This protocol it helps us understand how Microtubule Cytoskeleton behavior occurs in plants. At the same time, it also helps us understand how these structural components that influence cell and organ shape, respond to changes in mechanical forces. Although, this method requires little sophistication, it provides robust, quantitative read-after, the mechanical response differences between different genotypes and conditions.
Begin by growing arabidopsis seeds expressing microtubule binding domains fused with green fluorescent protein in soil, at 20 and 6 degrees Celsius under long day conditions for one week. After germination, transfer the seedlings into new pots with sufficient growth space to allow robust vegetative growth, and place the plants at 20 and 16 degrees Celsius under short day conditions. After three to five weeks, transfer the plants back to long day conditions at the same temperature until the plants bolt, allowing the inflorescence to grow up to two to five centimeters long.
For Shoot Apical Meristem dissection, putt the inflorescence, and use sharp forceps to peel the flowers at the base of the peduncles, until it is difficult to see the peduncles with the naked eye to remove the older flower buds. Use the forceps to create a slit in the agros in a dissecting dish, and plant the inflorescence base into the thick Augur. Place the dish under a dissecting microscope.
Starting with the oldest flower bud, push with the forceps to remove the flower bud from the plant. The shoot apical meristem is usually exposed when older flowers up to stage six to seven are removed. And use the forceps to push each flower bud down until the shoot apical meristem is visible.
As soon as the shoot apical meristem is exposed, let the freshly dissected sample in growth medium in an ethanol-sterilized rectangular plastic hinged culture box with the shoot apical meristem just exposed above the medium surface. Add a few drops of sterile deionized water to the edges of the culture box and close the lid to maintain the humidity inside the box. Wrap the box with micropore tape, and place the growth box under long or continuous day conditions at 22 degrees Celsius for 12 to 24 hours.
When the plant has recovered from the dissection procedure, cover the sample with sterile deionized water and check for air bubbles under the dissecting microscope. Transfer the culture box to an upright confocal microscope stage, and select the 40 or 60X water dipping lens. Lower the objective into the water and check for air bubbles on the front lens of the object.
To remove bubbles, lower the stage and gently wipe the lens with an optical tissue. Then use a Pasteur pipette to add a small volume of water to the front lens of the objective before re-immersing the lens in the water. Next, use the GFP filter and epi-illumination module of the confocal microscope to adjust the XY controller to locate the sample.
Adjusting the position of the oculars, position the shoot apical meristem directly under the light source, and focus along the Z-axis until the apex is located. Use a laser capable of exciting GFP to illuminate the sample, and adjust the optical zoom of the microscope, so that the entire shoot apical meristem and the stage one floral primordia are in the field of view. Then, adjust the power of the laser output and the gain settings to obtain an optimal signal to noise ratio.
After allowing the sample to settle for two to five minutes, acquire confocal Z stacks of the sample at 0.25 to 0.5 micrometers Z slice intervals, adding approximately 0.3 micro meter pixel size resolution. At the end of the acquisition, immediately remove the water and transfer the culture box back to the growth chamber. For Micromechanical Shoot Apical Meristem Perturbation, acquire pre-ablation image stacks of the cortical microtubule organization as just demonstrated, and decaf the water from the culture box into a culture dish.
Transfer the shoot apical meristem under a dissecting microscope, and slowly approached the shoot apical meristem with a clean 0.4 by 20 millimeter syringe needle. Briefly contact the periphery of the dome of the shoot apical meristem with the needle tip to confirm that the ablation has been completed. And refill the culture dish with sterile deionized water.
Then add 10 micrograms per milliliter of propidium iodide to the dish and immediately acquire image stacks as demonstrated every two hours for six hours, returning the culture dish to the incubation between each time point. For data analysis, generate surface projections of the image stacks using an appropriate image analysis software program, and perform extraction of the cortical microtubule anisotropy using the Fibril Tool Macro in Fiji. Here, typical projection images obtained from microtubule binding domain GFP lines with cells at the center of the dome containing disorganized cortical microtubules, cells at the periphery, having a circumferential distribution and boundary domain cells containing cortical microtubules aligned parallel to the cell's long axis can be observed.
Time-lapse imaging, shows cortical microtubule alignment changing from a highly disordered array to a more organized array within six hours of ablation. Tensors could be superimposed on the cortical microtubule images, and the extracted information could be represented by plotting the mean anisotropy over time. When adjusting laser output power and gain settings, we should ensure it clear observation of the filaments and try to avoid overexposure, as over saturated signals could bias the tensile direction in later analysis.
Atomic force microscopy can also be performed to assess how the changes in the mechanical forces actually affect the physical status of the cell wall.
