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Abstract
Plant cell walls are known as a highly complex and dynamic structure that encloses each cell in a plant. As of now, most research on cell walls is performed using biochemical analysis of fractionated walls and immunolabeling of fixed tissues. These techniques are difficult to apply to the study of temporal and developmental changes in plant cell walls. Antibodies that recognize plant cell wall polysaccharides have been developed as probes for cell wall analysis in vitro because they are intrinsically specific toward polysaccharides structures. In theory, if the genes encoding the antibodies can be obtained, they should be easy for heterologous expression and simple for modification with fluorescent protein markers. ScFvs are single polypeptide fragments of antibodies that are composed of the variable regions of the heavy (VH) chain and the light (VL) chain with a flexible peptide linker between. ScFvs may be more easily applied to the labeling of plant cell walls in vivo, if the they can show to retain the binding specificity and affinity of its parent antibody. In the work reported here, scFvs were generated from genes encoding several plant cell wall glycan-directed monoclonal antibodies. In vitro test showed the scFvs were similar but not identical in binding specificities compared to the parent antibodies. Heterologous expression of these scFv genes yielded proteins that could be used to label cell walls in sectioned plant tissues. Plant cell walls are known as a highly complex and dynamic structure that encloses each cell in a plant. As of now, most research on cell walls is performed using biochemical analysis of fractionated walls and immunolabeling of fixed tissues. These techniques are difficult to apply to the study of temporal and developmental changes in plant cell walls. Antibodies that recognize plant cell wall polysaccharides have been developed as probes for cell wall analysis in vitro because they are intrinsically specific toward polysaccharides structures. If the genes encoding the antibodies can be obtained, they should be easy for heterologous expression and simple for modification with fluorescent protein markers. ScFvs are single polypeptide fragments of antibodies that are composed of the variable regions of the heavy (VH) chain and the light (VL) chain with a flexible peptide linker between. ScFvs may be more easily applied to the labeling of plant cell walls in vitro if they are able to retain the binding specificity and affinity of their parent antibody. In the work reported here, scFvs were generated from genes encoding several plant cell wall glycan-directed monoclonal antibodies. Enzyme-linked immunosorbent assay, or ELISA showed that twelve of the heterologously expressed scFvs have largely similar binding specificities, although perhaps weaker binding affinity than their parent antibodies. One expressed scFv had no activity at all. The expressed scFv genes also yielded proteins that could be used to label cell walls in sectioned plant tissues. The immunolabeling results confirmed the weaker binding affinity of scFvs.Heterologous expression of scFvs in planta resulted in modulations of polysaccharide functions in plant cell walls. The in vivo expression of scFv-M1:YFP in Arabidopsis plants resulted in hypoplasia at the stages of seedling and/or vegetative growth, and withering in the early stage of floral budding. Transgenic scFv-M140:YFP plants displayed a stem-lodging phenotype, suggesting that although the scFv-M140:GFP displayed no activities in vitro it still affected the plant cell wall polysaccharides network to effect plant growth and developmental processes. These results indicate that expression of a specific scFv in Arabidopsis may lead to changes in the composition and/or structure of the cell wall, which consequently, may cause an impact on the development and/or morphological phenotype of the plant.