Dynamic Actin Controls Polarity Induction de novo in Protoplasts

Abstract

Cell polarity and axes are central for plant morphogenesis. To study how polarity and axes are induced de novo, we investigated protoplasts of tobacco Nicotiana tabacum cv. BY-2 expressing fluorescently-tagged cytoskeletal markers. We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages. The synthesis of a new cell wall marks the transition to the first stage of regeneration, and proceeds after a long preparatory phase within a few minutes. During this preparatory phase, the nucleus migrates actively, and cytoplasmic strands remodel vigorously. We probed this system for the effect of anti-cytoskeletal compounds, inducible bundling of actin, RGD-peptides, and temperature. Suppression of actin dynamics at an early stage leads to aberrant tripolar cells, whereas suppression of microtubule dynamics produces aberrant sausage-like cells with asymmetric cell walls. We integrated these data into a model, where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis. Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments, and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

Plant regeneration from leaf-derived protoplasts within the Daucus genus: effect of different conditions in alginate embedding and phytosulfokine application

“…Generally, a higher rate of cell colony formation was noted in the case of protoplasts embedded before culture, especially in alginate matrix, as compared to non-embedded protoplasts. Such a positive effect of embedding on cells may be explained by membrane stabilization through lipid peroxidase inhibition, the prevention of leakage of cell wall precursors or other metabolites, lower ethylene level (Bajaj 1989), or improved signaling cascade before the first cytokinesis (Sheahan et al 2007;Zaban et al 2013). Moreover, it was documented that protoplast separation ensured a better oxygen supply, resulting in avoidance of browning (Majewska-Sawka et al 1994;Pati et al 2005;Eeckhaut et al 2013).…”

Plant Cells Use Auxin Efflux to Explore Geometry

“…Only 12% of the cells did not regenerate a wall and remained arrested in stage 0, although maintaining viability. Although this fraction of non-regenerating cells is higher than the 5–7% found for axis regeneration outside a microfluidics environment9, the fact that almost 90% of viable cells underwent normal axis formation shows that conditions have been tuned close to the technically possible optimum.…”

“…In our previous work, we have followed the role of the cytoskeleton for the symmetry break in the regenerating protoplast9. Interestingly, the first phase of regeneration is characterized by extensive nuclear movements.…”

“…This consideration motivated us to design a system, where generation of axis and polarity can be generated de novo in single cells. To return to such a tabula-rasa state, we eliminated axis and polarity by removal of the cell wall in tobacco BY-2 cells, and then triggered the formation of a new axis and polarity9. By fluorescently tagged transgenic marker lines we could follow the behavior and the role of the cytoskeleton during this phenomenon.…”

Progress in plant protoplast research

“…This enhances division through better nuclear positioning (Sheahan et al 2007). In the model proposed by Zaban et al (2013), the cytoskeleton interacts with this nuclear positioning and thus controls activation and release of molecules involved in cell wall synthesis. Subsequently, dynamic actin filaments are indispensable for the induction of a new cell pole, required for elongation.…”