Analysis of microtubule-associated proteins specifically associated with xylem vessel formation

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Xylem is the vascular tissue responsible for conducting water and minerals between roots and leaves. The xylem 'vessels' are made of conducting cells called tracheary elements (TEs). Before these cells undergo programmed cell death and the end walls dissolve to form a hollow tube, the cortical microtubules (MTs) bunch together to form transverse 'hoops and helices' that act as a template for the cell wall thickenings that will prevent the hollow xylem from collapsing. I will investigate the protein involved in the key MT-bunching phase.The formation of TEs and their typically sculptured walls has been studied in differentiating cell cultures - mainly mesophyll cells isolated from leaves of Zinnia elegans that transdifferentiate into TEs. More recently, however, Arabidopsis thaliana suspension cultures have been induced to form TEs and this exciting development allows the Arabidopsis genome to be exploited for studying proteins involved in TE differentiation. The applicant has visited/worked in the Japanese labs that pioneered this technique and now proposes a logical extension to his training by working in a lab that has developed methods for the proteomic identification of microtubule-associated proteins (MAPs) from Arabidopsis. I will extend my experience on the Zinnia TE differentiation system to the published Arabidopsis suspension system and will use two complementary approaches to gain expertise on the plant cytoskeleton. I will identify those MAPs that are upregulated during TE differentiation using the proteomic strategy developed in the host laboratory on undifferentiated Arabidopsis cells. I will also examine the role of specific MAPs (including one novel MAP family) already identified in the host laboratory. Targetted proteins will be analyzed during TE differentiation by gene expression and localization studies using antibodies and GFP fusion proteins.

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