We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula × tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.
Most of the glycosyltransferases (GTs) that catalyze the formation of plant cell wall carbohydrates remain to be biochemically characterized. This can be achieved only if specific assays are available for these enzymes. Here we present a protocol for in vitro assays of processive and nonprocessive membrane-bound GTs. The assays are either based on the use of radioactive nucleotide sugars (NDP sugars; e.g., UDP-[U-(14)C]glucose) and the quantification of the radiolabeled monosaccharides incorporated into soluble or insoluble carbohydrates, or on the coupling of the GT reaction with that of pyruvate kinase (PK) and the oxidation of NADH by lactate dehydrogenase (LDH). The radiometric assays are more suitable for exploratory work on poorly characterized enzymes, whereas the spectrophotometric assays require the availability of highly enriched GTs. Both assays can be performed within 1 d, depending on the number of fractions to be assayed or reaction mixtures to be tested.
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