Abstract
In addition to synthesizing and secreting copious amounts of pectic polymers (Young et al., 2008), Arabidopsis thaliana seed coat epidermal cells produce small amounts of cellulose and hemicelluloses typical of secondary cell walls (Voiniciuc et al., 2015c). These components are intricately linked and are released as a large mucilage capsule upon hydration of mature seeds. Alterations in the structure of minor mucilage components can have dramatic effects on the architecture of this gelatinous cell wall. The immunolabeling protocol described here makes it possible to visualize the distribution of specific polysaccharides in the seed mucilage capsule.
Keywords: Arabidopsis thaliana, Plant cell wall, Immunolabeling, Carbohydrate, Fluorescence, Seed coat, Microscopy
Background
Since the first comprehensive immunofluorescence analysis of pectin-rich mucilage in Arabidopsis seed coat epidermal cells (Young et al., 2008), additional types of polysaccharides have been detected in this specialized cell wall (Voiniciuc et al., 2015a; 2015b and 2015c). To handle more samples in parallel, I adapted the original protocols (performed in 1.5-ml microcentrifuge tubes; Young et al., 2008; Harpaz-Saad et al., 2011) to a 24-well plate format. I recommend counterstaining seeds with Pontamine S4B, a fluorescent dye that is more specific to cellulose than previous stains (Anderson et al., 2010). By testing for cross-talk between multiple fluorophores and setting clear guidelines for image acquisition and processing, this protocol yields reproducible mucilage phenotypes that can be reliably interpreted.
Materials and Reagents
Equipment
Software
Procedure
Recipes
Note: The sterile solution is stable for at least 1 year.
Note: Make this solution fresh every time.
Acknowledgments
This protocol was briefly described by (Voiniciuc et al., 2015a), and was modified from the (Young et al., 2008; Harpaz-Saad et al., 2011) methods. I performed this work in the laboratory of Dr. Björn Usadel at Forschungzentrum Jülich with support from the Natural Sciences and Engineering Research Council of Canada (NSERC PGS-D3 grant).
References
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