学位論文 EFFECTS OF WATER TURBULENCE AND MEAN FLOW ON GROWTH, METABOLISM AND ULTRA-STRUCTURAL VARIATIONS IN AQUATIC MACROPHYTE; ELODEA NUTTALLII

KEERTHI SRI SENARATHNA ATAPATHTHU

内容記述
指導教員 : 淺枝隆
Contents ........................................................................................................................................... iList of tables ................................................................................................................................... ivList of figures .................................................................................................................................. vACKNOLEDGEMENT ................................................................................................................ viiABSTRACT ................................................................................................................................. viiiChapter 1. Introduction ................................................................................................................... 11.1. Overall objective .................................................................................................................. 31.2. Specific objectives ............................................................................................................... 3Chapter 2. Literature Review ......................................................................................................... 42.1. Abiotic stress on aquatic plants............................................................................................ 42.2. Water movements induced abiotic stress in aquatic plants ................................................ 11Chapter 3. Materials and method.................................................................................................. 143.1. Experiment-I ...................................................................................................................... 143.1.1. Sub Experiment 1 ........................................................................................................ 143.1.2. Sub Experiment 2 ........................................................................................................ 163.1.3. Experimental plant and growing conditions ............................................................... 193.1.4. Growth measurements and sampling .......................................................................... 203.1.5. Pigment extraction, chlorophyll fluorescence and plant stress ................................... 203.1.6. Sample preparation and stress assays ......................................................................... 203.1.7. Enzyme assays ............................................................................................................ 213.1.8. Field investigations ..................................................................................................... 233.2. Experiment-II ..................................................................................................................... 263.2.1. Microscopic study ....................................................................................................... 273.2.2. Metabolome analysis with CE-MS ............................................................................. 283.2.3. Determination of starch content .................................................................................. 283.3. Experiment -III................................................................................................................... 293.4. Statistical Analysis ............................................................................................................. 29Chapter 4. Results ......................................................................................................................... 304.1. Experiment-I ...................................................................................................................... 304.1.1. Sub experiment-1 ........................................................................................................ 304.1.2. Sub Experiment 2 ........................................................................................................ 314.2. Field investigations ............................................................................................................ 384.2.1. Field study-1 ............................................................................................................... 384.2.2. Field study-II ............................................................................................................... 404.3. Experiment -II .................................................................................................................... 494.3.1. Plant growth and cell ultra-structure ........................................................................... 494.3.2. Amino acid metabolism .............................................................................................. 544.4. Experiment-III.................................................................................................................... 574.4.1. Plant growth and ultra-structural variations ................................................................ 574.4.2. Effects of water flow on plant metabolism ................................................................. 62Chapter 5. Discussion ................................................................................................................... 645.1. Experiment 1 ...................................................................................................................... 645.1.1. Selection of suitable nutrient condition for E. nuttallii............................................... 645.1.2. Stress responses of E.nuttallii against turbulence and mean flow .............................. 645.2. Stress response of M. spicatumagainst turbulence in field condition (Field study-II) ....... 675.3. Experiment 2 ...................................................................................................................... 715.3.1. Effect of turbulence on plant growth and ultra-structure of plant cells ...................... 715.3.2. Effect of turbulence on major metabolites .................................................................. 735.4. Experiment-III.................................................................................................................... 755.4.1. Effect of mean flow on plant growth, ultrastructure and metabolism ........................ 75References ..................................................................................................................................... 81Appendix ....................................................................................................................................... 92
The array of functions provided by aquatic plants is essential to maintain the ecological balance of aquatic ecosystems. Aquatic plants experience various disturbances in aquatic systems for two reasons: the highly dynamic nature of the systems and the sedentary nature of the plants. The productivity of the plants is highly dependent on environmental stresses and a wide range of fluctuations in specific abiotic factors. Among the abiotic factors, water movement is the most important for submersed aquatic plants because the flow driven drag and lift forces have either a positive or a negative effect depending on the magnitude. The mechanisms governing flow-plant interactions are not yet fully understood, and the plant response to mean flow (without turbulence) against turbulence is largely unknown. Acquiring knowledge and clarifying the interactions between aquatic plants and environmental factors are important to understand how the functions of plants would benefit from the management of ecosystems. Although the stress responses of submersed plants to turbulence were reported previously, information on the plant responses to mean flow is scarce, to our knowledge. Further, effects of water movements on the ultra-structure of plant cells and amino acid metabolism are yet to be explored. This study hypothesized that, the water movements significantly impact to the plant metabolism, stress response and the ultrastructure of plant cells. Therefore in this study was designed to evaluate the variations in metabolism, stress responses and ultra-structures of aquatic plants responding to water movements. There were three laboratory experiments coupled with field observations.In the first experiment, growth and stress responses of Elodea nuttallii exposed to either turbulence or main flow were compared to control plants grown in stagnant waters. Turbulence and main flow were generated using a vertically-oscillating horizontal grid and a specially designed re-circulating system, respectively. At the end of the experiment after three weeks, growth, chlorophyll fluorescence, concentrations of indole acetic acid, H2O2, chlorophyll, cellulose, lignin and antioxidant enzyme activities of plants exposed to turbulence or main flow were compared with control plants. A decrease in shoot elongation coupled with an increase in radial expansion was observed in plants exposed to turbulence and mean flow. The effects on the plants exposed to turbulence were further accompanied by significant increases in cellulose and lignin. Significant elevations in H2O2 and antioxidant enzyme activities were observed in turbulence-stressed plants, and the lowest stress was observed in control plants. The effects of main flow-induced stress were similar to characteristics of control plants. Turbulence reduced total chlorophyll by approximately 40% compared to plants in the control and main flow. Mechanical stress induced by turbulence leads to increased oxidative stress and tissue rigidification in E. nuttallii and that turbulence triggered stress is more severe than that induced by main flow.The second experiment investigated growth, metabolism and ultra-structural changes in response to turbulence in aquatic macrophyte; Elodea nuttallii, after exposure to turbulence for 30 days. The turbulence was generated with a vertically oscillating horizontal grid. The turbulence reduced plant growth, plasmolyzed leaf cells and strengthened cell walls, and the plants exposed to turbulence accumulated starch granules in stem chloroplasts. The size of the starch granules increased with the magnitude of the turbulence. Using capillary electrophoresis–mass spectrometry (CE-MS), an analysis of the metabolome found metabolite accumulation in response to the turbulence. Asparagine was the dominant amino acid that was concentrated in stressed plants, and organic acids such as citrate, ascorbate, oxalate and γ-Amino butyric acid (GABA) also accumulated in response to the turbulence.The third trial was conducted to study the effect of mean flow on plant functioning and structure. E. nuttallii were exposed to flowing (~10 cms-1) and stagnant waters for 30 days. At the end of the experiment, plant growth, chlorophyll and ultra-structural variations were compared. Final shoot length was not significantly different among two treatments while the plants grew in flowing waters consisted of comparatively longer internodes. Although the chlorophyll content of plants in the latter treatment was less than that of control group, the difference was insignificant. Electron microscopy observed thin, elongated chloroplasts in leaves of flowing treatments while, control plants with wide chloroplasts. Structure of leaf cells were affected by the water flow as the diameter of cells in control plants was bigger than that of flowing treatment. These results indicated that turbulence caused severe stress that affected plant growth, cell architecture and some metabolic functions of E. nuttallii than the mean flow.A field investigation was conducted at Moto Arakawa, a tributary of the Arakawa River, Japanto study the consequences of turbulence induced stress response of aquatic macrophytes in natural condition. Six study sites were selected and the velocity fluctuations inside macrophytes stands were measured using a two dimensional electromagnetic current meter. Transects were significantly different (p<0.05) in terms of turbulence velocity. The location having the lowest turbulence was considered as the control turbulence. Plant stress responses to turbulence were compared by measuring the antioxidant activities (peroxidase, catalase and ascorbic proxidase), H2O2content and indole acetic acid (IAA). Antioxidant productions were significantly higher in plants exposed to higher magnitudes of turbulence compared to those grew in the control transect. Compared to control transect, an elevated level of H2O2 and low concentration of IAA were also measured in plants grown in turbulent environments. Decrease in IAA content was detected in response to increasing turbulence. Lignin and cellulose accumulation were significantly higher in plants exposed to elevated level of turbulence compared to control plants. Taken together, field observations suggest that mechanical stress induced by turbulence led to increase oxidative stress and tissue rigidification in M. spicatum and the laboratory findings were in consistent with field observations.Findings of this study revealed that turbulence make significant negative impacts to plant functioning and cell ultra-structure, and the severity of the impacts increase with the magnitude of turbulence. Even though the effect of mean flow is stressful for aquatic plants, the impacts were small compared to that of turbulence. All together, present findings demonstrates the causes of water movements to aquatic plants and provides insights to clarify the interactions between aquatic plants and water movements to the benefit of aquatic ecosystem management.
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