Abstract: Background and Objectives: The aquatic macrophytes (hydrophytes) can be used as biological indicators in addition to physical and chemical parameters for determining and monitoring water quality. Hence, the main aim of this study were to examine the phytoremediation of three types of hydrophytes (floating, emergent and submerged) and to investigate the effect of metals accumulation in protein and DNA of these three species. Methodology: Nine studied hydrophytes collected from two governorates El-Dakahlia and Damietta in Egypt in the spring of 2017 as follow: floating (Eichhornia crassipes, Nymphaea lotus, Pistia stratiotes), emergent (Echinochloa stagnina, Ludwigia stolonifera, Persicaria salicifolia), submerged (Ceratophyllum demersum, Myriophyllum spicatum, Potamogeton pectinatus), in addition to water and hydro soil samples for these samples. Heavy metals in plant, hydro-soil (sediments) and water samples were carried out and the concentrations in ppm were recorded. The SDS-PAGE was done using the seeds of investigated taxa. In addition, two molecular markers RAPD and ISSR using five primers in each were used based on fresh young leaves of the investigated taxa. Results: This present investigation confirmed that, emergent hydrophytes had the ability to accumulate the highest mean content of different heavy metals (Zn, Cu, Co, Cd and Pb) as 7.67, 4.06, 1.93, 2.97 and 6.65 ppm, respectively with the metals concentration uptake ordered as Zn>Pb>Cu>Cd>Co. Persicaria salicifolia collected from Kafr-Al-Tawila (Talkha), Dakahlia accumulated the highest content of mean values of heavy metals as Zn 8.40, Cu 4.62, Co 2.21, Cd 3.40 and Pb 7.73 ppm. The RAPD marker had the highest polymorphism percentage (85.71%) than ISSR (55.17%). Conclusion: This study concluded that Emergent hydrophytes can accumulate content of heavy metals higher than floating and submerged. The higher accumulation for heavy metals was recorded in Persicaria salicifolia.
INTRODUCTION
Hydrophytes are defined as water plants with perennating buds below the water surface and helophytes as marsh plants with perennating buds at or just above the soil surface1. Herbaceous hydrophytes classified according to Hess and Hall2 into wet land hydrophytes and aquatic hydrophytes. The wet land types grow in soils saturated with water during a major part of the growing season e.g., Echinochola crusgalli, E. stagnina and Persicaria salicifolia. On the other hand, the aquatic types usually occur in soils covered with water during a major portion of the growing season, these are subdivided into: (a) Floating hydrophytes e.g., Lemna gibba, Pistia stratiotes and Eichhornia crassipes, etc., (b) Emergent hydrophytes e.g., Cyperus articulates, Phragmites australis and Ludwigia stolonifera, etc., (c) Submerged hydrophytes e.g., Ceratophyllum demersum, Myriophyllum spicatum and Potamogeton pectinatus, etc3,4.
Phytoremediation (the use of plants to remove or break down toxic contaminants in the environment) is a great research interest. Plants that can absorb and store heavy metals can be used to remove those pollutants from an ecosystem. Aquatic plants can accumulate elements through their roots, stems and leaves5. Various species showed different capacities for metal uptake and the use of these species for bioremediation has numerous economic and ecological benefits including low cost, high efficiency, energy saving and prevention of secondary pollution6. Pistia stratiotes L. (water lettuce) can be used for treatment of eczema ulcers, stomach disorder and mouth inflammation in addition to its antifungal and antimicrobial properties against harmful diseases. Pistia stratiotes has ability for absorption of heavy metals without any toxicity and reduction in growth due to metal accumulation and has wide range of tolerance to metals7.
Floating Eichhornia crassipes can be used as a bioindicator of water quality in rivers and lakes. It is used as sedative, cooling, febrifuge and diuretic and in addition to having phytochemical compounds as steroids, phenalene and humic acid. From its flowers anthocyanins are produced8. Nymphaea Lotus is aquatic plant used in traditional medicine as analgesic, cardiotonic, anodyne and anti-inflammatory agent oxidants9. Nymphaea lotus is rich in phytochemicals and a good source of natural products10. The extract of emergent Ludwigia stolonifera has a potential affect in antidiabetic and immunosuppressive activities. The L. stolonifera contain triterpene acids and flavonoids in its aerial parts11. Persicaria salicifolia is emergent hydrophytes has phytochemical compounds e.g., flavonoids and glycosides. It has highest activity against prostate carcinoma in addition to its antioxidant activity12. Submerged Myriophyllum spicatum is a preferred aquatic plant in phytoremediation and is tolerant to a wide range of water quality conditions and can be regarded as a pioneering species. It can quickly colonize polluted waters that are unsuitable for other species13. Potamogeton pectinatus has antioxidant activity and accumulate heavy metals in large amounts14. Molecular markers, DNA fingerprints based on ISSR and RAPD methods are generally used to effectively indicate genetic relationships. Emission of toxic metals entering the biosphere in many places worldwide is becoming very dangerous, nowadays, resulting in water, soil and food contaminations. So, the main aim of the present work were to investigate the phytoremediation of hydrophytes and studying the effect of metals accumulation in protein and DNA of some different hydrophytes including floating, submerged and emergent hydrophytes.
MATERIALS AND METHODS
Nine samples of hydrophytes plants including three species from each floating, emergent and submerged hydrophytes collected from different drainage (two governorates) in Egypt (Table 1).
Heavy metal analysis: Aerial parts of plants (above ground) were collected and washed under running water then they were air dried for 1 week and grinding it into powder. Plant digestion occur using nitric acid, then different heavy metals were determined by using GSC on the ECA flow 150 GLP device. Samples of hydro-soil in addition to water were analyzed using the same method.
Biochemical analysis (protein SDS-PAGE): The method of discontinuous SDS-PAGE technique was based on Laemmli15 to identify the relationships between the different types of hydrophytes.
Molecular analysis: The bulked DNA extraction was performed using DNeasy Plant Mini Kit (QIAGEN). Polymerase chain reaction (PCR) condition for RAPD.
The DNA amplifications were performed using five primers of RAPD (Table 2) in an automated thermal cycle (model Techno 512) programmed for one cycle at 94°C for 4 min followed by 45 cycles of 1 min at 94°C, 1 min at 37°C and 2 min at 72°C. The reaction was finally stored at 72°C for 10 min.
| Table 1: | List of hydrophytes plants collected from different localities in Egypt |
| Table 2: | List of RAPD and ISSR primer name and the sequences used in this study |
RAPD: Random amplified polymorphic DNA, ISSR: Inter simple sequence repeat | |
Polymerase chain reaction (PCR) condition for ISSR: The DNA amplifications were performed using five primers of ISSR in an automated thermal cycle (model Techno 512) programmed for one cycle at 94°C for 4 min followed by 45 cycles of 1 min at 94°C, 1 min at 57°C and 2 min at 72°C. The reaction was finally stored at 72°C for 10 min (Table 2).
Data analysis: All gels were photographed and analyzed using Bio-Rad video documentation system, Model Gel Doc 2000. The presence or absence of each band was treated as a binary character in a data matrix (coded 1 and 0, respectively). Cluster analysis and Biplot mapping were conducted to generate the possible relationships among 10 taxa based on biochemical and molecular attributes using the SYSTAT version 7.0 program16.
RESULTS
Heavy metals: The higher mean content of heavy metals (Zn, Cu, Co, Cd and Pb) in plant leaves were found in emergent than floating and submerged hydrophytes as 7.67 Zn, 4.06 Cu, 1.93 Co, 2.97 Cd and 6.65 Pb. Emergent hydrophyte (Persicaria salicifolia) collected from Kafr-Al-Tawila (Talkha), Dakahlia accumulates the highest content of mean values of heavy metals as Zn 8.40, Cu 4.62, Co 2.21, Cd 3.40 and Pb 7.73. The lowest value of heavy metals accumulated by hydrophytes was observed in Ceratophyllum demersum collected from New Damietta as Zn 5.01, Cu 2.20, Co 1.00, Cd 1.69 and Pb 3.61. Comparing the heavy metals uptake by hydrophytes tissues, the uptake of heavy metals was ordered as Zn > Pb > Cu> Cd >Co (Table 3).
Biochemical analysis: Protein electrophoresis analysis was performed for 9 hydrophytes using SDS-PAGE method. The banding patterns of SDS-PAGE gel was represented in Fig. 1, while the band pattern for the hydrophytes is illustrated in Table 4. Eight total bands were generated from the protein gel with molecular weight ranged from 11-127 KDa. In floating hydrophytes the total bands were 7 bands with 4 monomorphic and 3 polymorphic bands. For submerged hydrophytes, there were 7 total bands classified into 4 monomorphic and 3 polymorphic bands, two bands from polymorphic bands were unique for Potamogeton pectinatus with molecular weight of 11 and 115 KDa. Regarding for emergent hydrophytes, 7 total bands were recorded with 6 monomorphic bands. The highest percentage of polymorphism 42.85% was reported in floating and submerged, while the lowest percentage of polymorphism 14.28% was found in emergent hydrophytes as shown in Table 4.
Molecular analysis (RAPD and ISSR): For RAPD and ISSR molecular marker, five primers were used in each marker. The amplification profiles of five RAPD primers are shown in Fig. 2. For this purpose, total 9 taxa were studied. Similarly, the amplification profiles of five ISSR primers for 9 taxa can be observed in Fig. 3.
For RAPD molecular, The highest percentage of polymorphism was reported by OP-D03 in floating hydrophytes (80%), while the lowest percentage was observed by OP-A15 in floating aquatic plants (33.33%) (Table 5). For ISSR marker, out of a 29 total bands with 13 monomorphic bands and 16 polymorphic bands were produced.
| Table 3: | Values of some heavy metal contents in hydrophytes plants, sediments and water from its location in ppm |
| Fig. 1: | Polyacrylamide gel illustrating Leaf protein bands of nine studied taxa: (M) Marker |
1: Eichhornia crassipes, 2: Nymphaea lotus, 3: Pistia stratiotes, 4: Ceratophyllum demersum, 5: Myriophyllum spicatum, 6: Potamogeton pectinatus, 7: Echinochloa stagnina, 8: Ludwigia stolonifera, 9: Persicaria salicifolia |
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These bands with molecular weight size of 150-1370 bp, the polymorphism of ISSR marker was 55.17%. The highest polymorphism was detected by primer HB-10 while the lowest was observed by primers (14A, Hb-12 & Hb-14) was 50% (Table 2). The floating hydrophytes regarding the higher percentage of polymorphism (60%) by using primer HB-10 (Table 6). The floating hydrophytes showed the higher percentage of polymorphism (60%) by using primer HB-10 (Table 7).
Data analysis: Results in Table 8 showed similarity indices between the studied aquatic plants, the lowest similarity value (0.291) was recorded between Eichhornia crassipes and Echinochloa stagnina, while the highest similarity value between Ludwigia stolonifera and Persicaria salicifolia (0.921).
Biplot showed the importance of primers OP-A15, OP-D03, Hb-10 and protein polymorphism to distinguish all 9 taxa into three separate groups. The first group included floating species (Eichhornia crassipes, Nymphaea lotus and Pistia stratiotes). The second group included submerged species (Ceratophyllum demersum, Myriophyllum spicatum and Potamogeton pectinatus), while the third group includes emergent species (Echinochloa stagnina, Ludwigia stolonifera and Persicaria salicifolia) as shown in Fig. 4.
| Fig. 2(a-e): | Amplification profiles of five RAPD primers for 9 studied taxa, (a) OP-A5, (b) OP-A15, (c) OP-C01, (d) OP-D03 and (e) OP-E15, (M) Marker |
1: Eichhornia crassipes, 2: Nymphaea lotus, 3: Pistia stratiotes, 4: Ceratophyllum demersum, 5: Myriophyllum spicatum, 6: Potamogeton pectinatus, 7: Echinochloa stagnina, 8: Ludwigia stolonifera, 9: Persicaria salicifolia |
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| Table 4: | Electrophoretic protein banding pattern by SDS-PAGE for the 9 studied taxa |
1: Eichhornia crassipes, 2: Nymphaea lotus, 3: Pistia stratiotes, 4: Ceratophyllum demersum, 5: Myriophyllum spicatum, 6: Potamogeton pectinatus, 7: Echinochloa stagnina, 8: Ludwigia stolonifera, 9: Persicaria salicifolia | |
| Fig. 3(a-e): | Amplification profiles of five ISSR primers for 9 studied taxa, (a) 14A, (b) 44B, (c) HB-10, (d) HB-12 and (e) HB-14, (M) Marker |
1: Eichhornia crassipes, 2: Nymphaea lotus, 3: Pistia stratiotes, 4: Ceratophyllum demersum, 5: Myriophyllum spicatum, 6: Potamogeton pectinatus, 7: Echinochloa stagnina, 8: Ludwigia stolonifera, 9: Persicaria salicifolia |
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| Table 5: | Polymorphism (%) and the type of bands generated from RAPD primers of the 9 selected taxa |
RAPD: Random amplified polymorphic DNA | |
Cluster analysis in Fig. 5 illustrates possible relationships among the 9 studied taxa combined in all matrix data. Investigated taxa were divided into two groups at 9.87. The first group includes floating plants contain three species.
| Fig. 4: | MDPREF (Biplot) analysis showing results of biochemical markers (SDS-PAGE technique) and molecular markers (RAPD and ISSR techniques) |
1: Eichhornia crassipes, 2: Nymphaea lotus, 3: Pistia stratiotes, 4: Ceratophyllum demersum, 5: Myriophyllum spicatum, 6: Potamogeton pectinatus, 7: Echinochloa stagnina, 8: Ludwigia stolonifera, 9: Persicaria salicifolia. MDPREF: Multi dimensional preference scaling |
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| Table 6: | Number of total bands, polymorphic bands and percentage of polymorphism of each primer generated |
RAPD: Random amplified polymorphic DNA, ISSR: Inter simple sequence repeat | |
| Table 7: | Polymorphism (%) and the type of bands generated from ISSR primers of the 9 selected taxa |
ISSR: Inter simple sequence repeat | |
| Table 8: | Spearman correlation matrix among the 9 selected taxa |
| Fig. 5: | Dendogram of 9 selected species with cluster analysis using biochemical markers (SDS-PAGE technique) and molecular markers (RAPD and ISSR techniques) |
1: Eichhornia crassipes, 2: Nymphaea lotus, 3: Pistia stratiotes, 4: Ceratophyllum demersum, 5: Myriophyllum spicatum, 6: Potamogeton pectinatus, 7: Echinochloa stagnina, 8: Ludwigia stolonifera, 9: Persicaria salicifolia |
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The second group is further divided into two subgroups at a distance of 4.09, the first subgroups includes two submerged species (Ceratophyllum demersum and Myriophyllum spicatum), where the second subgroup includes emergent (Echinochloa stagnina, Ludwigia stolonifera and Persicaria salicifolia) in addition to submerged Potamogeton pectinatus.
DISCUSSION
Wide range content of heavy metals accumulated in hydrophytes indicated the different extent of sites pollution. Li et al.17 reported that roots tend to absorbed more metals than leaves. The pH was negatively correlated with the ability of the aquatic plant to absorb zinc, copper, cadmium, cobalt and lead. Moreover, pH can play a vital role in heavy metals accumulation by plants. The enrichment mechanism is related to the surface area of plant exposed to the surface area of the plant exposed to water, which helps in the selection of the suitable aquatic plants for absorption of heavy metals from polluted water. In this study, emergent hydrophytes (Persicaria salicifolia) accumulated higher average content of heavy metals than submerged and floating. These results are in line with Jamnicka et al.18, who proved that different abilities of aquatic plants to accumulate heavy metals depends on individual plants and with difference in accumulation concentrations can effect in difference between groups of hydrophytes (submerged, emergent and floating). The accumulation of plant to heavy metals was higher for Zn and Pb comparable to other metals that agree with Fawzy et al.19. Water concentrations of Zn were significantly higher than the other metals. This was mainly due to the sewage discharges from sanitary domestic and agricultural effluents metals in all the studied sites and attained its maximum20. Guilizzoni21 stated that some rooted submerged plants may absorb metals directly from water when they are not readily available in sediments and/or in high concentration in the surroundings. In this research, emergent hydrophytes had the ability to uptake and accumulate heavy metals from sediment or water than submerged. The results proved that SDS-PAGE analysis exhibited distinctive qualitative alterations in electrophoresis SDS-proteins. Electrophoretic analysis of protein profile (SDS-PAGE) of the investigated study of hydrophytes plants showed the total number of the bands was 8 with the molecular weight ranging from 11-127 KDa. There were 4 monomorphic bands in floating and emergent, while 6 monomorphic band was detected in submerged hydrophytes. These alterations are dependent on variations in molecular weights and intensities of polypeptides bands as well as gain or loss of protein bands that lead to high levels of proteins polymorphism. Each polypeptide band represents the final products of transcriptional and translational events occurring due to active structural genes22. The differences in protein polymorphism in the present study may be resulted from insertions or deletions between mutated sites of protein bands and could be used as biomarkers as discussed by Mondini et al.23. The advantage of molecular markers over phenotypic data is the possibility to compare genotypes, even if they are sampled in different environment, type of tissue or stage of development. Another advantage is the theoretical possibility to detect DNA polymorphisms through the entire genome24. This paper recommends using emergent hydrophytes especially Persicaria salicifolia in bioremediation and determining water quality due to is ability to accumulate high concentrations of heavy metals.
CONCLUSION
This study revealed that emergent hydrophytes can accumulate content of heavy metals higher than floating and submerged. So, this paper recommends using emergent aquatic plants on a large scale in phytoremediation, especially, Persicaria salicifolia which can be considered as the best bio indicator model for pollution and a good phytoremediation model for accumulation of heavy metals and appropriate for wastewater treatment.
SIGNIFICANCE STATEMENT
This study discovered the importance of hydrophytic plants in accumulation of heavy metals. It was found that emergent hydrophytes have higher accumulation ability than submerged and floating hydrophytes. This study will help the researchers to uncover the critical areas of phytoremediation and waste water treatment that many researchers were not able to explore. Thus a new theory on Persicaria salicifolia may be arrived at.