Abstract
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 x 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.
Highlights
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 3 tremuloides)
The expression pattern of PttMAP20 is remarkably similar to three previously identified xylem-associated PttCESA genes (Djerbi et al, 2004). To confirm this pattern of expression, high-resolution quantitative PCR expression analysis of PtMAP20 and PtCESA1, PtCESA3-2, and PtCESA9-2 was conducted across the wood-forming tissues in Populus tremula
PtMAP20 expression was very low in functional phloem tissues and primary walled cambial cells, while it increased in expanding cells and most dramatically at the onset of secondary wall biosynthesis (Fig. 1B, top)
Summary
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 3 tremuloides). In Arabidopsis, all three secondary cell wall-associated CESA proteins colocalize with bands of cortical microtubules in older xylem vessels (Gardiner et al, 2003), and it has been suggested that the microtubules guide the deposition of the cellulose synthase complexes in the plasma membrane. There are data to suggest that organized microtubules are required to establish rather than to maintain the pattern of cellulose microfibril deposition (Sugimoto et al, 2003; Roberts et al, 2004; Wasteneys, 2004), perhaps to correctly position CESA protein complexes in the plasma membrane (Gardiner et al, 2003; Wightman and Turner, 2008). Owing to the extreme complexity of the microtubular networks, it is likely that many more plant MAPs remain to be discovered (Gardiner and Marc, 2003; Oda and Hasezawa, 2006)
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