A nuclear magnetic resonance study of plant-water relationships

Water is one of the most important constituents of a plant. It is the medium in which many biological reactions take place and nutrients are transported throughout the plant in aqueous solutions. Because it serves as a hydrogen donor In photosynthesis water can be considered as one of the building blocks of the plant structure. Water plays an essential role in plant temperature regulation and is an important supporting structure, especially in non-woody plants. The study of plant-water relations is an important discipline in plant physiology, and many methods are available to study water content, water potential and water flow in plants. Chapter 1 contains a summary of the most frequently used techniques. All techniques measure only a single variable: water content, water potential or water flow. Most of the techniques are destructive and invasive, which does not apply to Nuclear Magnetic Resonance (NMR).For over 25 years NMR has been used to obtain information on the water content of biological tissue. At the Department of Molecular Physics the Repetitive Pulse (RP) NMR method has been used as a technique to measure water flow since 1974. The method has shown to be successful when applied to cucumber, pumpkin, and gherkin plants.NMR has the following advantages:- It is non-destructive and non-invasive;- It permits almost simultaneous measurements of linear water flow, volumetric water flow and water content over long periods of time with a time resolution of a few seconds;- from a combination of volumetric and linear water flow the effective total cross-sectional area available for flow can becalculated.The intrinsic low sensitivity of NMR and high costs of the equipment are disadvantages of the method. Provided sufficient attention is paid to data acquisition and processing, the method can be made sensitive enough to obtain physiologically relevant data.The goal of the work presented in this thesis was to evaluate the significance of the NMR techniques in the field of plant physiology, in particular plant-water relations.Chapter 2 contains NMR theory, and defines the concepts used in describing the water status of plants, as well as the combination of NMR and plant-water relations.Materials and methods are described in Chapter 3. Particular attention has been paid to a home-built NMR probe and stabilization system for the main magnetic field ('lock').Experiments with respect to the magnetic properties of the plant material are described in Chapters 4 and 5. These experiments were carried out for further understanding of the possibilities and limitations of the method, as well as improvement of the NMR technique and equipment.When applied to plants with narrow xylem vessels, the NMR flow technique proved to be unsuccessful. This is due to a too short transverse relaxation time (T 2 ) in such vessels. A model relating T 2 to the xylem vessel diameter is presented in Chapter 4. It is assumed in this model that the xylem vessel walls contain 'relaxation sinks'. This means that the protons of the water molecules loose their magnetization (= relaxation) created In the experiment upon contact with the vessel wall, which makes them undetectable with the RP NMR technique. The effectiveness of this process is proportional to the value of a parameter H. The probability for a water molecule to reach the wall is determined by diffusion processes. A larger distance to the wall results in a longer time required for a molecule to reach the wall. The overall result is that T 2 of water in narrow vessels is shorter than in wide vessels. Using this information, it was possible to calculate in Chapter 6 that a well defined flow signal can only be observed in xylem vessels with a diameter of at least ca. 90 μm, using the experimental conditions described in this thesis.The magnetic properties of the plant stem as a whole are discussed in Chapter 5. The rationale behind this was that a signal of stationary water was observed during a flow experiment. Theoretically this should not be so, because the magnetization of the stationary water in the upper coil half should be counterbalanced by that of the lower half.This, however, was not found to be completely correct for plants, resulting in the presence of a background signal in addition to the flow signal. The observed NMR spectra of cucumber stem segments were found to be dependent on the orientation of the stem with respect to the magnetic field direction. The angular dependence could be explained by the presence of local magnetic susceptibility differences within the stem. These result in local magnetic field gradients, leading to a background signal due to staionary water in a flow experiment, and a decrease of T 2 . The latter effect increases the lower boundary of the diameter of xylem vessels in which flow signals can be detected. The line widths of the spectra showed a quadratic dependence on the main magnetic field. This does not agree with literature data, and is not yet understood. In the design of NMR flow meters this effect should be taken into account.In Chapter 6 the Influence of different flow profiles on the NMR flow curves is discussed. Because a plant contains many xylem vessels with different diameters, the flow profile is different from that of a system consisting of capillaries with equal diameters, which is used for calibration. The influence on calibration constants and possible adjustments are considered. The influence of relaxation times (see Chapter 4) and stationary water on the flow curves as derived from computer simulations is discussed as well. It was shown that variations in linear flow velocity of more than 5-10%, variations in volumetric flow velocity of more than 10-20%, and variations in water potential down to ca. 1 bar could be reliably detected by NMR. Experimental procedures to obtain quantitative information on flow rate and T 2 are described as well.In Chapter 7 water balance studies carried out on the stem of water cultures of intact cucumber and gherkin plants are described. In some experiments NMR results are compared with those obtained with a dendrometer. The following environmental conditions were varied: relative humidity, light intensity, CO 2 concentration of air and root medium, root temperature, and root area in contact with surrounding medium.Using the NMR techniques it was possible to discriminate between parameters affecting water uptake by the root and transpiration at the leaf surface. If the resistance for water uptake increased more that for transpiration, both water flow and content decreased. By contast, if the resistance for transpiration Increased more than for water uptake, the water content increased and the water flow decreased. This can also be concluded from the model for water balance in plants, which was formulated in Chapter 7, on the basis of experimental data.