The overall goal of this procedure is to provide a simple and safe method for the study of bacterial response to ethylene. This method will answer key questions regarding plant-microbe interactions, such as the role that ethylene plays in bacterial adhesion and colonization of plants. The main advantage of this technique is that it does not require any specialized equipment and it's much safer than handling gaseous ethylene in the lab.
Although this method can provide insight into how bacterial cellulose production is affected by ethylene, it can also be applied to other plant-associated microorganisms for the in vitro study of how ethylene-mediated processes are involved in plant colonization. In preparation, make 100 milliliters of growth medium for the Arabidopsis seedlings and make 100 milliliters of bacterial growth medium for the CEPA decomposition. Once the arabidopsis medium has tempered, divide 40 milliliters out to a sterile flask and supplement the aliquot with 40 microliters of 10 millimolar ACC.
Now, load the plate quadrants with five milliliters of medium and allow the agar to solidify. Opposing quadrants of all plates have bacterial growth medium and arabidopsis medium. Use three different medium configurations.
The CEPA treatment will occur later in the protocol. Prepare each plate configuration in triplicate. Once the agar has cooled, evenly distribute approximately 50 seeds onto each quadrant of arabidopsis medium.
Then, incubate the plates in the dark at four degrees Celsius for three to four days. Later, after incubating the seeds, expose them to fluorescent light for two hours. Then, spread 10 microliters of 500 millimolar CEPA stock solution onto the bacterial agar of one plate configuration.
The final CEPA concentration in the agar will be one millimolar. Now, seal all the plates with laboratory film and wrap them in foil. Then, place the plates in a dark environment at 23 degrees Celsius with the agar side down and let them incubate for three days to germinate the seeds.
After the seeds germinate, use flame sterilized forceps to remove seedlings corresponding to each treatment. Select 30 seedlings from each quadrant for a total of 180 per treatment. Image the seedlings under a dissecting microscope or with a high resolution digital camera against a black background next to a ruler for scale.
Then, measure their hypocotyl length using ImageJ software. Using the straight tool, measure 10 millimeters and select Set Scale under the Analyze tab to set the scale. Then, use the Segmented tool under the Straight Line tool to measure each hypocotyl and press M to calculate their lengths.
The pellicle assay is a standard analysis tool for bacterial cellulose production. PH and morphological analysis are detailed in sex protocol. In preparation, harvest cells from starter cultures in triplicate and quantify the cells using a Petroff-Hauser Counting Chamber.
Then, prepare four medium master mixes with different CEPA concentrations. For each master mix, eliquate 60 milliliters of pH 7 SH medium and add 120 microliters of either zero, five, 50 or 500 millimolar CEPA stock solution to obtain final CEPA concentrations of zero, 01, 0.1, or 1.0 millimolar. Mix these media by swirling the flasks.
One set of media is needed for each of the triplicates and a fourth set is needed to function as a sterile control. So, divide each 60 milliliter medium preparation into four 14 milliliter eliquates. Then, pre-cool the 14 milliliter eliquates before inoculating with their respective starter cultures for a final concentration of 100, 000 cells per milliliter.
Keep all the inoculated tubes on ice to prevent cellulose production. Use the remaining 14 milliliter eliquates for sterile control wells. Now, from each 14 milliliter master mix, load two milliliter eliquates into six wells of a sterile 24 well plate for a total of four loaded plates per starter culture analyzed.
Carefully seal the plates with paraffin film and incubate them statically for seven days at 30 degrees Celsius. During the incubation, incubate a 25 milliliter liquid culture in parallel to periodically measure the medium's acidity. If the acidity drops below a PH of five, then the organism's growth will inhibit the decomposition of CEPA to ethylene and is thus incompatible with this assay.
A week later, harvest and measure pellicle wet weight, thickness, dry weight and crystallinity. To collect a pellicle from the plate, depress one side of the pellicle to elevate the opposing edge and pick it up with forceps. While retaining grip, place them on a fresh paper towel for three seconds to remove excess medium.
Then, weigh each pellicle and record this as the wet weight. Next, align the pellicles along a ruler and photograph them from the side using a high resolution digital camera. Analyze these photos for the pellicles'thicknesses.
Next, individually transfer the pellicles into the wells of a six well plate. Treat them with 12 milliliters of 0.1 normal sodium hydroxide at 80 degrees Celsius for 20 minutes to lice the cells. Then, remove the sodium hydroxide and neutralize the treated pellicles by thoroughly washing them with 12 milliliters of ultra pure water for 24 hours with agitation in a six well plate.
During this incubation, change the water every six hours. When the incubation is over, the pellicles should be white. Then, place the pellicles on silicon mats and dry them at 50 degrees Celsius for 48 hours to prepare them for dry weight measurement.
The pellicles'crystallinity can be analyzed using Fourier-transform infrared spectroscopy. Dark grown arabidopsis thaliana seedlings exhibited the triple response phenotype of a short, thick hypocotyl with an exaggerated apical hook in the presence of ACC and in the presence of ethylene produced through the decomposition CEPA on SH medium, but not under untreated conditions. Clearly, the hypocotyl length of the treated seeds was significantly less than the untreated controls.
Thus, ethylene was released from CEPA on SH medium at a physiologically relevant concentration. All concentrations of CEPA derived ethylene significantly decreased pellicle wet weight, but did not affect pellicle thickness, and all concentrations of CEPA derived ethylene significantly increased pellicle dry weight. Furthermore, pellicle hydration was reduced by all concentrations of CEPA derived ethylene.
While attempting this procedure, it's important to measure the PH of the growth medium to ensure that the PH does not drop below five so that CEPA degradation into ethylene is not inhibited. Remember that powdered CEPA is extremely hazardous to mucous membranes. Proper care should always be taken when performing this protocol.
This technique will enable researchers who are interested in plant bacteria interactions to safely explore ethylene mediated responses in vitro. This will help shed light on how plant signals mediate bacterial colonization and adhesion. Currently, this area has been under studied.
