A rapid and quantitative safranin-based fluorescent microscopy method to evaluate cell wall lignification.
Fabien Baldacci-CrespCorentin SprietLaure TwyffelsAnne-Sophie BlervacqGodfrey NeutelingsMarie BaucherSimon HawkinsPublished in: The Plant journal : for cell and molecular biology (2020)
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.
Keyphrases
- cell wall
- ionic liquid
- high resolution
- induced apoptosis
- quantum dots
- arabidopsis thaliana
- living cells
- gene expression
- optical coherence tomography
- high throughput
- multidrug resistant
- stem cells
- signaling pathway
- high speed
- single cell
- genome wide
- fluorescent probe
- nitric oxide
- endoplasmic reticulum stress
- bone marrow
- dna methylation
- sensitive detection
- mesenchymal stem cells
- cell therapy
- human health
- tissue engineering
- wild type
- fluorescence imaging