Redox Reactions in Hydrocharis morsus-ranae L. under Industrial Impacts

H. morsus-ranae is a cosmopolitan perennial plant with numerous roots hanging down into the water column. The leaf blade is rounded; the aerenchyma is not developed. The plant has a high growth rate and vegetative reproduction, prefers water bodies with an average trophic status, grows best in low-flow habitats such as swamps, river backwaters, lakes, and reservoirs (Polechonska, Dambiec, 2014; Polechonska, Samecka-Cymerman, 2016).

Sampling of surface waters, sediments, and plant material was carried out in the Chelyabinsk Region (South Ural, Russia) in July 2018-2019. Samples were collected from two sites: the reference site (Site 1) and the impacted site (Site 2). The coastal water of the Irtyash Lake (55°52'22'' N 60°42'16'' E) was used as the reference site (Table 1). This reservoir lake is located in the Kaslinsky district of the Chelyabinsk Region. According to its primary productivity and nutrient content, the Irtyash Lake is a mesotrophic water body.

The Egoza River backwater (55°44'03'' N 60°30'57'' E) near the town of Kyshtym was used as an impacted site (Table 1). There are several industrial facilities in Kyshtym, and due to their activities, various pollutants, including HMs, enter the river. In addition, the increased contents of many HMs are associated with the presence of serpentinite rocks, which contain high concentrations of Ni, Fe, Cr, and Co (Tripti et al., 2021).

Table 1. The pH value, specific electrical conductivity, and total index of toxic load in the study sites

Data presented as Mean ± SE; asterisk (*) indicates significant differences between the study sites atp < 0.05.

The pH and specific electrical conductivity of the water were measured using a pH meter/ conductometer (Hanna Instruments, Germany).

To determine the HM content, water and sediment samples were taken at four points of each site and mixed to prepare a composite sample. The content of metals was determined in three replicates by inductively coupled plasma atomic emission spectrometry (iCAP 6500 Duo, Thermo Fisher, U.S.A.) after wet digesting with 70 % HNO3. As an integrated indicator of water and sediment pollution, the total index of toxic load (Si) was used, which was calculated using the formula (Bezel et al., 1998):

where, Cj is the concentration of metal in water/ sediments of the impacted site; Creference is the concentration of metal in the water/sediments of the reference site; n is the number of metals studied.

Physiological and biochemical characteristics such as the rates of lipid peroxidation, content of carotenoids, free proline, phenolic compounds, soluble thiols and proteins were measured in the leaves of H. morsus-ranae spectrophotometrically on a PD-303UV (APEL, Japan), according to standard methods.

The rate of lipid peroxidation was determined by the reaction of malonic dialdehyde (MDA) with thiobarbituric acid (TBA). Absorbance was measured at 532 and 600 nm (Heath, Packer, 1968). The contents of photosynthetic pigments (chlorophylls and carotenoids) were measured at 470, 647, and 663 nm after extraction in 80 % acetone. The carotenoid content was calculated according to Lichtenthaler (1987). Free proline content was determined after extraction in hot water (95 °C) and boiling in a water bath for 20 min (Kalinkina et al., 1990). For staining, a mixture of ninhydrin reagent with glacial acetic acid (1:1) was used; absorbance was measured at 520 nm. The total content of soluble phenolic compounds was determined at 760 nm using the Folin-Chiocalteu reagent after preliminary extraction with 70 % ethanol solution for 24 hours. Gallic acid was used as a standard (Singleton et al., 1999). The extraction and determination of protein and non-protein thiols were performed as described by Borisova et al. (2016). The total content of soluble thiols was determined after reaction with Elman's reagent (5.5'-dithiobis (2-nitrobenzoic) acid) at 412 nm. The content of protein thiols was calculated by subtracting the amount of non-protein thiols previously obtained by precipitation of proteins with 50 % trichloroacetic acid from the total soluble fraction. Reduced glutathione was used as a standard (Borisova et al., 2016). The content of soluble protein was determined at a wavelength of 595 nm according to Bradford (1976). Bovine serum albumin was used as a standard. Determination of physiological and biochemical parameters was carried out in 4 biological and 3 analytical replicates (n = 12). All parameters were measured on fresh plant material and then calculated as per one g of dry weight (DW).

The significance of differences was assessed using the nonparametric Mann-Whitney test at a significance level of p < 0.05. The tables and figures show the mean values (Mean) and their standard errors (SE); asterisks indicate significant differences between the study sites.

Results and discussion

The study sites did not differ significantly in water pH, while the specific electrical conductivity of the surface waters in the impacted site (the Egoza River) was 1.4 times higher than in the reference one (the Irtyash Lake, Table 1).

The total index of toxic load at the impacted site (Site 2) was calculated from the contents of four metals (Fe, Zn, Mn, and Ni). The toxic load for water was 1.7 times higher than for sediments. The average value of this index for surface waters and sediments was 2.7 (Table 1).

The maximum difference between sites in the contents of metals was found for Fe (5.6 times), Zn (2.4 times), Mn (2.2 times), Ni (3.0 times), and Co (4.0 times) in water, and for Ni, Pb, and Zn in sediments (4.2, 2.7, and 1.6 times, respectively) (Table 2).

Most of the concentrations of metals in water exceeded the maximum permissible concentrations (MPC) for fishery water bodies (Water quality standards ..., 2016). The highest excess of MPC was noted for mercury in the water of Site 2 (66 times), while for zinc, iron, and manganese it was 5 times higher on average. Among the metals studied, the most toxic to plant organisms are Hg, Co, Ni, Cu, and Pb (Kabata- Pendias, Mukherjee, 2007). Their contents in Site 2 were higher both in water and in sediments. The exception was Pb, whose concentration in the water of the impacted site was slightly lower than in the reference one (Table 2).

As is well known, an excess of HMs in the habitat causes inhibition of photosynthesis in plants, impaired transport of assimilates and mineral nutrition, growth inhibition, and changes in the water and hormonal status. Increased concentrations of HMs (especially of such redoxactive ones as copper, iron, and manganese) can promote the generation of reactive oxygen species (ROS) (Blokhina et al., 2003; Gill, Tuteja, 2010). Lipid peroxidation, whose rates are assessed by the MDA content, is a reaction indicating damage to cell membranes (Blokhina et al., 2003; Sharova, 2016).

An assessment of the lipid peroxidation rate in the leaves of H. morsus-ranae showed that the MDA content in plants in Site 2 was 1.2 times higher than in Site 1 (Fig. 1a). An increase in the MDA content in the plants under anthropogenic load indicates the development of oxidative stress, despite the fact that most metals accumulate in the H. morsus- ranae roots.

The concentration of ROS formed in the cell is maintained at a sufficiently low level by a multicomponent antioxidant defense system, the state of which largely determines plant resistance to stress (Blokhina et al., 2003; Sharova, 2016). ROS and antioxidants interacting with them are presently considered as redox-active molecules, which are the main participants in redox processes (Pradedova et al., 2017). As is well known, ROS in plants are involved in the regulation of many vital processes. At the same time, antioxidants play a leading role in the understanding of the physiological functions of ROS (Sharova, 2016; Pradedova et al., 2017).

Table 2. The contents of heavy metals in water and sediments of the study water bodies. Metal content in water, gg/L Metal content in sediments, mg/kg

Data presented as Mean ± SE; asterisk (*) indicates significant differences between the study sites atp < 0.05.

Carotenoids are the multifunctional compounds of plants. Not only do they take part in the absorption of light energy and the protection of green pigments from photodegradation, but they also have antioxidant activity (Strzalka et al., 2003). The content of carotenoids in H. morsus-ranae in Site 2 was significantly (1.4 times) lower than in Site 1 (Fig. 1b). A decrease in the carotenoid content in a habitat contaminated with metals was also noted in our previous study of the helophyte Typha latifolia L. (Maleva et al., 2019). Carotenoid molecules have double bonds, and they are oxidized when interacting with ROS (Strzalka et al., 2003). Thus, a decrease in the content of carotenoids in plants from contaminated habitats is apparently a consequence of their oxidative degradation.

Fig. 1. Contents of malondialdehyde (a), carotenoids (b), free proline (c), and soluble phenolic compounds (d) in the leaves of H. morsus-ranae. Data presented as Mean ± SE; asterisk (*) indicates significant differences between the study sites atp < 0.05

Proline is a proteinogenic heterocyclic amino acid, which plays an important role in the plant cells. It is involved not only in osmoregulation, but also in the stabilization of proteins, membranes, and subcellular structures. Proline is also able to chelate HMs, maintain cellular redox potential, and participate in ROS neutralization (Hare, Cress, 1997; Sharova, 2016). The free proline content in H. morsus- ranae from Site 2 increased by 26 % compared to Site 1 (Fig. 1c), which indicates its active role in the adaptation of the macrophyte to the environmental pollution.

Many phenolic compounds are known to have antioxidant properties. Due to OH groups, phenols can participate in the detoxification of ROS (Sharova, 2016). Phenolic substances readily interact with reactive oxygen species. Initially, they are oxidized to phenoxyl radicals, the further oxidation of which leads to the formation of quinones. They can also chelate HMs and stabilize membranes, which limits the diffusion of free radicals and reduces the rate of lipid peroxidation (Michalak, 2006). As a rule, under stress, the synthesis of phenolic compounds is enhanced (Pourcel et al., 2007; Sharova, 2016). However, the present study demonstrated that the content of soluble phenolic components in the leaves of H. morsus-ranae in Site 2 was 17 % lower than in Site 1 (Fig. 1d). It can be assumed that, as a result of the high level of toxic load, the rate of destruction of phenols was higher compared to its synthesis reactions. Interestingly, our previous study of the helophyte plant Typha latifolia L. revealed an opposite trend for phenolic compounds (Maleva et al., 2019). That species demonstrated higher tolerance to industrial pollution than H. morsus- ranae. At the same time, they share similar trends in changes of the contents of other non-enzymatic antioxidants, which indicates a major role of these secondary metabolites in the formation of tolerance to HMs.

Compounds containing SH-groups (thiols), which can be divided into protein and nonprotein ones, play an important part in the antioxidant protection of plants. Thiols can both bind HMs and act as antioxidants, participating in the neutralization of ROS formed during oxidative stress (Cobbett, 2000; Sharova, 2016). A previous study demonstrated that the contents of soluble thiols in different species of aquatic plants correlated with the accumulation of HMs (Borisova et al., 2016). The present study showed that the content of soluble thiols in the contaminated site (Site 2) was significantly (1.2 times) higher than in the reference site (Fig. 2a). Moreover, protein thiols prevailed over nonprotein ones: their content was about 86 % of the total amount of soluble thiols.

Many proteins are involved in the antioxidant defense system of plants. They are capable of both directly chelating HMs and acting as enzymes, catalyzing the reactions of ROS neutralization (Kulaeva, Tsyganov, 2011). Determination of the total content of soluble protein revealed an increase in its amount in plants from Site 2 compared to the reference ones (by an average of 24 %, Fig. 2b).

Fig. 2. Contents of soluble thiols (a) and protein (b) in the leaves of H. morsus-ranae. Data presented as Mean ± SE; asterisk (*) indicates significant differences between the study sites atp < 0.05.

Thus, the study of the H. morsus-ranae redox reactions to environmental pollution showed that the development of oxidative stress was accompanied by the accumulation of proline, soluble thiols, and proteins in cells, suggesting activation of their synthesis under anthropogenic load.

Conclusion

Comparative analysis of the redox reactions of the floating macrophyte H. morsus-ranae from natural habitats with different levels of industrial impact revealed some adaptive features of the plant. The increased level of lipid peroxidation products in the leaves of H. morsus- ranae from the impacted site indicates oxidative stress. At the same time, the macrophyte demonstrated a fairly high resistance to the pollution of the habitat by heavy metals, which was probably due to the increased synthesis of such non- enzymatic antioxidants as proline and soluble protein thiols.

References

Bezel V.S., Zhuikova T.V., Pozolotina V.N. (1998) The structure of dandelion cenopopulations and specific features of heavy metal accumulation. Russian Journal of redox reactions hydrocharis, 29(5): 331-337

Blokhina O., Virolainen E., Fagerstedt K. V. (2003) Antioxidants, oxidative damage and oxygen deprivation stress: a review. Annals of Botany, 91(2): 179-194

Borisova G., Chukina N., Maleva M., Kumar A., Prasad M. N.V (2016) Thiols as biomarkers of heavy metal tolerance in the aquatic macrophytes of Middle Urals, Russia. International Journal of Phytoremediation, 18(10): 1037-1045

Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2): 248-254

Brekhovskikh V. F., Volkova Z. V., Savenco A. V. (2009) Higher aquatic vegetation and accumulation processes in the delta of river Volga. Arid Ecosystems [Aridnye ekosistemy], 15(3): 34¬45 (in Russian)

Cobbett C. S. (2000) Phytochelatins and their roles in heavy metal detoxification. Plant Physiology, 123(3): 825-832

Galczynska M., Mankowska N., Milke J., Busko M. (2019) Possibilities and limitations of using Lemna minor, Hydrocharis morsus-ranae and Ceratophyllum demersum in removing metals with contaminated water. Journal of Water and Land Development, 40: 161-173

Gill S. S., Tuteja N. (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 48(12): 909-930

Hare P. D., Cress W. A. (1997) Metabolic implications of stress-induced proline accumulations in plants. Plant Growth Regulation, 21(2): 79-102

Heath R.L., Packer L. (1968) Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics, 125(1): 189¬198

Kabata-Pendias A., Mukherjee A. B. (2007) Trace elements from soil to human. Heidelberg, Springer-Verlag, 550 p.

Kalinkina L.G., Nazarenko L. V., Gordeeva E. E. (1990) Modified method for extraction of free amino acids and their determination in amino acid analyzer. Soviet Plant Physiology [Fiziologiya rastenii], 37: 617-621 (in Russian)

Kulaeva O.A., Tsyganov V. E. (2011) Molecular-genetic basis of cadmium tolerance and accumulation in higher plants. Russian Journal of Genetics: Applied Research, 1(5): 349-360

Lichtenthaler H.K. (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology, 148: 350-382

Maleva M. G., Borisova G. G., Shiryaev G. I., Kumar A., Morozova M. V. (2019) Adaptive potential of Typha latifolia L. under extreme technogenic pollution. AIP Conference Proceedings, 2063: 030013

Michalak A. (2006) Phenolic compounds and their antioxidant activity in plants growing under heavy metal stress. Polish Journal of Environmental Studies, 15(4): 523-530

Polechonska L., Dambiec M. (2014) Heavy metal accumulation in leaves of Hydrocharis morsus- ranae L. and biomonitoring applications. Civil and Environmental Engineering Reports, 12(1): 95-105

Polechonska L., Samecka-Cymerman A. (2016) Bioaccumulation of macro- and trace elements by European frogbit (Hydrocharis morsus-ranae L.) in relation to environmental pollution. Environmental Science and Pollution Research, 23(4): 3469-3480

Polechonska L., Samecka-Cymerman A., Dambiec M. (2017) Changes in growth rate and macroelement and trace element accumulation in Hydrocharis morsus- ranae L. during the growing season in relation to environmental contamination. Environmental Science and Pollution Research, 24(6): 5439-5451

Pourcel L., Routaboul J. M., Cheynier V., Lepiniec L., Debeaujon I. (2007) Flavonoid oxidation in plants: from biochemical properties to physiological functions. Trends in Plant Science, 12(1): 29-36

Pradedova E. V., Nimaeva O. D., Salyaev R. K. (2017) Redox processes in biological systems. Russian Journal of Plant Physiology, 64(6): 822-832

Sharova E. I. (2016) Antioxidants of plants. St. Petersburg, Publishing House of St. Petersburg University, 140 p. (in Russian)

Singleton V. L., Orthofer R., Lamuela-Raventos R.M. (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods in Enzymology, 299: 152-178

Strzalka K., Kostecka-Gugala A., Latowski D. (2003) Carotenoids and environmental stress in plants: significance of carotenoid-mediated modulation of membrane physical properties. Russian Journal of Plant Physiology, 50(2): 168-173

Tripti, Kumar A., Maleva M., Borisova G., Chukina N., Morozova M., Kiseleva I. (2021) Nickel and copper accumulation strategies in Odontarrhena obovata growing on copper smelter-influenced and non-influenced serpentine soils: a comparative field study. Environmental Geochemistry and Health, 43(4): 1401-1413

Water quality standards for fishery water bodies, including standards for maximum permissible concentrations of harmful substances in the waters of fishery water bodies: Appendix to the Order of the Ministry of Agriculture of Russia dated December 13, 2016 N552 (as amended on March 10, 2020). http://docs.cntd.ru/document/420389120 (in Russian)

Размещено на Allbest.ru