Structure and function of pit membranes in water conduction pathways of plants: combining novel microscopy techniques with xylem hydraulic experiments

Minute openings (pits) in the secondary cell wall of water conducting elements play an important role in water transport in living plants. They allow the flow of water and nutrients from one element to another, linking water uptake in roots with transpiration in leaves. Recent advances in the field of wood physiology have stimulated a renewed interest in the anatomy of pits. These studies highlight the functional importance of the pit membrane, which is the dividing structure between two complementary pits of adjacent cell walls, and illustrate that pit membranes may affect flow resistance and vulnerability to air entry into the transpiration stream (cavitation). Since this feature affects not only the movement of sap in living trees, but also the penetration of liquids, preservatives and gases in timber, research on pit membranes provides applications in the field of wood technology, including the paper and pulp industry. Nevertheless, our understanding of pit membranes has been hampered by difficulties in dissecting their structure in a native state without creating preparation artefacts and their full anatomical variation remains poorly documented in many plant groups. Given the potential variation in pit membrane structure, relationships between pit membrane characters and pit function, especially when considering trade-offs between conductive efficiency, vulnerability to cavitation and mechanical strength, are frequently noisy, complex and need careful testing based on a wide selection of plants. This project aims to carry out novel and progressive research on the structure and function of pit membranes in woody plants selected to represent both a broad phylogenetic sample and a range of differing pit anatomy and differing resistance to cavitation. The same selection of plants (sampled from the excellent Kew living collections) will be used as much as possible for all objectives. By carrying out novel microscopy techniques and hydraulic experiments on both vessel-based wood of flowering plants and tracheid-based wood of conifers the following questions will be addressed: 1. What can we learn about structural characteristics of pit membranes using atomic force microscopy, which is a surface scanning instrument, in addition to transmission and scanning electron microscopy? Atomic force microscopy provides promising opportunities to study pit membranes in their native state. Based on pilot results of previous work, the main challenges will be: (1) to corroborate that cellulose microfibrils in wet pit membranes are arranged in a more open pattern than was previously believed, and (2) to investigate the occurrence of a non-fibrillar layer on the surface of pit membranes. 2. What is the size variation of pores in pit membranes, and how can we understand this in terms of safety and efficiency of water transport? The size of pores in pit membranes as based on microscopy will be compared to measurements using perfusion experiments with colloidal gold particles of known diameter. Also, measurements of the pressure required to force air bubbles through pit membranes will allow us to study possible relationships between porosity and vulnerability to cavitation while the membrane is under mechanical stress due to stretching and deflection. 3. How can we link the distribution and structure of pit membranes with the complex network of water conduction pathways? This problem will be addressed by quantifying hydraulic connections in wood based on X-ray computed microtomography, which is a non-invasive and accurate technique to explore the internal three-dimensional structure of wood. This method will allow us to determine the average area of overlap between vessels and the total pit area per unit contact area, as these are truly important parameters for relating anatomical data to pit function. In addition, visualisation of water distribution in living trees at the cellular level will be examined using dye-injection experiments.