The overall goal of this procedure is to measure gene expression from reporter constructs after introducing them into barley aleurone cells by particle bombardment. This method can help answer key questions in plant hormone regulated gene expression such as how specific signaling molecules act to regulate abscisic acid into barley induced genes. The main advantage of this technique is that it provides higher throughput than previous methods, allowing a large number of gene constructs and incubation conditions to be tested.
Demonstrating the protocol will be Grace, a student in my lab. First, place a few embryoless grains into the lid of a 90 millimeter plastic petri dish containing 20 milliliters of imbibing solution and 20 microliters of chloramphenicol. Using sterile forceps, gently remove the transparent seed coat from each grain.
Peeling the seed coats from the barley grain is a critical step. If the seed coats are not fully removed, the gold microcarriers will be prevented from entering the aleurone cells. But if the peeling is done too roughly, the aleurone cells can be damaged.
After peeling the seed coat from all of the grains, transfer them from the petri dish to a vermiculite plate. Then, place the peeled embryoless grains in an incubator at 24 degrees Celsius for 16-20 hours. Now, label a one point five milliliter tube from each treatment that will be included in the bombardment experiment.
To each tube, add two point five micrograms of UBI luciferase internal control plasmid, two point five micrograms of a GUS reporter plasmid and the desired amount of an effector plasmid. Add ultra-pure water to each tube to bring the total volume to five microliters. For each treatment, add 50 microliters of microcarriers suspended in 50%glycerol into each one point five milliliter tube containing plasmid DNA.
At the final step of the microcarrier coding procedure, pipette eight microliters of suspended microcarriers onto each of the five macrocarriers and allow them to air dry. Set up the particle bombardment apparatus by turning on the vacuum pump, opening the helium valve, and then turning on the particle delivery system. Place a 1550 psi rupture disk into the retaining cap and mount it on the particle delivery system.
Place a macrocarrier containing the desired plasmid combination with a stopping screen into the microcarrier launch assembly. Insert the assembly into the top slot of the bombardment chamber. Next, arrange eight embryoless grains in a tight radial pattern with the thinner ends pointing inward on a filter paper circle that is taped down flat to the lid of a 90 milliliter petri dish.
Place the dish with the grains into the bombardment chamber at a distance of six centimeters from the stopping screen. Close the door and press the vacuum switch to evacuate the bombardment chamber to 95 kilopascals. Quickly turn the vacuum switch to the hold position.
Then hold down the fire switch to bombard the sample. After releasing the vacuum, transfer the bombarded grains to a labeled 60 millimeter petri dish containing four milliliters of imbibing solution with one milligram per milliliter of chloramphenicol. Repeat until all of the bombardments have been completed.
Then, incubate the bombarded grains on a shaking platform at 100 rpm at 24 degrees Celsius for 24 hours. Using forceps, remove the eight bombarded grains from the 60 millimeter petri dish and blot them dry on a paper towel. Divide the grains into two labeled two milliliter tubes, each containing 800 microliters of grinding buffer and a five millimeter stainless steel bead.
After repeating the previous step for all bombarded grains, place up to 48 two milliliter tubes into two racks of a bead homogenizer. Then mount the racks on the homogenizer and shake the samples at 30 hertz for three minutes. When finished, remove the racks from the homogenizer and place them on ice for five minutes to re-cool the samples.
Now, mount the racks on the homogenizer in the opposite direction. Shake the samples again at 30 hertz for three minutes. After removing the racks from the homogenizer, place them on ice for five minutes to re-cool the samples.
Following this, centrifuge the grain extracts at 16, 000 G at four degrees Celsius for 10 minutes. Immediately after centrifugation, decant the clear supernatants into a second set of microcentrifuge tubes. Briefly vortex each tube and store on ice.
After transferring the supernatant recover the stainless steel beads from the pellets so they can be washed and reused. Now add 200 microliters of GUS assay buffer to each well of a 96-well plate. Place 50 microliters of each grain extract into one of the wells and mix with the pipette tip.
Then, seal the top of the plate with sealing film. Place the 96-well plate in an incubator in the dark at 37 degrees Celsius for 20 hours. Following incubation, centrifuge the 96-well plate at 4, 000 G at four degrees Celsius for 10 minutes.
When finished, place the plate on ice. Now, add 250 microliters of zero point two molar of sodium carbonate to each well of a black 96-well plate. Add six point two five microliters of each centrifuged GUS assay mixture into each well of the black plate and mix with the pipette tip.
Include wells with six point two five microliters of 10-40 at 100 micromolar, four methylumbelliferone as standards. Place the black plate into a plate fluorometer. Read the fluorescence of methylumbelliferone setting the gain of the instrument, such that a well containing six point two five microliters of 40 micromolar, four methylumbelliferone and 250 microliters of zero point two molar sodium carbonate give a reading of 100 fluorescence units.
In this experiment, an HVA1 GUS reporter construct was introduced into aleurone cells together with a UBI TaABF1 effector construct. While a TaABF1 HVA1 ratio of just zero point zero one was sufficient to cause a three-fold induction of the HVA1 reporter gene in the absence of exogenis absyssic acid or ABA. Higher levels of the TaABF1 construct resulted in progressively greater HVA1 expression in a dosed dependent manner.
Both exogenis ABA and TaABF1 could strongly induce expression of the HVA1 GUS reporter construct. In either case, the presence of one butanol strongly inhibited the HVA1 induction, which suggests that phospholipase D could play an important role in the activation of HVA1 gene expression by ABA through TaABF1. ABA was shown to strongly inhibit gibberellin induced expression for the Amy32b GUS reporter construct.
While neither hydrogen peroxide nor sodium nitroprusside caused any significant reduction. These results, therefore, do not support a role for nitric oxide and hydrogen peroxide in aleurone cell ABA responses. Once mastered, this technique can allow many different samples to be measured for gene expression in a relatively short time.
For example, bombardment of 200 different grain samples can be done by two investigators in a couple of hours. Then, on the following day, a single investigator can prepare all of the protein extracts in two hours. The modified GUS assay carried out in 96-well plates also requires much less labor than other protocols that use in the future tube assays.
