These progresses will promote future researchers of not only AMOR but Plant glycobiology. First, monoclonal antibodies that can recognize AMOR disaccharide structure including the Modification of AMOR by Organic Chemical SynthesisBased on the structure-activity relationship of AMOR, we succeeded in adding polyethylene glycol as a linker to the galactose of the AMOR disaccharide, while maintaining the ring-closed structure, in collaboration with Dr. Shinya Hagiwara and Dr. Shuhei Kusano in RIKEN (Figure 3). For the production of monoclonal antibodies, about 10 molecules of AMOR were added to one molecule of carrier protein (KLH, BSA) by a click reaction. In addition, multimeric (dimeric, trimeric, and tetrameric) AMORs were also produced by joining linkers to each other.Establishment of AMOR monoclonal antibodiesAs the progress after the granted period, we continued to develop AMOR monoclonal antibodies by using the chemically modified AMOR molceules. In order to select hybridoma producing antibodies that recognize the presence or absence of the terminal methyl group, which greatly enhances AMOR activity, we generated monoclonal antibodies against the above two antigens as well as the molecules without the methyl group. As a result, six hybridoma strains producing monoclonal antibodies that specifically recognize AMOR disaccharide structure (with methyl group) were successfully established (Figure 3B). The titer of the antibodies obtained from the six hybridoma strains differed from each other. All hybridomas are stored at IBBP of Japan. In addition, the conditions for immunostaining were examined and it was confirmed that the signal was obtained in Torenia ovules. Further analysis is underway to determine how the signal is altered in mutants of glucuronyltransferases and methyltransferases in Arabidopsis and Torenia.Analysis using multimeric AMORIt has been suggested that natural AMOR is more active than disaccharide AMOR. One possible reason for this is that arabinogalactan sugar chains are generally branched, and thus there may be many methylglucuronosylgalactose termini in one molecule. Since the multimeric AMOR is expected to mimic this structure, its activity was examined. The results using Tetra-AMOR (Figure 3A) showed that the activity of multimeric AMOR depended on the number of molecules of AMOR terminal structure in solution, but the activity per molecule increased, and tetrameric AMOR was found to be sufficiently active at 3.4 nM. Action mechanism of bio-active sugar chain AMORTetsuya HigashiyamaFigure 2. AMOR assay by micromanipulation of an ovuleSequential images of AMOR assay are shown. Time (MM:SS) in each panel indicates the time after the placement of an ovule (unfertilized seed tissue) of Torenia, which has a unique protruding egg apparatus. Arrows indicates an attracted pollen tube toward the tip of the protruding egg apparatus, suggesting that this pollen tube is competent by receiving AMOR in the medium. Scale bar: 20 μmwas also successful to show that glucuronidation is reduced in these mutants. However, the phenotype was not stable and clear possibly due to redundancy of glucuronosyltransferase genes. The importance of analysis during the fertilization process in Trenia and the importance of monoclonal antibodies (which can distinguish the presence or absence of the methyl group important for AMOR activity) to easily examine the distribution and amount of AMOR glycan terminal structures was again suggested5).From genomic and transcriptomic analyses in Torenia, candidate glucuronosyltransferases and methyltransferases were identified by gene homology and an assay using an expression system in tobacco leaves. We found eight genes that are homologous to the Arabidopsis glucuronosyltransferase GlcAT14A-C and expressed in the pistil organ. As a homolog of Arabidopsis methyltransferases AGM1 and AGM2, we identified a sole homolog which is expressed in the entire body including ovules. In addition, we could identify lectin receptor-like kinases of Torenia specifically expressed in the pollen tube, which might be potential candidates of AMOR receptor. Genetic analysis of these genes in Torenia has been in progress, by genome editing using CRISPR/Cas96). Therefore, we could establish a basis for further functional analysis of AMOR in Torenia. Moreover, we reported development of microdevices to quantitatively assess pollen tube attraction to ovules in Torenia7-8), which are also powerful tools for further functional analysis of AMOR.64
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