In this study, dynamic changes in structural polysaccharide deposition on the plasma membrane and cortical microtubules (CMTs) behavior were monitored in protoplasts isolated from white birch callus using confocal laser scanning microscopy and atomic force microscopy. We focused on the influence of an environmental stimulus on cell wall regeneration in protoplasts by employing an acidic culture medium containing a high concentration of Ca2+ (the stress condition). Under the non-stress condition, cellulose microfibrils and callose were initially synthesized, and thereafter deposited on the plasma membrane as “primary cell wall material”. Under the stress condition, callose micro-sized fibers were secreted without cell wall regeneration. Behavior of CMTs labeled with mammalian microtubule-associated protein 4 with green fluorescent protein in transgenic protoplasts was monitored by time-lapse video analysis. Under the non-stress condition, CMTs behavior showed a linear arrangement at a fixed position, whereas unfixed manner of CMTs behavior was observed under the stress condition. These findings indicate that excessive Ca2+ affects cellulose synthesis and CMTs dynamics in plant protoplasts. Current study first demonstrated dynamics of cell wall regeneration and CMTs in woody protoplast, which provides novel insight to aid in understanding early stages of primary cell wall formation in plants.
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