B Development of monoclonal antibodies for AMOR. The top and bottom panels show the result studies would be the future direction. Genome editing is possible in Torenia, however, we realized in the granted period that developing a novel model closely relating to Torenia fournieri would be powerful if the new model species have properties as a model plant species. For this purpose, we are now focusing on Torenia crustacea, which is much smaller than Torenia fournieri and produces seeds by auto-pollination (self-pollination). In Torenia crustacea, disaccharide AMOR showed activity and AMOR assay was possible. Breakthroughs in plant glycobiology are expected by using novel molecular tools and model plant species.Figure 3. Progresses based on chemically modified AMOR A Tetrameric AMOR produced by conjugation of four AMOR disaccharide molecules with a linker. methyl residue was achieved for the first time (Figure 3B). Localization of 4-Me-GlcA-β-(1→6)-Gal in the entire plant body will be clarified, which will significantly contribute to finding novel functions of AMOR. Second, bio-active AMOR disaccharide with a linker was produced chemically. We used this to prepare the antigen for AMOR monoclonal antibody and to producing multimeric AMOR that showed higher activity per molecule (Figure 3A). Visualization of AMOR by conjugating fluorescent compounds, as well as adding crosslinkers, would be powerful to search for the unknown receptor of AMOR. Combining these molecular tools with genetic/physiological ReferencesThe AMOR disaccharide part is shown in red and blue.of ELISA for a monoclonal antibody using methylglucuronosylgalactose (AMOR; with methyl residue) and glucuronosylgalactose (without methyl residue) antigens, respectively. Closed circles, open circles, and cross marks indicate results of purified antibodies, supernatant, and column-through, respectively.1) Mizukami, AG et al. The AMOR arabinogalactan sugar chain induces pollen-tube competency to respond to ovular guidance. Curr. Biol. (26): 1091-1097, 20162) Okuda, S et al. Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature (458): 357-361, 20093) Jiao, J, Mizukami, AG, Sankaranarayanan, S, Yamguchi, J, Itami, K, Higashiyama, T. Structure-activity relation of AMOR sugar molecule that activates pollen-tubes for ovular guidance. Plant Physiol. (173): 354-363, 20174) Sankaranarayanan, S, Higashiyama, T. Capacitation in Plant and Animal Fertilization. Trends Plant Sci. (23): 129-139, 20185) Su, S, Higashiyama, T. Arabinogalactan proteins and their sugar chains: functions in plant reproduction, research methods, and biosynthesis. Plant Reprod. (31), 67-75, 2018.6) Xiao, W, Su, S, Higashiyama, T, Luo, D. A homolog of the ALOG family controls corolla tube differentiation in Torenia fournieri. Development (146): dev177410, 20197) Yanagisawa, N, Higashiyama, T. Quantitative assessment of chemotropism in pollen tubes using microslit channel filters. Biomicrofluidics (12): 024113, 20188) Yanagisawa, N, Kozgunova, E, Higashiyama, T. Pulsatile reverse flow actuated microfluidic injector: toward the application for single-molecule chemotropism assay. RSC Adv. (11): 27011-27018, 202165
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