To begin, using a dropper, plate 250 Arabidopsis seeds for each group of conditions on filter paper placed on a Murashige and Skoog Basal Media agar plate without sucrose, supplemented with 1.2%phyto agar. Wrap the plate with aluminum foil to block light and place it in an incubator at four degrees Celsius for two days to induce vernalization. After two days, unwrap the foil and transfer the vernalized seeds to a growth chamber.
Allow the plants to grow for five days. Next, seal the plate containing five-day-old seedlings with waterproof adhesive tape. Place the plate in a 40 degree Celsius water bath for one hour for heat stress treatment.
Immediately after heat treatment, cool the plate down and transfer it to a growth chamber set at 22 degrees Celsius for two hours to allow recovery. Harvest 250 heat treated or untreated seedlings into 1.5 milliliter microcentrifuge tubes. Freeze the collected samples immediately in liquid nitrogen.
Prepare the sucrose gradient solution with concentrations of 12.5%24.4%36.3%48.1%and 60%To prepare a discontinuous sucrose density gradient, first load 2.2 milliliters of 60%sucrose solution at the bottom of the tube. Then, place the tube in a 80 degrees Celsius freezer to freeze the sucrose solution to a solid state. After solidification, load 2.2 milliliters of each of the other sucrose concentrations in the series from the bottom to the top.
Store the prepared sucrose gradient at 80 degrees Celsius until use.
