Using Carbohydrate-Binding Modules as tools for in vivo visualization/modulation of plant cell wall polysaccharides
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Plant cell walls are highly complex and dynamic cell compartments. Until now, most knowledge of cell wall is derived from the biochemical analysis of fractionated walls and immunolabeling of fixed tissues. Consequently, the majority of temporal and developmental information is lost. Recently, Carbohydrate-Binding Modules (CBMs) from microbial cell wall polysaccharides hydrolases have been developed as probes for cell wall analysis in vitro. CBMs are particularly attractive as in vivo cell wall probes because of their intrinsic specificities toward polysaccharides, ease of heterologous expression, and convenience of modification with fluorescent protein markers. Preliminary research found xylan-binding CBM is more suitable to be utilized as in vivo tagging xylan tool since expression of fluorescent protein mCherry tagged CBM2b-1-2 has no deleterious effect on plant morphology or development. The heterologously expressed fluorescent protein-tagged CBM2b-1-2 selectively labels cell walls that contain xylan, including those of xylem vessels and interfascicular fibers associated with phloem in wild-type Arabidopsis plants. As a functional in vivo visualization tool, we were interested in testing whether fluorescent protein-tagged CBM2b-1-2 would allow us observe effects that arise from mutations in genes that affect the synthesis and deposition of xylan in vivo. Mutation of a WRKY transcription factor causes ectopic xylan deposition in pith cells of Arabidopsis. The plant line carrying a mutation in this WRKY gene was crossed with the stable CBM2b-1-2:mCherry expression line. Examination of F2 plants resulting from this cross demonstrated that the ectopic deposition of xylan can be observed simply and conveniently via hand sectioning. These results suggest that the fluorescent protein-tagged xylan-binding CBM2b-1-2 is a functional in vivo visualization tool to follow xylan dynamics during plant development. In contrast, heterologous expression of cellulose-binding CBM3a affected plant cell wall polysaccharides network and modulated plant growth and developmental processes. Transgenic CBM3a:mCherry plants displayed a dwarfed phenotype, had reduced cellulose and xylan content, and showed changes in glycan epitope extractability patterns. These results indicate that expression of a specific CBM in Arabidopsis may lead to changes in the composition and/or structure of the cell wall, which in turn, may have an impact on the development of the plant.