Visualising Differential Growth of Arabidopsis Epidermal Pavement Cells Using Thin Plate Spline Analysis

Background

Understanding the spatial pattern of growth of a cell provides insight into how plant cells form different shapes. Epidermal pavement cells of Arabidopsis thaliana cotyledons and leaves are a good model system for investigating how complex cells grow as their cell wall boundaries develop multiple undulations of different size from boundaries that were initially simple arcs (Armour et al., 2015; Fu et al., 2005). Growth of plant cells has been measured by fixing externally applied markers to cells such as algal Nitella internodes (Green et al., 1970), root cells (Shaw et al., 2000), and trichomes (Schwab et al., 2003). However measurement of cell growth from externally applied landmarks is sometimes not feasible such as when the strong fluorescence of externally applied fluorescent markers would obscure fluorescently labeled cytoskeletal elements within cells (Armour et al., 2015). Thin plate spline analysis which visualizes the changing positions of a defined number of homologous landmarks over time or between different objects has previously been used to analyze changes in the three dimensional size and shape of objects such as hominid skulls (Rosas and Bastir, 2002; Gunz et al., 2009), or the two dimensional shapes of insect wings (Börstler et al., 2014) and leaves (Polder et al., 2007). This protocol describes how to use thin plate spline analysis to visualize size and shape changes of individual cells. This technique is relatively easy to utilize on a range of cell images as it uses the input of the outline of a cell at sequential times to approximate the relative growth rate and growth direction of different areas of the cell wall.