Measuring Spatial and Temporal Ca²⁺ Signals in Arabidopsis Plants

The overall aim of this procedure is to demonstrate a method of monitoring the calcium signals at both the cellular and whole plant level. This is accomplished by first growing seedlings on the MS agar plate or directly within the imaging chamber. The second step is to transfer the seedlings onto an adhesive film or to keep them in the imaging chamber.

Next stimuli applied to the seedlings to induce calcium signals. Ultimately, lifestyle luminescence or fluorescence imaging is used to show whole seedling or cytosolic nuclear calcium response respectively to different stimuli. The main advantage of this technique over existing methods like single plant occurring as say, is that film adhesive seedlings assay is simple, sensitive, and robust.

Therefore, this technique can be adapted to the high throughput screening for mutants with altered calcium signal responses. In addition, compared to flat based calcium imaging, using GFP based genetically encoded calcium indicator avoids complicated microscopic settings and calculations. This method provides insight into calcium signaling during the stress response in Arabidopsis, but it can also be used in other contexts such as during development or defense in other plant models.

Demonstrating this method will be shiny. A student from our lab To begin this procedure sterilize the seeds of a rabbit opsis plants expressing a quain with 10%bleach solution containing 0.01%Triton 100, sow the seeds on a square plate containing full strength MS 1%sucrose and 1.2%agar. Next, place the plates vertically in a growth chamber after stratification of four degrees Celsius for two days.

Afterward, transfer the seedlings onto a film. Place an adhesive film on top of the seven to 10 day old seedlings growing on the plate. Gently push the film by hand to ensure that the seedlings are adhere to the film.

Then peel the film gently so that the seedlings remain a adhere to the film. Next, incubate the seedlings with the cofactor by placing the A adhere seedlings onto a square plate containing 15 milliliters of two micrograms per milliliter HCTZ in water. Incubate the seedlings at room temperature for four hours to overnight.

To prepare for luminescence imaging, take the film outta the HCTZ solution and cut it down the middle to form two pieces. Place the two pieces of film with the seedlings facing up in two different plates. Then leave the plates in the dark for five minutes to acquire luminescence images in the dark.

Place two plates next to each other on the stage of the luminescence imaging system. Acquire images immediately upon adding 20 milliliters of stimuli solution to the plates simultaneously. To analyze the luminescence images, choose the same display range for all luminescence images, crop the ROI and generate the images as jpeg files.

Alternatively, export the images as SPE format files and import them into the image J image analysis software. Set the measurements for calculating the mean gray value of ROI select the same size of ROI area. Then measure the mean gray value and present data as bar graphs.

To prepare seedlings for confocal imaging. Sterilize the seeds of the opsis plants expressing case 12 with 10%bleach solution containing 0.01%Triton. So the sterile seeds on a plate containing full strength MS salts, 1%sucrose and 1.2%agar.

Then place the plates vertically in a growth chamber after stratification at four degrees Celsius for two days. In the meantime, assemble an imaging chamber with a slide and a cover slip. Place a piece of water soaked cotton wool on the top of the slide.

Then transfer one or two five day old seedlings from the plate onto the middle of the slide. Then stick small pieces of clay to each corner of the cover slip and place the cover slip on top of the seedlings to create a gap between the cover slip and slide. After that, connect one end of the polyethylene tube to a one milliliter syringe and place the other end of the tube immediately adjacent to the chamber.

Hold the tubing in place with tape to assemble an imaging chamber with a plexiglass chamber. Spread a thin layer of silicon grease around two polyethylene tubes and press the coated tubes into the channels on each side of the plexiglass chamber. Then spread a thin layer of silicon grease on the surface of the chamber that contains the tube grooves and place a cover slip onto the grease to seal one side of the chamber opening.

Next, transfer a five day old seedling from the plate into the chamber and place piece of cotton wool soaked with water on top of the seedling. Spread the silicone grease onto the other surface of the chamber and press another cover slip on top to seal. Afterward, connect the end of one of the tubes to a one milliliter syringe and leave the end of the other tube open.

To prepare an imaging chamber with a chambered cover glass first sterilize the chambered cover glass with 70%ethanol and leave it in the hood until dry at 0.6 milliliters or 0.2 milliliters of full strength MS.Medium containing 1%sucrose and 0.5%fighter gel into each well of a two well chamber or an eight well chamber respectively. Then sterilize the seeds and sow them directly on a chambered cover glass containing a thin layer of clear gel and let them grow vertically for five days. For image acquisition.

Using a confocal microscope applies stress stimuli by slowly injecting 200 microliters of 150 millimolar sodium chloride, ice cold water, or one millimolar hydrogen peroxide solution into the chamber just prior to acquiring images. Capture the images immediately after applying the stimulus solution. Using an inverted confocal laser scanning microscope with a 20 x water immersion lens.

Collect a time series of images at four second intervals with excitation and emission wavelengths of 4 88 nanometers and 500 to 550 nanometers respectively, and a AIX resolution of five 12 by five 12 or image analysis using Nikon elements. ROIs are drawn around each cell of interest. Total intensity within each ROI is measured over time using the time measurement dialogue box.

Then total intensity measurements are exported and processed in data graph shown. Here is the comparison of the spatial temporal calcium response of 10 day seedlings. The different stress stimuli here is the time series of luminescence, images of seedlings subjected to 400 millimolar mannitol and 400 milli molar ole plus one millimolar copper chloride, 75 millimolar sodium chloride and 75 millimolar sodium chloride plus one millimolar copper chloride and one millimolar hydrogen peroxide or one milli molar hydrogen peroxide plus one millimolar copper chloride.

The upper and lower panels are the luminescence images acquired during the first 40 seconds and second 40 seconds respectively. And here is the bar chart of the average luminescence intensity of seedlings in response to the indicated stress stimuli shown here as the cytosolic and nuclear calcium oscillation. In response to salt stress, the confocal images show that case 12 is expressed in the cytoplasm and nuclei of the root cells and the leaf cells of the transgenic plants expressing case 12.

This image is emerged, image of the red, autofluorescence of the chloroplasts and the green fluorescence of case 12. These two plots show the salt stress induced calcium oscillation in the cytosol, a nuclei of the root cells. While attempting this procedure, it's important to remember to use well grown seedlings.

After watching this video, you should have a good understanding of how to monitor dynas at both cellular and the whole plant level.