Background

The structure of primary plant cell walls plays a key role in determining cell wall biomechanical properties and regulating plant cell growth and morphogenesis (Cosgrove, 2005 and 2016). To visualize cell wall organization, common methods include transmission and scanning electron microscopy (TEM, SEM), light microscopy and AFM (McCann et al., 1990; Marga et al., 2005; Anderson et al., 2010; Ding et al., 2012; Abraham and Elbaum, 2013; Zhang et al., 2014; Zheng et al., 2017). In recent years AFM has enabled the imaging of soft biological samples in fluid, thus allowing studies of plant cell walls at nm-resolution in a close-to-native state, without dehydration, harsh chemical treatment, embedding or sectioning. Compared to EM techniques, which typically require dehydrated samples for high resolution imaging, AFM avoids dehydration artifacts and simplifies the sample preparation procedure while achieving high resolution imaging at the nanometer scale (Zhang et al., 2016). Combining AFM imaging with enzyme treatments, mechanical testing or use of cell wall mutants (Xiao et al., 2016; Zhang et al., 2017), we can test the hypothesized roles of specific cell wall components in cell growth, mechanics and cell wall structure.

For this protocol we focus on the onion scale epidermal cell wall, which has been the subject of numerous mechanical, spectroscopic and microscopic studies (Wilson et al., 2000; Kerstens et al., 2001; Hepworth and Bruce, 2004; Vanstreels et al., 2005; Loodts et al., 2006; Suslov et al., 2009). A key difference between our preparation method and that of previous authors is that our method splits open the epidermal cell layer, separating the outer epidermal cell wall from the remainder of the epidermal cell. After a brief wash to remove membranes and cellular debris, the newly-synthesized surface is ready for direct imaging by AFM. This is possible because the abaxial epidermis (on the convex or outer surface of the onion scale) adheres tightly to the underlying parenchyma tissues, so the peeling procedure splits open the epidermal cells. In previous studies with onion, whole-cell epidermal layers were peeled from the adaxial (inner or concave) surface of the onion scale, i.e., this cell layer adheres weakly to the underlying tissues and so separates intact at the interface (the middle lamella) with the underlying tissue. This difference in peeling procedure is of course important for surface-imaging methods such as AFM and SEM.