| Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides
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Author:
Date:
2017-06-05
[Abstract] In addition to synthesizing and secreting copious amounts of pectic polymers (Young et al., 2008), Arabidopsis thaliana seed coat epidermal cells produce small amounts of cellulose and hemicelluloses typical of secondary cell walls (Voiniciuc et al., 2015c). These components are intricately linked and are released as a large mucilage capsule upon hydration of mature seeds. Alterations in the structure of minor mucilage components can have dramatic effects on the architecture of this gelatinous cell wall. The immunolabeling protocol described here makes it possible to visualize the distribution of specific polysaccharides in the seed mucilage capsule.
[摘要] 除了合成和分泌大量的果胶聚合物（Young等人，2008）外，拟南芥种皮表皮细胞产生少量的二级纤维素和半纤维素细胞壁（Voiniciuc等，，2015c）。这些组分复杂连接，并在成熟种子水合时作为大胶囊释放。在较小的粘液组分的结构中的改变可以对该凝胶状细胞壁的结构产生显着的影响。这里描述的免疫标记方案使得可以可视化种子胶囊中特定多糖的分布。
背景 自从拟南芥种皮表皮细胞（Young等人，2008）第一次富含果胶的胶质综合免疫荧光分析以来，在这种特殊的细胞壁中已经检测到另外类型的多糖（Voinicucucum，等等。，2015a; 2015b和2015c）。为了平行处理更多的样本，我修改了原始方案（在1.5 ml微量离心管中执行; Young等人，2008; Harpaz-Saad等人，2011年）到24孔板格式。我建议用Pontamine S4B（一种比以前的污渍更具体的纤维素荧光染料）来重新研磨种子（Anderson等人，2010）。通过测试多个荧光团之间的串扰，并为图像采集和处理设定明确的指导，该方案产生可重复的粘液表型，可以可靠地解释。
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| Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds
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Author:
Date:
2016-05-05
[Abstract] The Arabidopsis thaliana seed coat epidermis produces copious amounts of mucilage polysaccharides (Haughn and Western, 2012). Characterization of mucilage mutants has identified novel genes required for cell wall biosynthesis and modification (North et al., 2014). The biochemical analysis of seed mucilage is essential to evaluate how different mutations affect cell wall structure (Voiniciuc et al., 2015c). Here we describe a robust method to screen the monosaccharide composition of Arabidopsis seed mucilage using ion chromatography (IC). Mucilage from up to 48 samples can be extracted and prepared for IC analysis within 24 h (only 4 h hands-on). Furthermore, this protocol enables fast separation (31 min per sample), automatic detection and ...
[摘要] 拟南芥种皮表皮产生大量的粘液多糖（Haughn和Western，2012）。 粘液突变体的表征已经鉴定了细胞壁生物合成和修饰所需的新基因（North等人，2014）。 种子粘液的生物化学分析对于评估不同突变如何影响细胞壁结构是必要的（Voiniciuc等人，2015c）。 在这里我们描述了使用离子色谱（IC）筛选拟南芥种子粘液的单糖组成的有力方法。 在24小时内（仅4小时手动）可以提取并准备来自多达48个样品的粘蛋白并进行IC分析。 此外，该协议使快速分离（每个样品31分钟），自动检测和定量的中性和酸性糖。
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| Quantification of the Mucilage Detachment from Arabidopsis Seeds
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Author:
Date:
2016-05-05
[Abstract] The Arabidopsis thaliana seed coat produces large amounts of cell wall polysaccharides, which swell out of the epidermal cells upon hydration of the mature dry seeds. While most mucilage polymers immediately diffuse in the surrounding solution, the remaining fraction tightly adheres to the seed, forming a dense gel-like capsule (Macquet et al., 2007). Recent evidence suggests that the adherence of mucilage is mediated by complex interactions between several cell wall components (Griffiths et al., 2014; Voiniciuc et al., 2015a). Therefore, it is important to evaluate how different cell wall mutants impact this mucilage property. This protocol facilitates the analysis of monosaccharides in sequentially extracted mucilage fractions, and quantifies the ...
[摘要] 拟南芥种子产生大量的细胞壁多糖，其在成熟干种子水合时从表皮细胞中膨胀。 虽然大多数粘液聚合物立即在周围溶液中扩散，但剩余部分紧密粘附于种子，形成致密的凝胶状胶囊（Macquet等人，2007）。 最近的证据表明粘液的粘附由几个细胞壁组分之间的复杂相互作用介导（Griffiths等人，2014; Voiniciuc等人，2015a）。 因此，重要的是评估不同的细胞壁突变体如何影响这种粘液性质。 该协议有助于分析顺序提取的粘液部分中的单糖，并且量化每种成分从种子中的脱离。
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