Department of Molecular Genetics and Center for Applied Plant Sciences, Ohio State University, Columbus, Ohio 43210, USA.
Department of Biological Sciences, University of Wisconsin, Milwaukee, Wisconsin 53211, USA.
Plant Physiol. 2021 Dec 4;187(4):2393-2404. doi: 10.1093/plphys/kiab426.
Formation of pollen wall exine is preceded by the development of several transient layers of extracellular materials deposited on the surface of developing pollen grains. One such layer is primexine (PE), a thin, ephemeral structure that is present only for a short period of time and is difficult to visualize and study. Recent genetic studies suggested that PE is a key factor in the formation of exine, making it critical to understand its composition and the dynamics of its formation. In this study, we used high-pressure frozen/freeze-substituted samples of developing Arabidopsis (Arabidopsis thaliana) pollen for a detailed transmission electron microscopy analysis of the PE ultrastructure throughout the tetrad stage of pollen development. We also analyzed anthers from wild-type Arabidopsis and three mutants defective in PE formation by immunofluorescence, carefully tracing several carbohydrate epitopes in PE and nearby anther tissues during the tetrad and the early free-microspore stages. Our analyses revealed likely sites where these carbohydrates are produced and showed that the distribution of these carbohydrates in PE changes significantly during the tetrad stage. We also identified tools for staging tetrads and demonstrate that components of PE undergo changes resembling phase separation. Our results indicate that PE behaves like a much more dynamic structure than has been previously appreciated and clearly show that Arabidopsis PE creates a scaffolding pattern for formation of reticulate exine.
花粉外壁外壁的形成是在发育中的花粉粒表面沉积的几种短暂的细胞外物质层的发育之前发生的。这样的层是初生外壁（PE），它是一种薄的、短暂的结构，仅存在很短的时间，并且难以可视化和研究。最近的遗传研究表明，PE 是外壁形成的关键因素，因此理解其组成和形成动力学至关重要。在这项研究中，我们使用拟南芥（Arabidopsis thaliana）发育中的花粉的高压冷冻/冷冻置换样品，对 PE 超微结构进行了详细的透射电子显微镜分析，涵盖了花粉发育的四分体阶段。我们还通过免疫荧光分析了来自野生型拟南芥和三个 PE 形成缺陷突变体的花药，仔细追踪了四分体和早期游离小孢子阶段中 PE 和附近花药组织中的几个碳水化合物表位。我们的分析揭示了这些碳水化合物可能产生的部位，并表明这些碳水化合物在四分体阶段在 PE 中的分布发生了显著变化。我们还确定了用于分期四分体的工具，并证明了 PE 的成分发生了类似于相分离的变化。我们的结果表明，PE 的行为比以前认为的要动态得多，并且清楚地表明拟南芥 PE 为形成网状外壁创造了支架模式。
