Research Article
Effects of Boron and NaCl on Antioxidant Defence Mechanisms in Duckweeds (Spirodela polyrhiza L.)
Department of Molecular Biology and Genetics, Institute of Science, Ibrahim Cecen University, Agri, Turkey
LiveDNA: 90.8716
In many regions, the shorter life of water sources used in plant irrigation has caused low qualification water, notably briny water and recycled wastewater. These water resources are often characterized by elevating saltiness and toxic elements such as Boron (B). This may be able to adversely affect crop and growth1.
Boron threatens all living organisms in terms of health as it is generally found in high concentrations in industrial wastewater, household wastewater, irrigation water and mine residue2,3. Though boron is a necessary micronutrition for plants, its overage might decline in plant upgrowth and product yield, destructive impacts may occur in the end4. B plays a crucial role in many physiological activities such as photosynthesis, glucose transference, cell cleavage, lignifications, the constructional and functional totality of the cell membranes, carbohydrate and RNA metabolisms and respiration etc.5. Because of their particular chemistry in biotic and abiotic constituents of ecosystems, B and its compounds cannot be degraded. So they are discharged to aquatic environments due to inartificial periods, for example, filtrating of salt layer and abrading minerals from rocks6. Owing to its excessive dissolvability and small dimension of unloaded molecules, the remotion of B from polluted effluent is excessively hard, therefore, several techniques like membrane filtration, B picky resins, reverse osmosis, ion exchangers, adsorption and precipitate-coagulation, are utilized for cleaning boron from water7. Although these systems provide wastewater to drainable standards, they need costly investment, advanced shuttle and mending expenditure and usage of chemicals8. So inexpensive and influential application technologies were researched. According to the report, phytoremedial techniques could remove contaminations from domestic, trading, industrial and mining effluent as an eco-friendly, inexpensive and alternating method. As a result of the studies, it has been determined that varied aquatic macrophytes such as duckweed species remove inorganic and organic pollutants in wastewater by accumulation.
Duckweeds, which belong to monocotyledons, are tiny floating water plants and the most well-known species of these water plants are Lemna, Spirodela, Wolffiella, Landoltia and Wolffia9-11. Spirodela polyrhiza, is a member of the Lemnaceae family we used in this study, is one of the most sought-after species in ecotoxicology and plant breeding studies12. This aquatic plant has been used as a sample herb to study nutrients and heavy metal ion uptake works for years due to its brief lifetime, outstanding biomass manufacture, large dispersion in native water ecosystems and persistence to environmental alterations13.
Salinity could negatively affect plant growth, photosynthetic ratio, elementary element contents, carbon assimilation, water relations, nutrition and various metabolic failures13,14. The rise in the quantity of water resulting from the deterioration in the homeostasis caused by salt stress can physiologically disrupt growth and improvement as it triggers the redox systems unbalance in plants15. Boron frequently existed in elevated concentrations in irrigation water and various salts16.
The current study's goal is to find out the productivity of S. polyrhiza in B removal below different salt stress in water. For that purpose, we have grown plants in waters containing 0, 10, 20, 40 and 80 mg L1 of Boron and ranging from 25-50 mM NaCl. We searched the switches in the activity of enzymes CAT, APX and SOD and chlorophyll amounts and the growth of S. polyrhiza. The foundation aim of this search was to appraise the potential of S. polyrhiza used to remove Boron from aquatic ecosystems under salt stress.
Study area: This study was conducted in Central Laboratory of Department of Molecular Biology and Genetics, Ibrahim Cecen University, Agri, Turkey from June, 2019-April, 2020.
Plant materials and experimental design: Spirodela polyrhiza L. was obtained from the manufacturers. After the plants were brought to the laboratory, they were settled in plastic pots containing Hoagland and grown up for 168 hrs (temperature 25±2°C, 115 μmol m2 s1 light with 16 hrs photoperiod) in the upgrowth chamber before the experiment. Spirodela polyrhiza was fixed with 0.1% Hoaglands nutrition solution including 2 mM MgSO4, 2 mM Ca(NO3)2, 1 mM NH4H2PO4, 10 mM KNO3 as macro nutrition, 0.55 μM H2MoO4, 46 μM H3BO3, 0.76 μM ZnSO4·7H2O, 9 μM MnCl2 ·4H2O, 0.32 μM CuSO4 as micronutrition and 78 μM Fe-EDTA like the iron resource17. Boron and NaCl applications were begun on the 169 hrs and went on for 120 hrs, by applying Hoagland’s solution comprising 0, 5, 10, 20, 40 and 80 mg L1 B (pH = 5.8±0.1) prepared using H3BO3 and NaCl (sodium chloride) concentrations ranging from 25-50 mM (Merck, purity>99). Previous experiments decided on boron concentrations. 168 hrs plants, which were identical dimensions as outer view (3±0.2 mm caliber), were chosen for the trial. Three test groups were composed for every concentration and the solutions were altered in each 48 hrs. After 120 hrs, the plants were harvested, cleaned with pure water, frozen at fluid nitrogen and stored at -80°C until further usage. Spirodela polyrhiza was dried at 70°C. Then plants were digested with 10 mL of intense HNO3, using a Milestone Start microwave (Milestone, Italy). Afterwards, the volume of every example was regulated to 25 mL usage deionized water18. Thermo ICP-MS achieved specification of the Boron amounts in real examples (triplicate).
Growth rate assessment: The growth of plants can be evaluated by looking at the number of fronds. Especially, whole fronds were manually counted daily during the 120 hrs test period. Fronds were dried between the towels for 2 min and their fresh weight was detected. Then, RGR (Mean relative upgrowth rate) was estimated according to the equality usage by Drost et al.19:
RGR (Mean relative upgrowth rate) is the mean private growth ratio for a definite experiment duration, Δt is the trial time from i to j, Ni and Nj are the fronds counts in the trial or control (at the time i and j).
Boron analysis of plant samples: The amount of boron in S. polyrhiza fronds was evaluated according to the method of Uruc Parlak. The amount of boron in S. polyrhiza fronds was measured according to Uruc Parlak20. Dried examples of S. polyrhiza fronds were disintegrated (Milestone Start Microwave) with 10 mL of HNO3. The volume of every example was then arranged to 25 mL using double distillate water. An Inductively Coupled Plasma Mass Spectrometer (Thermo Scientific X series 2 ICP-MS, Bremen, Germany) carried detection of the boron contents in real examples.
Determination of the total chlorophyll: The total chlorophyll amounts were detected considering the technique21. Fronds (200 mg) were homogenized in 10 mL acetone (80%). The absorbance of the supernatant fraction of the homogenates centrifuged at 3000 rpm was determined at 663.6 and 646.6 nm by a Spectrophotometer (Thermo Scientific Multiskan Go, Vantaa, Finland). Total chlorophyll pigment concentration was figured out using the equality declared previously22.
Determination of antioxidant enzymes
Extraction for enzyme studies: Fresh plant piece (0.2 g) was homogenized with 2 mL of 50 mM potassium phosphate buffer (pH 7.0) involving 1% polyvinylpyrrolidone and 1 mM EDTA. The homogenate was centrifuged at 15,000 g for 25 min at 4°C23. The supernatants acquired after centrifugation were utilized in enzyme detection studies. Furthermore, the total soluble protein ingredient of the supernatants was appraised concerning Bradford24,25.
CAT (catalase): CAT activity was carried out spectrophotometrically considering the method of Aebi26,27 at 240 nm in enzyme extract (about 50 mg of protein) having 15 mM H2O2 and potassium phosphate buffer (150 mM, pH 7).
APX (ascorbate peroxidase): The APX activity was detected by monitoring the absorption of ascorbate at 290 nm concerning Nakano and Asada's procedure28,29.
SOD (superoxide dismutase): SOD activity was detected by gauging the inhibition of the photochemical degradation of Nitroblue Tetrazolium (NBT)30,31.
Statistical evaluation of results: Entire tests were replicated in triplicate. Worthes shown in the figures indicated the mean worthes±Standard Error (SE) for every boron concentration. One-way analysis of variance (ANOVA) was made to verify the data's changefulness and the effectiveness of the conclusions and Tukey analyses were carried out to define substantial distinctions inter the practices. Unique letters demonstrated significantly dissimilar worths p<0.05.
Influence of B on the development of S. polyrhiza: In this experiment, a wide B dose range was used for S. polyrhiza. During the experimental period, low B concentrations did not significantly increase S. polyrhiza frond counts, whereas high B concentrations had a significant effect on frond counts. Such as seen in Fig. 1, at low B concentrations (10 mg L1), the number of fronds of S. polyrhiza increased from 52-273 duration of the trial. The effect of B and NaCl applied to S. polyrhiza at different concentrations on the Relative Growth Ratio (RGR) is also shown in Fig. 2. Low B and NaCl concentrations during the experiment period did not significantly increase the growth ratio of S. polyrhiza fronds (p>0.05). However, it was cleared that high B and NaCl concentrations have an essential effect on frond growth. For this reason, it can be proposed that frond count is a susceptible point to describe S. polyrhiza growth prevention. When the results were examined, it was concluded that the increase in exposure duration and extreme B and NaCl concentrations depressed the growth of plants during the trial period.
Fig. 1: | Affect of the B on fronds number of the S. polyrhiza |
Fig. 2: | Affect of the B and NaCl on RGR of the S. polyrhiza Asterisk (*) indicates important variation (p<0.05) of the average worth between the example and its corresponding control |
In this case, S. polyrhiza has an elevated phototoxic sensibility toward Boron and NaCl and so the progressively enhance B and NaCl levels can permits the pursuing of toxical impacts of Boron and NaCl with S. polyrhiza in the water ecosystem32.
Dry biomass amount of the S. polyrhiza afterwards 120 hrs of application are indicated in Fig. 3. It is observed that the dry biomass of S. polyrhiza step by step reduces with rising B and NaCl concentrations. Sodium is an element that is not necessary for plant growth but causes cellular damage. The high concentrations of salt (NaCl) in the aquatic ecosystem can cause oxidative stress in plants, reducing water's osmotic potential33,34. Toxicity investigations by different researchers have shown that biomass in plants diminishes significantly with augmenting B concentrations. The reason for this, maybe that S. polyrhiza's, exposing to higher B concentrations due to its capability to accumulate metals and metalloid, such as S. polyrhiza's B, contains more B than other S. polyrhiza grown at low concentrations. As a result of the evaluation, it can be said that S. polyrhiza is a suitable plant species and for bioindicator and toxicology studies.
Fig. 3: | Dry biomass of the S. polyrhiza in the test duration underneath divergent B and NaCl applications Asterisk (*) indicates important variation (p<0.05) of the average worth between the example and its corresponding control |
Fig. 4: | Boron accumulation in S. polyrhiza afterwards 120 hrs treatment Asterisk (*) indicates important variation (p<0.05) of the average worth between the example and its corresponding control |
Boron accumulation in S. polyrhiza: Considering the results of our study, it may be told that S. polyrhiza can be utilized in water lands involving B levels used under 20 mg L1. B accumulation in S. polyrhiza species rose in parallel with rising B concentrations in the experiment vessels. However, a systematic decrease in B accumulation was observed inversely with increasing NaCl concentration (Fig. 4). The maximum B accumulation in S. polyrhiza was detected as 28.77±0.69 mg L1 (only Boron in non-NaCl application). Some conclusions with L. gibba2,3,35, L. minor36,37 and Gerbera38 are in well concur with our results. Moreover, the results acquired in this study showed that high concentrations of B are toxic to S. polyrhiza. Statistical analyses show that there are essential differences in S. polyrhiza leaves accrued in 20, 40 and 80 mg L1 B applications compared to control. Böcük et al.2 demonstrated that B readily went inside the herbs and after that, the herbs indicated B toxicity signs and declining in biomass manufacture.
Boron effect on total chlorophyll content: The impact of B and NaCl on total chlorophyll amounts in S. polyrhiza were demonstrated in Fig. 5. As shown in Fig. 5, the total chlorophyll concentration gradually declined with increasing boron and salt concentration. When evaluated statistically, it was determined that there was no significant difference between 10 mg L1 B administration (including all salt applications) and the control group. Excessive NaCl and B can harm the chloroplast dice and thylakoid through raised manufacture of free radicals, ending in reducing total chlorophyll production39,40. The reduction in photosynthetic pigment amount due to B and NaCl toxicity may cause the diminish in biomass manufacture and growth ratio of S. polyrhiza plants used in our study37. The results of similar studies made with Lemna by Radic et al.41 verify our results. Therefore, it is thought that the growth ratios and chlorophyll amounts of S. polyrhiza species may depend on the B and NaCl concentrations in the waters.
Effects of boron on antioxidative enzymes: Typically, ROS (Reactive Oxygen Species) are unavoidable bi-products of metabolism in plants, respiration and photosynthesis. Antioxidant enzymes are activated to detoxify increased ROS production underneath environmental pressures such as air pollution, water pollution, drought, salt stress, ultraviolet radiation and heavy metals42. CAT activity in S. polyrhiza was linearly rising with increasing B and NaCl concentrations in the experiment vessels (Fig. 6). Furthermore, statistical analyses demonstrated essential distinctions between the control and administration groups throughout the test term (p<0.05).
The gradual increase in CAT activity in S. polyrhiza plants showed that a large amount of ROS is manufactured to harm proteins, membrane lipids, nucleic acid and pigments. Previous studies conducted by various researchers are in good conformance with our outcomes investigating the relation between CAT activities and Band NaCl toxicity in S. polyrhiza plants. De Morais et al.43 obtained data that support our results in their study with Lemna aequinoctialis species. The prior records are in well deal with our outcomes searching to correlate inter CAT activities and B toxicity in the S. polyrhiza35.
APX activity in S. polyrhiza rose gradually with increasing B and NaCl concentration until the test level reaching 80 mg L1 (Fig. 7). As demonstrated in Fig. 7, significant statistical differentiation was determined between nearly whole B and NaCl applications (except for 10 mg L1 application)and the control group (p<0.05). Our study results are consistent with previous research on aquatic plants44.
Fig. 5: | Total chlorophyll amount of S. polyrhiza afterwards 120 hrs treatment Asterisk (*) indicates important variation (p<0.05) of the average worth between the example and its corresponding control |
Fig. 6: | CAT enzyme activities in S. polyrhiza afterwards 120 hrs treatment Asterisk (*) indicates important variation (p<0.05) of the average worth between the example and its corresponding control |
Fig. 7: | APX enzyme activities in S. polyrhiza afterwards 120 hrs treatment Asterisk (*) indicates important variation (p<0.05) of the average worth between the example and its corresponding control |
Fig. 8: | SOD enzyme activities in S. polyrhiza afterwards 120 hrs treatment Asterisk (*) indicates important variation (p<0.05) of the average worth between the example and its corresponding control |
The hyperactivity of peroxidase underneath stress B and NaCl in S. polyrhiza showed its function in stationary detoxification of H2O2. Rise APX and CAT activities may show these enzymes' impressiveness on H2O2 detoxification and conservation toward oxidative harm45. Unlike CAT, APX is more interested in H2O2, so more specific places allow the removal of small amounts of H2O246. For this reason, in ecotoxicological research, the antioxidant capacity of S. polyrhiza can be determined by measurement APX activities.
SOD activity raised parallel with the rising Boron and NaCl concentrations in the experiment containers (Fig. 8). The highest SOD activity for S. polyrhiza was registered at 20 mg L1 (0.949±0.006). Since SOD is the initial enzyme of the defensive mechanism, SOD activity in the detoxification process shows an increasing S. polyrhiza subjected to low B and NaCl levels. Different researchers have obtained similar results concerning SOD activity in the face of toxic stress and confirmed that SOD constitutes the initial defensive to oxidative stress led to metalloids and metals20,35. Besides, essential distinctions amid S. polyrhiza subjected to application groups (except for 80 mg L1) and control group may promote this event statistically (p<0.05).
When we evaluate our results, insufficient antioxidant defensive mechanisms of S. polyrhiza lead to oxidative harm to plants when subjected to B and NaCl. At this point, antioxidant enzymes, described as practical preservative tools, come into play. Our experiment demonstrated that oxidative stress in S. polyrhiza and an increase in almost whole enzyme activities are due to B and NaCl concentration.
While B accumulations in S. polyrhiza increased with the B dose, it decreased inversely with the NaCl concentration. Moreover, the increase in both B and NaCl caused a decrease in the amount of dry biomass. This study's conclusions show that antioxidant enzymes like CAT, APX and SOD, which are thought to play a critical role in the antioxidant defensive mechanism of duckweed under B and NaCl toxicity, may serve as significant biomarkers for the environment.
This study explores plant species elements that can be useful in cleaning contaminated water with low levels of B and NaCl. Spirodela polyrhiza is also an appropriate selection for biomonitoring owing to elevated phototoxic sensibility opposite B and NaCl. As it is known, the phytoremediation method is cost-effective and environmentally friendly. Some studies have been done on B or NaCl with this plant before but their combined effects have not been examined. This study will help to fill the gap on how these two stress factors affect the plant at the same time.