One of the main characteristics of plant cells is the presence of the cell wall located outside the plasma membrane. In particular cells, this wall can be reinforced by lignin, a polyphenolic polymer that plays a central role for vascular plants, conferring hydrophobicity to conducting tissues and mechanical support for upright growth. Lignin has been studied extensively by a range of different techniques, including anatomical and morphological analyses using dyes to characterize the polymer localization in situ. With the constant improvement of imaging techniques, it is now possible to revisit old qualitative techniques and adapt them to obtain efficient, highly resolutive, quantitative, fast and safe methodologies. In this study, we revisit and exploit the potential of fluorescent microscopy coupled to safranin-O staining to develop a quantitative approach for lignin content determination. The developed approach is based on ratiometric emission measurements and the development of an imagej macro. To demonstrate the potential of our methodology compared with other commonly used lignin reagents, we demonstrated the use of safranin-O staining to evaluate and compare lignin contents in previously characterized Arabidopsis thaliana lignin biosynthesis mutants. In addition, the analysis of lignin content and spatial distribution in the Arabidopsis laccase mutant also provided new biological insights into the effects of laccase gene downregulation in different cell types. Our safranin-O-based methodology, also validated for Linum usitatissimum (flax), Zea mays (maize) and Populus tremula x alba (poplar), significantly improves and speeds up anatomical and developmental investigations of lignin, which we hope will contribute to new discoveries in many areas of cell wall plant research.

中文翻译：

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基于番红蛋白的快速定量荧光显微镜方法，用于评估细胞壁的木质化。

植物细胞的主要特征之一是位于质膜外部的细胞壁的存在。在特定的细胞中，此壁可以用木质素（木质素）增强，木质素是对维管束植物起关键作用的多酚类聚合物，可赋予疏水性以传导组织并为垂直生长提供机械支持。木质素已通过一系列不同的技术进行了广泛的研究，包括使用染料进行解剖和形态分析以表征聚合物的原位定位。随着成像技术的不断改进，现在有可能重新审视旧的定性技术，并使它们适应以获得高效，高度可分辨，定量，快速和安全的方法。在这个研究中，我们将重新探讨并利用荧光显微镜与番红O染色相结合的潜力来开发定量测定木质素含量的方法。所开发的方法基于比例发射测量和imagej宏的开发。为了证明我们的方法与其他常用的木质素试剂相比的潜力，我们证明了番红O染色用于评估和比较先前鉴定的拟南芥木质素生物合成突变体中木质素的含量。另外，对拟南芥漆酶突变体中木质素含量和空间分布的分析也为漆酶基因下调在不同细胞类型中的作用提供了新的生物学见解。我们基于番红花O的方法论也经过了亚麻（Linum usitatissimum）（亚麻）的验证，