The overall goal of this procedure is to screen the genes involved in callose-mediated plasmodesmal gating during tropic response. This method can help answer key questions in the field of plant developmental biology field, such as which unknown genes control cell-to-cell signaling by plasmodesmata during tropic response. The main advantage of this technique is that it is simple, works too, and repeat.
Generally individuals new to this method will struggle because the handling of the Arabidopsis etiolated hypocotyls without any damage during the whole process is challenging. We first had the idea for this method when were establishing the rule of GSL8, a glucose synthase in plasmodesmal gating. Visual demonstration of this method is critical as the procedures mentioned in the audio have not explained the critical steps which are difficult to execute because they needed special setup and handling.
Prepare Murashige and Skoog, or MS medium, one day before the experiment. Autoclave it, and fill 125-millimeter square plates with 50 milliliters of media per plate. The next day, dot seeds that have been sterilized with 1.1%sodium hypochlorite on semi-dried plates.
From a 1-millimeter micropipette tip, dot seeds with 200 microliters of water. Arrange the seeds in five rows, 1.8 centimeters apart, with the seeds being a minimum of 0.1 centimeters apart in the rows. Let some water evaporate before covering and sealing the plates with surgical tape.
Then wrap the plates in foil and place them within a box. Incubate the box at four degrees Celsius for three days. After three days of cold treatment, transfer the box of seeds to 22 degrees Celsius for three days.
After three more days, in a dark room, check the germination status of the seeds under green light. Usually, three-day-old Col-0 etiolated seedlings grow up to 1.6 centimeters in length. Under the green light, analyze the altered phototropic and gravitropic response.
Using a sterilized pick, select similarly-sized seedlings with straight, vertical growth. Gently lift the seedlings from the lowest part of their hypocotyl without touching any other parts. Then, carefully transfer the selected seedlings to a fresh MS agar plate.
On the new plate, orient the seedlings'hooks in the same way. Select at least 20 seedlings of each genetic background to make each row, and make a duplicate of every plate. Each plate contains two genotypes in two rows.
To check the phototropic response, position the plates in a black box so the seedlings are oriented vertically. Leave one side of the box open to the edge of the plates. Then, incubate the box in a plant growth chamber with unilateral white light.
To check the gravitropic response, wrap the plates with foil and put in a black box so the seedlings are horizontal. At several time points, usually beginning at 90 minutes and extending to 12 hours, image the plates under low light conditions. Use a scanner set to collect 600 DPI JPEG images.
To measure the bending angle of the seedlings, load their images in ImageJ software. Use the magnifying tool to zoom in, and scroll to a seedling to analyze. Then use the angle tool to measure the angle at which the seedling is bent.
To do this, double left-click on the original hypocotyl growing direction point A, and drag the mouse to the bending initiation point B.Then, use a left-click to draw the first line. Now, drag the mouse from point B to the bending end, point C, and use a left-click to draw the second line, forming an angle, A.The true bending angle is angle B, which is equal to 180 degrees minus angle A.Now, measure and record angle A by pressing M on the keyboard. The result file will open separately.
Repeat the previous step for measuring the rest of the seedlings, and measure the average angle of two different backgrounds using the data set spreadsheet file. Preparation of the HPTS agarose blocks needed for this assay is covered in the text protocol. Begin with preparing the plant samples.
In this example, mutants with an altered plasmodesmata size exclusion limit are analyzed. First, create a platform for the assay. Place a microscope cover slide on a new MS medium plate.
Next, from MS plates with three-day-old etiolated seedlings, use sharp surgical scissors to carefully excise the seedlings from the base of the hook, and transfer them to the cover glass. Transfer at least five seedlings per genetic background. Orient the seedlings so only 0.5 centimeters of hypocotyl from the excision position remains on the cover slide and the rest of the hypocotyl touches the medium.
Then, place an HPTS agarose block on top of the excised seedlings so that the excised area is in contact with the HPTS block. Make certain that the contact is made. Let the block sit on the hypocotyls for five minutes.
Then, transfer the hypocotyls to a 10 millimeter petri dish containing double-distilled water, and wash them with shaking for 15 minutes. Next, transfer the washed seedlings to a slide. Place each mutant next to a control to make direct comparisons.
Cover the hypocotyls with 100 to 150 microliters of double-distilled water and apply a cover slide. Now, analyze the slide using confocal microscopy as described in the text protocol. For this assay, use freshly-prepared dye that is less than two-days old and kept in the dark.
To inhibit de novo callose synthesis, add 1.5 millimolar of 2-Deoxy-D-glucose to the dye. Begin this assay by selecting three-day-old etiolated seedlings for callose staining, with or without tropic response. Cut the seedlings from their hook region using thin surgical scissors.
Then, using the tip of a toothpick, transfer the seedlings in a small petri dish containing two milliliters of staining buffer. Incubate the seedlings in the staining buffer in the dark for two hours with continuous shaking at 30 RPM. After staining, wash the seedlings with double-distilled water for two minutes to remove the excess staining buffer.
Once washed, select at least five seedlings of each background for confocal analysis. Measure the callose signal intensity with ImageJ software. Open the confocal microscopy file already saved in JPEG format in the imaging software.
Next, using the rectangular selection tool, drag the cursor over the picture, and select the area to be examined. Then, measure and record the numerical values for signal intensity by pressing M.The result file will open separately. In the result file, the term Mean"indicates the signal intensity of the selected area, not the mean of several areas.
One by one, analyze the signal in this manner for each seedling. Repeat the above step for measuring the rest of the seedlings, and measure the average angle of two different backgrounds using the data set spreadsheet file. Using the described protocols, dexamethasone inducible RNAi lines of AtGSL8 were analyzed.
DsGSL8 seedlings were clearly defective in phototropism and gravitropism. They exhibited no bending under the influence of either force. Alterations in symplasmic movement showed the HPTSA dye movement was substantially more extensive in dsGSL8 RNAi knockdowns than in either controls.
Callose is one of the key regulators of plasmodesmata size exclusion limit, and AtGSL8 is a known plasmodesmata callose synthase. So, callose aniline blue staining was carried out. In the control, callose accumulated after six hours, as marked by yellow arrows.
However, after six hours, the mutants had persistently low plasmodesmata callose levels. This absence of callose synthesis was measurably less as soon as three hours after phototropism. After watching this video, you should have a good understanding of how to do rapid screening of genes in variant controlling plasmodesmal gating due to tropic response through modulating purely callose.
Once mastered, this technique can be done in approximately six hours after three days'germination of Arabidopsis seedlings. While attempting this procedure, it's important to remember to avoid damaging seedling while transferring them. Also, it's important to make a proper contact between the agar and the hypocotyl during the loading assay.
After this development, this technique paved the way developmental biology to explore genes, new genes, that play a role in controlling symplasmic conductivity during tropic
