Water potential measurement Tissue tension

Transcription

1 Water potential measurement Tissue tension (L, M) Authors: Niels Sievertsen, Stefan Zeyen, Connie Müller, Melanie Weis, Assistant: Yvonne Steinbach, 07. June Objective The goals of the two experiments are to study the fundamental importance of water to plants and to show important basics of water metabolism. 2 Introduction Water is one of the most important resources to plants and crucial for all metabolic processes. Most of the absorbed water (roughly 97 %) is released from the leaves in a process called transpiration, the evaporation on a leaf s surface. Remaining 3 % are used in biomass production and metabolic processes within the plant. The water potential is calculated using the below equation and depends on the three factors pressure potential (Ψ p ), gravity potential (Ψ w ), and solute potential (Ψ s ). Ψ s represents the potential of solutes in water, which lower its free energy. Therefore, Ψ s is also referred to 1

2 3 METHODS as Ψ π or the osmotic potential. It relates to the law of van t Hoff as seen in the equation. Ψ w = Ψ p + Ψ g + Ψ s = Ψ p + Ψ g R T c s (1) R is the universal gas constant ( J K 1 mol 1 ) and T is the temperature in K. c is the molality of the dissolved substance. Molality is not to be confused with molarity, which refers to a concentration per volume. The pressure potential Ψ p is the pressure the water experiences due to more or less inelastic cell walls between the inside and the outside of a cell. The cell wall is important, because it stops already completely filled (turgescent) cells from taking up even more water. Otherwise, they would be at risk to burst and be destroyed. The gravitational potential Ψ g can be ignored for plant heights of less than approximately 5 m. When no flow of water to the inside or outside of a cell can be observed, the potential of both the inside and outside solutions are equal and said to be isotonic. Certainly, water molecules can still pass through the membrane, but the system has reached an equilibrium. A potential is a directed phenomenon and therefore has a flow direction. The water always flows from environments of high potential to those with a low potential. The reason why plants take the biggest fraction of water from the soil is because moist soil has a very high osmotic potential, helping the plant to take up water. 3 Methods 3.1 L - Water potential measurement A dilution of a 1 M saccharose solution with the concentrations from 0.1 M to 0.5 M was prepared, whereas the water was used for the dilutions. The final volume of each sample added up tp 10 ml. One test tube was filled with 10 ml of distilled water as a control. 2 mm thick cylinders were cut out of a potato. As a tool, a certain cork screw was used. After determination of the precise weight, 2 g of 2 mm thick potato slices were added to each of the 6 test tubes. The samples were closed with Parafilm and shaken from time to time. After two hours, the slices were taken out, carefully dried on a piece of paper, and weighed again. The weight differences indicate the osmotic potential Ψ s with one specific saccharose concentration leading to both the cell lumen and the outside to be isotonic. The overall methodology can be seen in figure 1. 2

3 3.2 M - Tissue tension 3 METHODS Figure 1: Experimental setup for experiment L as seen in the course material. 3.2 M - Tissue tension The methodology used for this experiment can be seen in figure 2. Segments of the hypocotyl stretching zone right under the cotyledon node with were cut at a length of 1 cm from one week old sunflowers. The segments were incubated in distilled water. After two hours of incubation, one 2 mm stripe of the epidermis was removed with a pair of tweezers. The lengths of the hypocotyl and the epidermis were measured. In the second part of this experiment, segments of 2 cm were sliced and 20 individual segments were prepared in our experiment. With the help of a pair of tweezers, the entire epidermis of 10 segments was removed. The segments with and the ones without an epidermis were incubated separately for two hours and their length was then measured and interpreted. 3

4 3.2 M - Tissue tension 3 METHODS Figure 2: Experimental setup for experiment M as seen in the course material. 4

5 4 RESULTS & DISCUSSION Figure 3: A graph showing the weight change in percent from the starting weight at different concentrations and osmotic potentials with the interpolated c 0 and the 95% confidence intervals. 4 Results & Discussion 4.1 L - Water potential measurement The potato weight was measured before and after incubation in the different saccharose solutions. The percentage loss or gain of weight was calculated and the values were plotted in graph 3. In this graph, a zero in weight change means, that parenchyma cells and the outside solution are isotonic. This is the case at c 0 = M, which has been determined by performing an interpolation of the molality. The 95% confidence interval is evident, as well. As a consequence no net water flow occurs, because water potentials of both liquids are the same. If the concentration of solute molecules is lower than c 0 water diffuses into the cell because the surrounding solution has a higher water potential than the parenchyma. This kind of solutions are called hypotonic. This diffusion based on osmosis can be seen by increased potato weights. If the solute concentration is higher than c 0, which is then called a hypertonic solution, water diffuses out of the cell, because the water potential of the surrounding area is lower than inside the cell. This is visible by the lower weight of the potato after treatment and incubation. As it is 5

6 4.2 M - Tissue tension 4 RESULTS & DISCUSSION evident in the graph, the measurement using a 0.4 M saccharose solution is at the equilibrium value, too. This could be probably because the balance which was not very precise and only showed one decimal after the comma. Additionally, it has to be kept in mind, that only one measurement for each concentration was done and each of the potato slices were not exactly of the same size. However, the expected trend could clearly be shown. It is possible to approximate the water potential Ψ s of parenchyma cells with the obtained c 0 value via van t Hoff s law. Ψ w = R T c 0 = 0.839MP a (2) For the temperature T 293 K were used, the variable R was considered with 8.32 J mol 1 K 1. All solutions have a negative water potential because they are lower than the one of pure water. 4.2 M - Tissue tension Confirmation of epidermis stretching The average value of the hypocotyl segment lengths was measured at 1.08 cm after incubation. The mean value of the epidermis, which were stripped away of the hypocotyl was measured at 1.01 cm. This value correlates with our expectation of an epidermis elongation, even though it is not a big difference. Instead the hypocotyl stretch more visible. The epidermis should stretch, because it has direct contact to the surrounding water. In contrast, the hypocotyl only has contact to the cut surface or else water has to diffuse first through the epidermis. Nevertheless, the epidermis increases less than the hypocotyl. This could be, because it was quite difficult to peel of the epidermis and afterwards it curls up, instantly. Other than that, it could be because epidermis cells can not store as much water as hypocotyl cells Confirmation of inner tissue compression The average length of the unpeeled sunflower hypocotyl was 2.12 cm after incubation in distilled water. The mean value of the length of peeled hypocotyl segments was 2.27 cm. According to these results, the hypocotyl elongation of the peeled segments increased much more than the unpeeled pieces. This effect was expected and can be explained. The unpeeled hypocotyl is shielded 6

7 4.2 M - Tissue tension 4 RESULTS & DISCUSSION by the epidermis, the cells in this tissue have strong cell walls, and are covered with the cuticula wax coat. However, the peeled hypocotyls do not have this shield anymore and are entirely exposed to the water of which it can take up a larger amount in the same time. Additionally, the epidermis compresses the inner tissues which raises the pressure within the cells. This is called tissue tension. It raises the water potential Ψ w and less water enters the affected cells. 7