Abstract
Maize leaves grow in a gradient. The base of young maize leaves contains a high proportion of symmetrically and asymmetrically dividing cells, making the leaf an effective model system for the study of cell division. Here, we describe a double staining method to visualize actin filaments and microtubules in fixed maize leaf tissues, using immunofluorescent staining for microtubules and fluorescent phalloidin staining for actin filaments. This method provides a technique for the study of the cytoskeleton during maize cell division.
Keywords: Maize, Actin filament, Microtubules, Double staining, Division zone
Background
Immature growing maize leaves mature in a gradient. The developing leaf blade, which is distal to the pre-ligular band or ligule, has three primary developmental zones. Proximal to the base or ligule are undifferentiated cells undergoing symmetric divisions, followed by differentiating cells undergoing asymmetric divisions, and finally cells that are no longer dividing but undergoing rapid cell expansion (Sylvester et al., 1996; Facette et al., 2013). This makes the monocot leaf an excellent model for studying cell division and aspects of leaf development. The developmental progression observed in monocot leaves has been used to study photosynthetic and vascular bundle formation in maize (Li et al., 2010); stomatal divisions in Brachypodium distachyon (Raissig et al., 2016), rice (Wu et al., 2019) and maize (Facette et al., 2015); and cell division in maize (Hunter et al., 2012; Nelissen et al., 2012). Maize leaves have large cells excellent for imaging these processes. Live cell imaging of the immature leaf using fluorescent markers is powerful (Rasmussen, 2016), however this requires that plants express transgenic markers, which can be limiting. We present a whole-mount procedure that does not require difficult and time-consuming sectioning, to observe the actin and microtubule cytoskeleton in the developing maize leaf epidermis.
Materials and Reagents
Equipment
Procedure
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Recipes
Acknowledgments
This protocol was adapted from and optimized from previous methods (Panteris et al., 2006; Cartwright et al., 2009). MRF acknowledges funding from NSF-IOS 1754665.
References
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Yes - thank you for catching this and our apologies. The fluor on the Phalloidin should be different than the fluor on the antibody. Thus, it should state AlexaFluor 488-Phalloidin is added (if using Goat anti-mouse AlexaFluor 568). These two colors can be reversed if you wish - we find the brighter actin staining easier to see - but either way it will work, as long as the microtubules and actin filaments are labelled with different colors.