Contributions Of Microtubules And Actin Filaments In Mechanoresponses Of Arabidopsis Cells

Mechanical perturbations exist throughout the plant life course. A large number of studies have confirmed that mechanostimuli, including wind, rain, and the turgor pressure within plant cells, play key roles in a series of physiological processes in plants and have important influences on plant growth and development. Correct understanding of the mechanisms underlying plant mechanosensing and mechanoresponse is helpful to human being in reasonably using and conserving agricultural and forestry resources. Based on researches in animal cells, two hypothetical plant mechanoperception/transduction pathways have been proposed. One is the"cell wall-plasma membrane-cytoskeleton"pathway and the other is the"mechanosensitive channel"pathway. However, there is no direct experimental evidence to test these two hypothetical pathways so far. In the present thesis, the link between cell wall and cytoskeleton, the dynamics of the cytoskeleton in response to mechanical stress and the influence of calcium channel blockers on cytoskeletal reorganization in response to mechanical stress were investigated by in vivo real-time visualization of microtubule (MT, labeled with GFP-MBD) and actin filament (AF, labeled with GFP-FABD2) structures using confocal laser microscope. The results provided direct experimental evidences to confirm that cell wall is connected with the cytoskeleton, helped us to understand the role of cytoskeleton in mechanosensing and improved the model of mechanotransduction in plant. In addition, different methods for real-time cytoskeleton observation under dynamic mechanical loading were designed, which provided new approaches for investigation of stimulus-response relationship of plant cells. Following are the major results and conclusions of the present thesis:GFP-FABD2 and GFP-MBD suspension cell culture systems were established and optimized so that no significant differences was preexisted in cellular morphology, growth and viability between wild-type. The optimized transgenic suspension cells'ability to response to low temperature, hyperosmotic stress and mechanical stress stimuli were then detected, and no differences between transgenic and wild-type were found. These results indicated that the transgenic suspension cells were ideal materials for stress response studies.The connections between cell wall and cytoskeleton are verified by the following researches. Cell wall of suspension cells were selectively digested with cellulase or pectinase, and both resulted in plasmolysed ratios lower than the control group, which suggested that the cell wall has contributions to osmotic response. Cell wall digestion with cellulase or pectinase caused sustained reducing of fluorescence intensity of AFs, while the same treatment did not cause any detectable change in fluorescence intensity of MTs, consisted with a possibility that AFs have direct connection with cell wall. To further understand the association between cytoskeleton and cell wall, suspension cells were pretreated with 0.5mM GRGDS peptide, and it was found that the plasmolysed ratios of treated cells were declined and MTs in treated cells were depolymerized and crystallized, but little change in actin cytoskeleton. In contrast, GRGDS peptide pretreatment did not affect the sustained reduction of fluorescence intensity of AFs, which was induced by adding cellulase or pectinase. Interestingly, CD treatment reduced the plasmolysed ratios, while Oryzalin treatment increased, suggesting that the effect of RGD or cell wall-digesting enzymes on plasmolysed ratios might be due to the depolymerization of cytoskeleton.It is well known that turgor pressure in plant cell varies with extracellular osmotic pressure. So, 1M mannitol, 0.55M mannitol or distilled water was used to increase or decrease the turgor pressure of embedded cells and the dynamics of cytoskeleton were observed at the same time. It was found that the dynamics of arrangements and fluorescence intensity of AFs and MTs displayed differential characteristics in response to the variation of turgor. These results indicated that dynamic arrangements of AFs and MTs are able to reflect the trend of turgor pressure change. Analysis by software Image Pro Plus and ABAQUS revealed that MT arrangements, cell morphology and cell wall surface stress generated by turgor have intrinsical linkage.To understand the correlation between calcium and cytoskeleton in plant mechanoresponses, we studied effects of calcium channel blockers on plasmolysed ratios and dynamics of cytoskeleton in response to turgor changes. It was found that pretreatment with La3+ caused reduction of plasmolysed ratios, while pretreatment with ruthenium red caused increasing of plasmolysed ratios. By image analysis, we found that the blockage of calcium channel leaded to increasing of fluorescence intensity of AFs and MTs, and altered the dynamics of cytoskeleton and fluorescence intensity in response to turgor changes.To further explored how the dynamics of cytoskeleton were disturbed by calcium channel blockers, the plasmolysed ratios, dynamics of cytoskeleton and fluorescence intensity in response to turgor changes were investigated after the suspension cells were incubated with phalloidin or taxol. The results showed that effects of phalloidin or taxol on plasmolysed ratios and fluorescence intensity were similar to the effects of La3+, suggesting that the changes of stress response patterns might due to the increasing of cytoskeleton stability induced by calcium channel blockers. Thus, calcium signaling pathways might connect with cytoskeleton signaling pathways in plants'response to turgor changes.In the present thesis, two mechanical loading systems were established, by which the real-time dynamics of cytoskeleton of single cells in response to controllable mechanical force could be achieved. In loading system 1, the atomic force microscope and confocal microscope were combined, by which the dynamics of cytoskeleton were investigated as the single living cell was being loaded by probe of atomic force microscope. Because the loading force is very small, this combined device provided an acceptable approach to study the threshold of the mechanical force to induce mechanoresponses of plant cells. In loading system 2, an empty cup made by polyethylene was fixed on the agar layer embedding suspension cells, and then an increasing pressure was loaded by injecting liquid of big density into the empty cup and real-time dynamics of cytoskeleton were recorded by confocal microscope. By this method, the dynamic loading and the plant cell response were recorded together at a fine-time scale, which provided an efficient approach for investigating the kinetics of plant cells'responses while they are experiencing a varying stress.