ABSOLUTE QUANTIFICATION IN SMALL PLANT RADIOTRACER STUDIES

The main objective of this dissertation research is to develop measurement and data-analysis tools for improving the quantitative accuracy of radiotracer studies of small plants, e.g., grasses in their early growth stages and tree seedlings. Improved accuracy is needed due to the thin nature of plant organs, e.g., leafs and stem. In addition, the methods developed in this thesis are applied to study the plant-environment interface of barley. Some of the approaches explored have potential to increase the statistical accuracy of counting data using PET imaging techniques. Improving the statistical precision of radionuclide tracking data will add to the analysis options. Another important goal is to measure the absolute photosynthetic rate. The standard approach in plant radiotracer experiments is to perform measurements of the relative distribution of radioactivity in various parts of the plant being studied. A limitation of this approach is that it does not take into account differences in the amount of radioisotope assimilated that are available for transport and allocation to the various sinks, that is, absolute CO2 uptake and photosynthetic rates are important factors in understanding the holistic physiological responses of plants to external conditions. For example, monitoring the movement of carbon-11 (11C) tagged carbohydrates in a plant requires an estimate of the average photosynthetic rate to determine the actual quantity of carbohydrates in each plant region (e.g. leaf, shoot, and root).

Radiotracing provides a method for real-time measurements of substance absorption, allocation and metabolic consumption and production in living organisms. Application of radioactive labelling in plants enables measurements associated with core physiological processes, e.g., photosynthesis, water uptake and nitrogen absorption and utilization. Plant uptake of radiotracers allows for tracking spatial and temporal distribution of substances, which enables studies of the plant-environment interface and the mechanisms involved in the allocation of resources (e.g., sugars, nutrients, and water). As such, these techniques are increasingly becoming an important tool for investigating the processes involved in the physiological responses of plants to changes in their local environmental conditions.

This dissertation has two major components: (1) development of experiment techniques for absolute photosynthetic rate measurements in plants using radio-isotope labeling, and (2) application of radioisotope tracing techniques to study the plant-environment interface in barley. The first component is covered in chapters one through three. The second component is presented in chapter four. An introduction into radio-tracing techniques is provided in chapter one. Chapter two describes radio-isotope production, radio-labelled compound preparation and delivery of labels to plant measurements. Chapter three outlines methods that can be employed to measure the absolute photosynthetic rate (µmol/m2/s) for a closed-loop system with [CO2] monitoring capabilities. Chapter four describes the background and results of our study on changing environmental conditions on a model system, barley seedlings. Chapter 5 will introduce the use of Monte-Carlo modeling for scaling the collected data to adjust the detected coincidence counts for losses due to positron escape from plant tissue. Chapter 6 describes the development of a novel imaging technique using direct positron detection that takes advantage of the high fraction of positrons escaping thin plant tissue.

In this dissertation, we have performed the most extensive measurements of carbohydrate allocation and translocation in a plant species using radio-isotope tracing techniques. A major practical limitation of studies based on radio-isotope labeling is the number of samples that can be measured in a single project. Our study on barley (Hordeum distichum) includes measurements on more than 30 plants. The short-lived radionuclide, 11C, was used to determine the real-time response to metabolite transport in barley. Sugars are photosynthesized and tagged with a positron-emitting radioisotope by flowing carbon dioxide (11CO2) tagged air over an active leaf. Data analysis of measurements taken in this dissertation indicates that the fraction of carbohydrates allocated to below ground sinks decreased, by 31% ± 9% in ambient [CO2] and by 37% ± 14% in elevated [CO2], when the nutrient conditions were rapidly changed from high to low nutrient.