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

次生细胞壁中的微小孔（纹孔）在植物体内的水分运输中起着重要的作用。它们允许水和养分从一个元素流向另一个元素，将根部的水分吸收与叶片的蒸腾作用联系起来。木材生理学的最新进展激发了人们对纹孔解剖学的新兴趣。这些研究突出了孔膜的功能重要性，孔膜是相邻细胞壁的两个互补孔之间的分隔结构，并说明孔膜可能会影响流动阻力和空气进入蒸腾流（空化）的脆弱性。由于这一特征不仅影响活树中树液的运动，而且影响液体、防腐剂和气体在木材中的渗透，因此对纹孔膜的研究在木材技术领域，包括造纸和纸浆工业中提供了应用。然而，我们对纹孔膜的理解一直受到阻碍，因为在自然状态下解剖它们的结构而不产生制备伪影的困难，并且它们的完整解剖变异在许多植物组中仍然记录不多。考虑到纹孔膜结构的潜在变化，纹孔膜特征和纹孔功能之间的关系，特别是当考虑传导效率、对空化的脆弱性和机械强度之间的权衡时，经常是嘈杂的、复杂的，并且需要基于广泛的植物选择进行仔细的测试。该项目旨在对木本植物中的孔膜的结构和功能进行新颖和渐进的研究，以代表广泛的系统发育样本和一系列不同的孔解剖结构和不同的抗空化能力。所有目标都将尽可能多地使用相同的植物选择（从优秀的基尤活体收集中取样）。通过对开花植物导管材和针叶树管胞材的显微镜观察和水力学实验，研究了以下问题：1。除了透射和扫描电子显微镜外，我们还可以使用原子力显微镜（一种表面扫描仪器）了解窝膜的结构特征吗？原子力显微镜提供了很有前途的机会，研究在其原生状态的坑膜。基于先前工作的试验结果，主要挑战将是：（1）证实湿纹孔膜中的纤维素微纤丝以比先前认为的更开放的模式排列，以及（2）调查纹孔膜表面上的非纤丝层的发生。2.纹孔膜中孔隙的大小变化是什么，我们如何从水运输的安全性和效率的角度来理解这一点？基于显微镜的孔膜中的孔的大小将与使用已知直径的胶体金颗粒的灌注实验的测量进行比较。此外，测量所需的压力，迫使气泡通过坑膜将使我们能够研究孔隙率和气蚀的脆弱性之间的可能关系，而膜是在机械应力下，由于拉伸和偏转。3.我们如何将纹孔膜的分布和结构与水传导途径的复杂网络联系起来？这一问题将通过基于X射线计算机显微断层扫描的木材中的液压连接进行量化来解决，这是一种探索木材内部三维结构的非侵入性和准确的技术。这种方法将使我们能够确定血管之间的平均重叠面积和每单位接触面积的总窝面积，因为这些是将解剖数据与窝功能相关联的真正重要的参数。此外，可视化的水分布在活的树木在细胞水平将使用染料注射实验。

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.