Dynamic Monitoring And Study Of Mechanical Properties Of Model Plant Cells By QCM Technique

Plant development is the result of three important processes of cell growth,division and differentiation,all of which have their unique mechanical characteristics.The interaction between turgor and cell wall is the basis of plant cell growth.The mechanical properties of plant cells mainly depend on the viscoelasticity of cell wall,the viscoelasticity of cytoskeleton and the turgor pressure.However,there is currently no satisfactory single technique being able to monitor and reveal the complex cellular processes in a non-destructive,dynamical,real-time and long-term way.In this study,we used quartz crystal microbalance(QCM)technique to monitor the suspension cells of tobacco BY-2 and rice,and study the mechanical properties(including the viscoelasticity of cell wall and cytoskeleton,and the cells-generated forces)of plant cells under different growth processes and external stimuli.The main experimental results are as follows:1.Based on the system of living plant cells to be detected,cell adhesion methods shitable for QCM detections were established.According to the negative charge characteristics of plant cell wall and cell membrane,we modified and compared the QCM chips with positively charged molecules suitable for the adhesion of plant cells.1%PDADMAC was suitable for the adhesion of plant cells,0.1% Poly-L-lysine was suitable for the adhesion of protoplasts.2.The cells-generated forces and viscoelasticity of BY-2 cells were measured using the established QCM detection system.As the QCM-D allows the simultaneous measurement of changes in frequency and damping at multiple overtones of the crystal,it is possible to test the viscoelastic characteristics of different cellular structures at different depths within the living cells.The dynamic changes of viscoelasticity of tobacco BY-2 suspension cells and protoplasts under the stresses of different osmotic pressures were monitored in real time.In addition,we observed and compared the morphological changes of BY-2 cells and the protoplasts under different osmotic pressures.It was found that the viscoelastic changes of cells and protoplasts are different,and the viscoelastic changes of cells at different overtone frequencies under the same osmotic pressure are also different.Double-resonator technique was used to monitor the dynamic changes of cells generated stresses of BY-2 cells and the protoplasts accompanied with the changes of osmotic stresses.During hypoosmotic conditions,the turgor pressure of plant cells is the main driving force for cells generated stress on the QCM chips,so the magnitude of the stress measured can reflect the magnitude of cell turgor.In hypertonic conditions,the cell wall-protoplast membrane-cytoskeleton continuum affects the viscoelasticity of cells.3.The effect of plant cell wall on cell mechanics was preliminarily explored.The dynamic changes of viscoelasticity and the process of enzymatic hydrolysis of BY-2 cell wall and the cell wall regeneration were monitored in real time by QCM technique.The results indicate that these dynamic changes are caused by a combination of the effects of cytoskeleton and cell expansion.In addition,we observed and compared the morphological changes during the enzymatic hydrolysis of BY-2 cell wall and the cell wall regeneration of BY-2 protoplasts by optical microscope.The enzymatic hydrolysis of cell wall is accompanied by the gradual disappearance of Hechtian strands,which is a process of cell hardening.As the cell wall regenerates and thickens,the cell hardens.These results suggest that cell wall and cytoskeleton play important roles in maintaining cell volume and swelling.Moreover,we also preliminarily monitored the viscoelastic changes accompanying the increase in the number of BY-2 cells in different cycles of cell division,and found that the viscoelastic changes in each cell division cycle showed similar viscoelastic fluctuations.4.The effect of cytoskeleton on plant cell mechanics was preliminarily investigated.In order to understand the different roles of cytoskeletal microtubules and microfilaments in plant cell’s mechanical properties,we performed real-time and dynamic monitoring of different concentrations of cytoskeletal drugs on the effects of rice suspension cells and BY-2protoplasts.Dynamic changes in cells’ viscoelasticity and cells generated stresses under the treatments of different concentrations of microtubule interacting drugs(colchicine,paclitaxel)and drug affecting microfilaments(cytochalasin D)were monitored.The depolymerization of microtubules made the cells softer.The polymerization of microtubules made the cells harder.The higher the concentration of paclitaxel,the faster the polymerization rate of microtubules,leading to increased degree of stiffening of the cells.In summary,this study verified the feasibility of non-destructive,dynamic,real-time,and long-term monitoring of plant cells’ mechanical parameters(changes in plant cells’ viscoelasticity and cells generated stresses)by wide frequency QCM technique of different overtones and double resonator technique.In turn,the dynamic changes of the cell wall and the cytoskeleton were obtained.QCM technology can be used as a new tool to test the mechanical properties of plant cells.