A programme for studying the role of microtubule-associated proteins in xylem formation

当嫩芽在大气中生长时，水和矿物质从根部输送到生长的叶子上。这发生在称为木质部的特殊运输组织中。木质部含有称为气管的细胞（因为早期的显微镜学家认为它们看起来像动物的呼吸管/气管）。气管元件通过用新壁材的螺旋状或环状增厚其壁形成导管；然后，它们通过程序性细胞死亡将其内容物掏空，端壁穿孔，允许细胞间的连续流动。主动传导的木质部存在于边材中，但后来，细胞变得更厚，形成不活跃的心材——木材的支撑材料。我们需要知道形成木材的细胞是如何形成的，以便合理地操纵这种材料。以前，通过研磨百日草的叶子，可以在试管中将一些细胞转化为木质部细胞。我们对百日草进行了研究，但它的遗传信息没有测序，这使得分子生物学研究非常困难。其他人已经证明，拟南芥（其基因组已经测序）的细胞也可以在试管中被诱导成木质部细胞，但这种情况发生的效率很低，而且是成团的。现在，我们已经找到了一种方法，可以一次又一次地将单个拟南芥悬浮细胞同步高效地转化为木质部细胞。这使我们处于重要的领先地位，在初步工作中，我们已经表明下列方案的所有部分都是可行的。在细胞死亡之前，木质部细胞的细胞质经历了一次惊人的重组，形成了与细胞壁增厚肋骨完全匹配的环状带。这是由于附着在细胞膜内部的微管（细胞的“支架棒”）聚集而成的。微管作为沿着细胞膜移动的酶的轨迹，将纤维素微原纤维挤出细胞壁。这解释了微管形成的图案与细胞壁增厚的肋骨之间的巧合。因此，气管元件的同步形成是跟踪微管如何聚集在一起的理想模型系统，特别是因为高度可见的壁增厚可以很容易地在系统筛选中监测到。我们想要筛选木质部细胞形成过程中影响微管组织的微管相关蛋白。我们建议研究拟南芥的整个基因集合，看看哪些基因在木质部细胞形成时被打开/关闭（“转录组学”）。然后，我们将这些与试管中结合微管的实际蛋白质进行比较（“蛋白质组学”）。通过检查所有已知的微管相关蛋白，并可能识别新的蛋白，我们将选择那些在细胞形成过程中活跃的蛋白。然后，我们将干扰这些基因，以验证我们的假设，即微管的行为和组织的变化将改变细胞壁增厚的模式。我们将使用激光显微镜来跟踪微管行为的变化，从而产生不同的墙壁雕刻。

As shoots grow into the atmosphere, water and minerals are transported from the roots to the advancing leaves. This occurs in specialized transport tissue called xylem. Xylem contains cells called tracheary elements (because early microscopists thought they look like animal breathing tubes / trachea). Tracheary elements form conducting pipes by thickening their walls with spirals or hoops of new wall material; they then hollow their contents by programmed cell death and the end-walls become perforated allowing continuous flow from cell to cell. Actively conducting xylem is found in sapwood but, later, the cells become more thickened to form inactive heartwood - the supporting material of wood. We need to know how wood-forming cells form in order to rationally manipulate this material. Previously, by grinding leaves of the plant, Zinnia, it was possible to convert some cells to xylem cells in the test tube. We have worked on Zinnia but its genetic information is not sequenced, making it very difficult to do molecular biology. Others have shown that cells of Arabidopsis (whose genome has been sequenced) can also be induced to become xylem cells in the test tube but this occurs with low efficiency and in clumps. Now, we have found a way of converting single Arabidopsis suspension cells into xylem cells synchronously, with high efficiency, time after time. This gives us an important lead and in preliminary work we have shown that all parts of the following programme are feasible. Just before cell death, the cytoplasm in xylem cells undergoes a striking reorganization, forming circumferential bands that exactly match the thickening ribs of cell wall. This is due to the bunching-up of microtubules (the cell's 'scaffolding rods') attached to the inside of the cell membrane. Microtubules act as tracks for enzymes that move along the membrane, extruding cellulose microfibrils into the cell wall. This explains the coincidence between the patterns formed by microtubules and the thickened ribs of cell wall. Synchronous formation of tracheary elements is therefore an ideal model system for following how microtubules bunch together, particularly since the highly visible wall thickenings can be easily monitored in systematic screens. We want to screen the microtubule-associated proteins that influence the organization of microtubules during xylem cell formation. We propose studying the entire collection of genes in Arabidopsis to see which ones are switched on/off as xylem cells form ('transcriptomics'). We will then compare these with the actual proteins that bind microtubules in the test tube ('proteomics'). By examining all known microtubule-associated proteins, and possibly identifying novel ones, we will select those that are active during cell formation. Then we will interfere with these genes to test our hypothesis that changes in the behaviour and organization of microtubules will change the pattern of thickening in the cell wall. We will user laser microscopy to follow the changes in microtubule behaviour that result in the different wall sculpturings.