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

多糖是植物细胞壁的主要成分。其中一些，即果胶，具有非常复杂和高度带电的结构。在果胶多糖中，有一个亚群，即apiogalacturonans (AGAs)，在整个绿色谱系中很少被描述。到目前为止，已知的AGAs仅来自浮萍和海草。这两个类群既不是单系的，也不是近亲，而是属于泽泻目。这一事实指出了共同的进化轨迹，并强调了在整个过程中进行系统分析以推断导致AGAs发生的影响因素的必要性。在第一步中，将通过从粗果胶馏分中提取AGA的存在和结构来研究该顺序的代表性成员。尺寸排除层析以及特定的酶裂解（使用最近描述的鼠李糖半乳糖醛酸- 1裂解酶）将有助于纯化被命名为AGA的apiosylated的均半乳糖醛酸支架。由于少数结构描述的AGAs（来自浮萍和海草）的现有结构数据在一定程度上是矛盾的，因此将采用经典的多糖分析方法对结构特征进行详细分析。不同生活方式和栖息地（淡水、微咸水、咸水和陆地）的植物物种被包括在内，代表了AGA生物合成的最可能触发因素。浮草中积累了大量的硼，硼通常是通过鼠李糖半乳糖醛酸- ii中的apiose残基交联的。因此，将采用离体Lemna培养和Zostera愈伤组织培养的实验装置来研究硼（ICP-MS测定）对AGA含量的影响。我们将检验AGAs的硼储存功能假说以及盐度对AGA表达程度的影响。单克隆抗体是植物细胞壁研究的重要工具。它们有助于研究植物中多糖表位的定位和存在。纯化的Zostera AGAs将作为抗原免疫小鼠，并产生针对apiose侧链结构的单克隆抗体。该抗体将为利用免疫荧光和透射电镜对AGA定位的研究提供有价值的工具，并将补充现有的针对果胶表位的抗体工具包。总之，拟议的AGAs项目将有助于以各种方式扩大对这一不寻常的果胶多糖群的现有知识。本研究将有助于从分子角度理解陆地植物中AGAs发生模式的刺激因子。

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.