Staining Maize Epidermal Leaf Peels with Toluidine Blue O

Background

Histological methods provide a reliable way to study plant cell morphology and anatomy with a focus on visualizing differences in cell wall composition. TBO, also known as Tolonium chloride (C₁₅H₁₆N₃S⁺), is a basic (cationic), dye that binds anionic groups in the cell and stains them distinct colors. Polyphenolic substances, such as lignin, are stained green-blue. Carboxylated polysaccharides, such as pectic acid, are stained pink-purple (Yeung, 1998). Nucleic acids are stained blue-purple. TBO has been used by plant biologists to understand the development, cellular anatomy and chemical makeup of cells for nearly one hundred years, but used more commonly after a landmark paper showed that it differentially stained different cell types and cell wall components (O’Brien et al., 1964). In Arabidopsis thaliana, TBO has been used on transverse sections of stems to identify xylem and phloem via differences in relative abundance of lignin in the secondary cell wall (Pradhan Mitra and Loqué, 2014; Verhertbruggen et al., 2017).

In Zea mays (maize), TBO has been used to understand the development, organization, shape and orientation of cell divisions (Smith et al., 1996; Gallagher and Smith, 1999; Cartwright et al., 2009; Wright et al., 2009; Frank et al.., 2003; Rosa et al., 2017). TBO has been used to characterize defects in maize cell division and development such as found in discordia1 (dcd1) and dcd2 mutants with abnormal asymmetric subsidiary cell divisions (Gallagher and Smith, 1999) or symmetric and asymmetric division defects in tangled1 mutants (Smith et al., 1996; Martinez et al., 2017). Developmental defects in maize leaves have been studied using TBO on mutant plants (Moose and Sisco, 1994; Chuck et al., 2011) such as crinkly4 (cr4), a receptor-like kinase (Jin et al., 2000) and the adaxialized leaves of the semi-dominant Rolled leaf1-O mutant (Nelson et al., 2002; Juarez et al., 2004). TBO was used to analyze defective kernel1 (dek1) mutants in kernel cross-sections for analysis of the maize embryo (Becraft et al., 2002). Cell wall composition of maize leaf epidermis in corn rootworm beetles1 (crw1) mutant plants was revealed to be different from wild-type using TBO (Venkata et al., 2013). A recent study on the narrow odd dwarf 1 (nod1) mutant used TBO to show delays in the juvenile to adult leaf transition and defects in stomatal cell organization (Rosa et al., 2017).

Importantly, polychromatic TBO staining can be used to describe cell anatomy in plants lacking molecular tools. The popular use of TBO is exemplified by the numerous examples of fixation, dissection, and TBO staining in different plants (Regan and Moffatt, 1990; Ye and Varner, 1991; Cheng et al., 2000; Gao and Showalter, 2000; Tavakol et al., 2015; Murgia et al., 2017). Many of the examples referenced above use whole-mount leaf tissue and also epidermal peeling followed by TBO staining. If the focus is to observe multiple cell layers, cross-sections of whole-mount leaf tissue are used. When whole-mount leaves are used to examine epidermal cells, careful observation is required to analyze just epidermal cells while avoiding the cells below. If the focus is to gather high-quality quantitative information about epidermal cells, which play a critical role in development and growth, this method provides reliable and clean epidermal cell micrographs for analysis. Our protocol shows step-by-step epidermal leaf peeling and common troubleshooting during TBO staining for maize researchers.