant enzymes possibly because they were short of sufficient photosynthetic carbon. Floating-leaved macrophytes have a better balance between phenolics and antioxidant enzymes. However, great interspecific disparities are noticed in the study attributed to the synthetic action of evolution and external environment.

6 DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.