Enigmatic apiogalacturonans: Investigations on structure, taxonomic occurrence, localization and responses to environmental factors will help disentangling this unusual group of pectic polysaccharides

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Polysaccharides are major components of the plant cell wall. Some of them, namely the pectins, possess a very complicated and highly charged structure. Among the pectic polysaccharides, one subgroup, the apiogalacturonans (AGAs), are only rarely described throughout the whole green lineage. Up to now, AGAs are only known from duckweeds and seagrasses. Both groups are neither monophyletic nor closely related, but belong to the order of Alismatales. This fact pinpoints a shared evolutionary trajectory and highlights the need of a systematized analysis throughout that order to infer influence factors, which lead to AGAs occurrence. In a first step, representative members of the order will be investigated for AGA presence and structure by extraction from the crude pectin fraction. Size-exclusion chromatography as well as specific enzymatic cleavages (usage of a recently described rhamnogalacturonan-I lyase) will help to purify the apiosylated homogalacturonan scaffold named AGA. As available structural data on the few structurally described AGAs (from duckweeds and seagrasses) are contradictory to some extent, a detailed analysis of structural features will be performed by classical polysaccharide analysis methodologies. Plant species of different lifestyles and habitats (freshwater, brackish water, saline water and terrestrial) are included to represent most possible triggering factors for AGA biosynthesis. Duckweeds accumulate high amounts of boron and boron is usually crosslinked by apiose residues in rhamnogalacturonan-II. Therefore, an experimental set-up with Lemna in-vitro culture and Zostera callus culture will be used to correlate influences of boron (determined by ICP-MS) on AGA content. The hypothesis of a boron-storage function of AGAs and influences of salinity on the degree of AGA expression will be tested. Monoclonal antibodies are an important tool for plant cell wall research. They help to investigate the localization and presence of polysaccharide epitopes in plantae. The purified AGAs of Zostera will be used as antigen to immunize mice and generate a monoclonal antibody directed against the apiose side chain structures. This antibody will provide a valuable tool to foster the research on AGA localization by immunofluorescence as well as transmission electron microscopy and will complement the available antibody toolkit against pectic epitopes. In conclusion, the proposed project on AGAs will help to expand the current knowledge on this unusual group of pectic polysaccharides in various ways. This research will hint towards a molecular understanding of the stimulating factors leading to AGAs’ occurrence pattern in land plants.