Final Activity Report Summary - XYLOSKELETON (Analysis of microtubule-associated proteins specifically associated with xylem vessel formation) The colonisation of land was a crucial step in the evolution of plants, requiring development of systems for projecting their photosynthetic and reproductive organs up into the atmosphere, away from the ground-held water. This depended absolutely on the structural and conducting tissue, xylem, which forms the major component of wood. One type of cell in xylem tissue is the tracheary element (TE) - so-called because its ribbed wall resembles our own trachea. The formation of TE cells involves the bunching-up of cortical microtubules into characteristic hoop-like patterns, the laying down of cellulose-rich secondary cell wall on top of those microtubules, programmed cell death to dispose of the cell contents, perforation of the end walls to make a continuous tube, and the further chemical hardening and water-proofing of the tube by lignin deposition. Because the wall thickenings exactly overlie the microtubule bundles, tracheary elements provide an unparalleled system for investigating the role of microtubules in wall formation. However, xylem formation is very difficult to follow in planta because its differentiation occurs fast, deep inside complex tissues. Culture systems do exist for the trans-differentiation of cells into TEs. Unfortunately, these methods either involve species whose genomes aren't known (and so the cells cannot be analysed by modern techniques involving stable genetic transformation) or else the xylem cells form deep inside clumps of cells that frustrate microscopical analysis. To overcome the difficulties associated to the study of TEs, I developed a novel in vitro system from the plant genetic model Arabidopsis thaliana. This new cell culture-based system allows TE to be specifically induced rapidly (with 3 days), efficiently (up to 40%), in large quantity (gram quantities) and as single cells. Using this system, I was able to better characterise TE differentiation. For the first time, I followed TE differentiation by live-time imaging and measured the speed and succession of TE secondary cell wall polymer synthesis. I confirmed using microtubule-related drugs that microtubules controlled the synthesis, positioning and patterning of TE secondary cell wall. I also identified novel microtubule associated proteins (MAPs) implicated in TE differentiation. I analysed several known MAP families by gene expression profiling and identified a solitary MAP that was both up-regulated upon, and specific to, TE differentiation. This was the plant-specific microtubule-associated protein, AtMAP70-5, which was co-regulated with cellulose synthesis genes specific for TE secondary cell wall formation. Another member of the AtMAP70 family, AtMAP70-1, is constitutively expressed in un-induced cells, co-localizing with AtMAP70-5 in cells and binding to it in vitro. Direct modulation of either of these genes, using RNA-dependent gene silencing or over-expression, alters the normal course of secondary cell wall formation in tracheary elements: the wall's banding pattern is abnormal and thick with cellulose even forming ectopic strands across the vacuole. As observed in vitro, plants constitutively silenced for MAP70-5 exhibited reduced stem height, fewer vascular bundles and no interfascicular fibres - all consistent with an alteration of xylem structure. This work establishes that AtMAP70-5, which is specifically upregulated during the differentiation of TEs, is essential for the normal banding pattern of secondary cell wall - a key step in wood formation.
