Abstract
Here we describe two experimental protocols to measure the biomechanical properties of primary (growing) plant cell walls, with a focus on analyzing cell wall epidermal strips of onion scales. The first protocol measures cell wall creep (time-dependent irreversible extension) under constant force. Such creep is often mediated by the wall-loosening action of expansin or selective endoglucanases. The second protocol is based on two consecutive stretches of the wall and measures the wall’s elastic and plastic compliances, which depend on cell wall structure. These two assays provide complementary information that may be linked to cell wall structure and expansive growth of cells.
Keywords: Cell walls, Mechanics, Elastic modulus, Plastic modulus, Creep, Onion epidermis
Background
The primary walls of growing plant cells are strong enough to resist the tensile forces generated by cell turgor pressure, yet can expand irreversibly during cell growth as a result of the selective loosening action of expansins or other catalysts necessary for irreversible cell wall enlargement (Cosgrove, 2016a and 2016b). Assessments of cell wall mechanical properties, such as elasticity and plasticity, are important for understanding cell wall structure and its modification during growth (cell enlargement) and after growth ceases. An important measure of wall mechanics is based on uniaxial tensile tests, as described below in our stress/strain assay, which measures elastic and plastic compliance (compliance is the reciprocal of modulus; modulus is a measure of stiffness). A second, complementary assay is based on the irreversible, time-dependent increase in length (creep) that occurs in primary cell walls when they are held at constant tension and continuously loosened by endogenous expansin or exogenous endoglucanase (Durachko and Cosgrove, 2009; Cosgrove, 2011; Cosgrove et al., 2017). In this protocol, we describe procedures for preparing epidermal cell wall strips from onion scales and testing them in these two assays. Onion epidermal cell walls provide a useful model to explore the connection between cell wall structure and biomechanics (Wilson et al., 2000; Suslov et al., 2009; Kim et al., 2015; Zhang et al., 2017; Zheng et al., 2017).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
We thank Ed Wagner, Dr. Sarah Kiemle and Xuan Wang for technical assistance. This work was supported by the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences (grant No. DE-SC0001090). This protocol is adapted from Zhang et al. (2017). The authors declare no conflicts of interest or competing interests.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Thanks for your query and we are happy to clarify. In the Materials and Recipes sections, the pH of the HEPES and acetate buffers were incorrectly amended during the last stage of bio-protocol publication, and we missed this editorial change. HEPES buffer should be pH 6.8 and acetate buffer should be pH 4.5, throughout. Sorry for the confusion and we will ask bio-protocol to correct these typos. About curling: indeed the peeled strips have a strong tendency to curl. As described in step A.8 and shown in Figure 4B, it is important to leave chunks of subepidermal tissue at the two ends of the peel (like barbells). This helps a lot with the handling. Also, when you float the peels on a solution, ALWAYS place them with cuticle side up (facing the air). If you place the cuticle side down, they will curl. The power of surface tension of water! With a fine pair of forceps they can be uncurled under a dissecting scope, but it is challenging. The issue with curling should have been made clearer in the protocol. The curling behavior is not likely related to the solution composition. Also, we note that the tendency to curl varies greatly among onions, so try more than one onion. Good luck.
Ok thanks a lot, putting them with the cuticle facing up fixed the curling problem. Staining with DAPI also seems to indeed show the absence of the nuclei in the peeled part. The nuclei nonetheless remain in the thicker bordering parts, which is good. Thanks for the advice ! William Nicolas