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

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.

Tracheary elements (TEs) are the cells that join end to end to form the xylem transport system. Xylem is the major structural tissue of woody plants and is therefore of great interest, not only academically but for understanding the structure of wood. Previously, it has been possible to study the transdifferentiation of Zinnia leaf mesophyll cells into TEs. However, the genome is not sequenced and Zinnia is not transformable. Reports suggest that Arabidopsis tissue culture cells can transdifferentiate into TEs in vitro, but with poor reproducibility and frequency. The postdoctoral scientist, Edouard Pesquet, has therefore spent the last year optimizing an Arabidopsis suspension cell system. We now have a robust system for inducing single Arabidopsis suspension cells to form TEs synchronously, reproducibly, with 40% efficiency. We have preliminarily tested the system, using RT-PCR to analyse candidate cytoskeleton genes predicted to be active during transdifferentiation, and shown that microtubule-associated proteins can be isolated at key stages, ready for proteomic analysis. Dr Pesquet's Marie Curie fellowship ends in January 2009 and we seek funding to fully exploit this exciting system. We are especially interested in studying the bunching up of cortical microtubules that determines the characteristically patterned secondary cell walls. We propose to analyse the expression patterns of >200 candidate proteins known to be associated with microtubules to see how they are regulated during transdifferentiation. Also, we will use quantitative proteomics to investigate those proteins that can be isolated by microtubule-pulldown over the differentiation process. Proteins of interest, together with selected candidate genes, will be investigated in overexpression and RNAi knockdown experiments to test their importance in the MT bunching process and the resulting effects on cellulose formation and lignification.