Dynamics of structural polysaccharides deposition on the plasma-membrane surface of plant protoplasts during cell wall regeneration

Satomi Tagawa,Ryo Funada,Ugai Watanabe,Yusuke Yamagishi,Tetsuo Kondo

doi:10.1186/s10086-019-1826-0

Abstract

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.

Highlights

The primary cell wall, which is a thinner membrane-type wall, is deposited on the cell surface during cell division and expansion
The current study demonstrates dynamics of structural polysaccharides deposition relating to cortical microtubules (CMTs) dynamics on the plasma-membrane surface of plant protoplasts during cell wall regeneration
Under the acidic stress condition, using a culture medium containing a high concentration of Ca2+, cell wall regeneration was suppressed in protoplasts of white birch callus, which was possibly due to inhibition of cellulose microfibril (CMF) deposition (Figs. 3, 5b)

Summary

Introduction

The primary cell wall, which is a thinner membrane-type wall, is deposited on the cell surface during cell division and expansion. Research on the structure of primary cell walls has been carried out mostly with a biochemical focus together with microscopic visualization. Studies on primary cell wall structure began with isolation. Agrobacterium tumefaciens was cultured overnight at 28 °C in liquid yeast extract-mannitol medium in the presence of 50 mg l−1 kanamycin, and this culture was used for transformation of birch calli. The calli were cultured on selective callus-induction medium [MS medium supplemented with 100 mg l−1 kanamycin, 500 mg l−1 carbenicillin, 2 mM NAA, 1 mM 4PU and 1% (w/v) agar]. After calli development for 2–3 months, the grown portions were transferred to fresh selective medium (MS medium supplemented with 50 mg l−1 kanamycin, 250 mg l−1 carbenicillin, 2 mM NAA, 1 mM 4PU, and 1% (w/v) agar). The transformed protoplasts were selected from the transformed calli by enzymatic treatment

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Conclusion