The widely-used probe CFDA, or short for carboxyfluorescein diacetate, is non-fluorescent. The two H3 groups and the 3'6'position on this compound render it membrane-permeant. Where once inside cells, internal cellular acetates will remove the acetate groups and release the new form of CFDA.
That's a carboxyfluorescein, or the CF, which is fluorescent. And because this new form is less membrane-permeant, the weight, all the cells is to move through the intercellular pores, or the plasmodesmata in plants, that's making this diacetate the ideal probe to study intercellular transport in phloem activity. In today's video, we're going to demonstrate the CFD loading into the root and hypocotyl of the Arabidopsis plants, respectively, and we will show how the two slightly different procedures will affect the bottom to top movement of this fluorescent probe.
Achieve staining results consistently, we store the harvested Arabidopsis seeds in four Celsius degree, 40%moisture cabinet. Newly harvested seeds stored in this condition for more than seven days are readily used for sowing. To ensure every plant is grown in a relatively similar space in the Petri dish, we are using a seed zone grade card.
Now, wetting the sterilized tip, dip in the sterilized seeds, and sow the seeds one-by-one on the designated position. After finish sowing, seal the Petri dish with parafilm and a sticky tape, put them on a Petri dish stand to let it grow vertically. Here are the tools we are going to use in this experiment.
Please note that we'd better load the dye as quickly as we can. Normally, you finish the whole process in 10 to 15 minutes. Before we perform the CFDA loading experiment, we always prepare fresh CFDA solution.
Here, dilute the one millimolar CFDA into five micromolar concentration with water before immediately use. Cut the parafilm into small pieces in three by three millimeter size. Because there is condensation on the lid and bottom of the Petri dish, we need to clear them out with paper towel to avoid dye flowing around.
A small parafilm piece is put under the root. Lift the plants and put them on the lid. Cut the roots about five to 10 millimeter below the hypocotyl.
Take the shoots back on the small pieces of parafilm. Now carefully apply one microliter CFDA onto the cutting end of the root. Try to avoid contaminating all the proto-plants.
Then cover the lid, and then leave them in the growth room for two hours. The Hypocotyl-pinching Method. The small parafilm piece, as previously prepared, is put under the root-hypocotyl junction.
We use a forecep to gently pinch the hypocotyl close to the root tissue, and carefully apply 0.1 microliter CFDA onto the pinched side and leave the plant for two hours. The photos from nine to 12 day after zone staining plants. They all show the vascular staining pattern in both the cotyledons and the true leaves.
Only in a very rare case, the cotyledon leaf showed a half-leaf staining pattern. So basically, the two methods did not make a difference in the CF staining pattern, however if we perform dye loading into more and more plants, we find the two procedures give out a significant difference deal in efficiency. With the root-cut method, about 70%of the plants show the fluorescent signal in the shoot.
But the efficiency can be increased to 91%using the hypocotyl-pinching method. We also tried the root-pinching method, but this method result in the even lower efficiency, it's about 30%Therefore, it seems the way to load the dye does not account for the staining difference. The difference most probably lies in the loading sites, which might be separated by a symplastic barrier.
We showed how we load the CFDA into the Arabidopsis roots and hypocotyl. We used two slightly different procedures, and these two slightly different procedures result in significant difference in this bottom to top probe movement. We suggest that this method could help us to identify a potential symplastic barrier for root-derived shootward signals.
