To examine phytochrome responses in specific plant cell types. GAL 4G FP enhancer trap, and U-A-S-B-V-R transgenic lines across to produce phytochrome deficient. U-A-S-B-V-R cross GA 4G FP progeny leaf protoplasts are isolated from plants.
Next, the protoplasts is sorted by facts to isolate GFP negative and GFP positive protoplasts in separate samples, isolated protoplasts are analyzed by confocal laser scanning microscopy. To confirm the collection of GFP positive Protoplasts, RNA is extracted from protoplasts and analyzed spectrally. Hi, I'm Sanka Pivar from the laboratory of BARDA Montgomery in the Department of Energy Plant Research Laboratory at Michigan State University.
Today we will show you a procedure for isolating leaf protoplasts from atop ANA for fluorescence assisted cell sorting and cell type specific gene expression analysis. We use this procedure in our laboratory to study tissue and organ specific phytochrome dependent responses. So let's get started.
The Arabidopsis ANA line used in this procedure was generated by a bipartite enhancer trap approach parents. One is from a library of enhancer trap lines with different patterns of gal or expression. Parent one also expresses GFP under the control of an upstream activation sequence or UAS.
In short, the transcription factor GA four activates any gene placed under the control of the UAS. In the parent one line GA four activates GFP parent one was crossed with parent two in parent two. The BVR gene is under the control of GAL four through UAS.
The line resulting from the cross is U-A-S-B-V-R cross G 4G FP.In the parental lines, the biosynthesis of phytochrome chromo four proceeds from a LA through heme to biver abbreviated bv. But in the progeny, the BVR gene product blocks the biogenesis of phytochrome chromophore by reducing the BV precursor. This enhancer trap BVR progeny line was confirmed and can be used to examine how a reduction in active phytochrome levels affects different plant tissues.
Begin this procedure by preparing soil and pots for growing plants in order to generate leaf protoplasts. So sterilized seeds around 2000 per sample per experiment on soil in pots. Water is added to the bottom of the tray containing the pots and the entire tray is covered with plastic wrap.
Grow the plants for five weeks under white illumination at 22 degrees Celsius and 70%humidity. After the first five to six days, germination of the seeds is confirmed and the plastic wrap is removed from the tray. In order to isolate protoplasts first prepare TEX buffer for one liter of buffer weigh out GA's B five salts MES, calcium chloride, ammonium nitrate and sucrose.
Dissolve the components completely in around 900 milliliters of deionized distilled water. Abbreviated DD H2O and bring the pH to 5.7 with one molar KOH. Then bring the final volume to one liter and filter sterilize.
Next, collect green healthy leaves from the plants. Collect around 250 milliliters of leaves loosely packed in a beaker. Rinse the leaves with 40 milliliters.
DD H2O four times followed by rinse in twice with sterile DD H2O using a number 20 scalpel. Cut the leaves into thin strips and divide tissue equally into two 50 sterile plastic tubes. One x leaves digestion mix is prepared by adding a five milliliter aliquot of 10 x stock solution prepared by dissolving 2%weight for volume mazy R 10 and 4%weight for volume cellulase on azua R 10 in TEX buffer to 45 milliliters of fresh TEX buffer.
Add around 25 milliliters of one x leaf digestion. Mix to the leaf strips in each tube so that all the leaf tissue is covered. Proceed to vacuum, infiltrate the leaf tissue in the digestion.
Mix in open tubes for one hour at room temperature using a vacuum desiccate connected to a water pump. Keep the cap tubes containing the leaf tissue and leaf digestion mix wrapped in aluminum foil to prevent exposure to light. Follow with a three hour incubation at room temperature on a rocker with gentle shaking.
After three hours, increase the rocker speed for two minutes to release the protoplasts. Filter the crude protoplasts suspension through two layers of sterile cheesecloth to remove abri and collect the filtrate in a sterile glass beaker. Filter the filtrate through a sterile 100 micrometer nylon mesh into a sterile Petri dish.
Collect the flow through and transfer it to a new sterile 50 milliliter tube. Use about 15 to milliliters of fresh TEX buffer to wash the sterile petro dish and collect any protoplasts during to the surface. Centrifuge the flow through at 100 times gravity at 10 degrees Celsius for 15 minutes using a swing bucket centrifuge with acceleration set to six and deceleration at zero when centrifugation is complete.
Use a sterile nine inch glass pasta per pet connected to a peristaltic pump to remove around 25 to 30 milliliters of the liquid below the floating protoplasts layer, which contains residual and pelleted debris without disturbing the floating protoplasts layer, leaving around 10 to 50 milliliters. After removing the pelleted debris, add fresh TEX buffer to a final volume of 40 milliliters. While gently resus suspending the protoplasts, repeat the removal of debris and resus suspension of the remaining floating protoplasts.
Two more times in order to remove as much cellular D debris as possible. The centrifugation time is reduced to 10 minutes in the first repetition and to five minutes in the final repetition. Finally, use a sterile one milliliter transfer pipette to aspirate the floating protoplasts and transfer them into a new 15 milliliter tube.
Proceed directly to sorting before sorting. Examine the protoplasts by confocal laser scanning microscopy or CLSM in short, using a 488 nanometer laser for excitation to confirm protoplasts integrity and the presence of GFP fluorescence in the protoplasts pool. Minimal day debris is necessary to avoid clogging the sorting nozzle sort isolated PROTOPLASTS in TEX buffer via fax.
Using a 200 micrometer nozzle on a macro sort head at event rates between 6, 000 and 15, 000 with a system pressure of around nine PSI following an adapted protocol, use wild type non GFP PROTOPLASTS to determine the autofluorescence thresholds to collect GFP positive protoplasts sort cells using an air called Argonne laser operated at 100 milliwatts on a 488 nanometer argon line. To identify GFP fluorescence using a 530 slash 30 band pass filter following the collection of GFP positive protoplasts. Again, examine the sorted protoplasts under the microscope as before the start of sorting for expression profiling.
Extract total RNA from the sorted protoplasts. CDNA can then be prepared for hybridization. The CD NA is then submitted for hybridization to a TH one OPSYS Ametri whole genome arrays.
Here are some representative results of protoplasts sorting the wild type control. Protoplasts lack any signals in the GFP positive gate. However, GFP positive signals are identified for protoplasts samples isolated from GAL 4G FP lines before sorting protoplasts isolated from wild type plants lack any GFP fluorescence in CLSM analyses.
Whereas a mixture of protoplasts lacking and exhibiting GFP fluorescence are detected in protoplasts samples from GAL 4G FP lines after facts. CLSM analysis indicates the isolation of GFP positive PROTOPLASTS from a representative gal 4G FP line RNA is extractable from isolated protoplasts and one milliliter yields RNAA sufficient quantity for detection by fluoro spectrometry. We have just shown you how to isolate leaf protoplasts from atopy ANA for fluorescence assisted cell sorting and cell type specific gene expression analysis.
When doing this procedure, it's important to remember to be gentle in the handling of the protoplasts and limit their exposure to light. So that's it. Thanks for watching and good luck with your experiments.
