Wiethölter Nicola, Graessner Barbara, Mierau Manfred, Willats William G T, Knox J Paul, Moerschbacher Bruno M
Institut für Biochemie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Hindenburgplatz 55, D-48143, Münster, Germany.
Carbohydr Res. 2003 Feb 14;338(5):423-31. doi: 10.1016/s0008-6215(02)00498-6.
In contrast to the typical type I cell wall of the dicot plants, the type II cell wall of the commelinoid monocot plants is known to be relatively poor in pectins. Assuming a critical role for the remaining pectins in terms of cell wall architecture and/or as a reservoir of signalling molecules, we have compared different protocols for the isolation of the main pectin polymer, homogalacturonan, from wheat leaf cell walls. Pectin was detected in these cell walls immunochemically using the monoclonal antibodies JIM5 and JIM7, and biochemically by monosaccharide analysis. The Ca(++)-chelators CDTA and imidazole extracted a pectin rich fraction from isolated cell walls which was however contaminated with significant amounts of hemicelluloses. Pretreatment of the cell walls with anhydrous hydrogen fluoride at controlled low temperatures followed by HF/ether- and water-extraction prior to imidazole-extraction of pectins yielded a purer homogalacturonan fraction. The near absence of rhamnosyl residues proved that the isolated homogalacturonan fraction was free of rhamnogalacturonans. If HF-solvolysis was performed at -23 degrees C, the resulting homogalacturonan had a degree of methyl esterification identical to that of the pectins in the initial wheat cell wall. The antibodies JIM5 and JIM7 as well as PAM1 and LM5 proved that the isolated homogalacturonan had a low methyl ester content, was polymeric and free of galactan side chains. We can thus isolate native homogalacturonan from the type II wheat cell walls with the original in muro pattern of methyl esterification still intact, to further investigate e.g., its degradability by plant or microbial pectic enzymes.
与双子叶植物典型的I型细胞壁不同，已知鸭跖草类单子叶植物的II型细胞壁中果胶含量相对较低。鉴于剩余果胶在细胞壁结构和/或作为信号分子储存库方面的关键作用，我们比较了从小麦叶细胞壁中分离主要果胶聚合物——同型半乳糖醛酸聚糖的不同方案。使用单克隆抗体JIM5和JIM7通过免疫化学方法在这些细胞壁中检测果胶，并通过单糖分析进行生化检测。Ca(++)螯合剂CDTA和咪唑从分离的细胞壁中提取了富含果胶的部分，但该部分被大量半纤维素污染。在低温控制下用无水氟化氢预处理细胞壁，然后在咪唑提取果胶之前进行HF/乙醚和水提取，得到了更纯的同型半乳糖醛酸聚糖部分。几乎不存在鼠李糖基残基证明分离出的同型半乳糖醛酸聚糖部分不含鼠李糖半乳糖醛酸聚糖。如果在-23℃进行HF溶剂解，得到的同型半乳糖醛酸聚糖的甲酯化程度与初始小麦细胞壁中的果胶相同。抗体JIM5和JIM7以及PAM1和LM5证明，分离出的同型半乳糖醛酸聚糖甲酯含量低、呈聚合物形式且不含半乳聚糖侧链。因此，我们可以从II型小麦细胞壁中分离出天然的同型半乳糖醛酸聚糖，其甲酯化的原始壁内模式仍然完整，以便进一步研究例如其被植物或微生物果胶酶降解的能力。
