The isolation of highly pure plastoglobules is critical to their study. A rapid and effective protocol for their isolation that facilitates their downstream biochemical investigation is presented here. The primary benefit of this method is the relative speed of procedure and its adaptability to numerous plant species and environmental conditions.
Particular care must be taken while sonicating thylakoid resuspensions to ensure sufficient force to strip plastoglobules from thylakoids and with subsequent extraction of plastoglobules from sucrose gradient. Demonstrating this procedure will be Dr.Elsinraju Devadasu, a postdoctoral researcher in my laboratory, and Febri Susanto, a PhD student in my laboratory. Begin by acquiring three-weeks-old seedlings of six healthy maize, having V5 growth stage and weighing approximately 120 grams.
Clip off all the leaves at the base of the stem and rapidly dunk them in an ice bath before transporting them to the cold room. Working under a green safety lamp, remove the maize leaves from the ice bath and snip them into smaller five-by-five centimeters pieces using scissors. Gently and thoroughly grind half of the clipped leaf tissue in 350 milliliters of the grinding buffer at a level lower than seven on a commercial blender.
Start and stop the blender five to six times to ensure that all the leaves are cut. Filter the homogenate through one layer of 25 micron nylon cloth on a large funnel to four 250-milliliter centrifuge bottles. After repeating the blending step with the remaining clipped leaf tissue, evenly divide the filtrate between the four bottles.
Remove an aliquot of the leaf filtrate and set it aside to be stored as a representative total leaf sample. Next, centrifuge the filtrate for six minutes at 1, 840 G and four degrees Celsius. Pour off the resulting supernatant and, with gentle swirling movements of the brush, resuspend the pellet in 12 milliliters of medium R 0.2, containing 0.2 molar sucrose.
After resuspending the pellet in one bottle, add the suspension to the next bottle and repeat the resuspension to pool the suspensions into one bottle. Distribute the pooled suspension between six three-milliliter ultracentrifuge tubes, reaching a maximum volume of 2.5 milliliters in each tube. Then use the tip sonicator at an amplitude of 100%to sonicate each tube four times for 10 seconds each.
Ensure to keep the sonicator horn submerged and away from the liquid surface to prevent frothing. Slowly move the sonicator horn up and down within the suspension during each round. While alternating the four tubes, return each tube to an ice bucket after each sonication to allow the sample to cool.
Once done, centrifuge the sonicated crude thylakoid suspension at 150, 000 G for 30 minutes at four degrees Celsius. Skim the cushion surface using a syringe and 22 gauge needle and harvest the resulting yellow and oily floating pad of crude plastoglobules from the sucrose cushion. Recover approximately 500 microliters from each tube and deposit them into a 2.0-milliliter tube.
Collect crude thylakoid aliquots before and after the sonication and release of plastoglobules. Once done, either continue the plant tissue processing or store the crude plastoglobules at minus 80 degrees Celsius and purify them later. Grow 50 milliliters of synechocystis species PCC 6802 culture in BG11 media for 7 to 10 days to reach the stationary phase.
Using a spectrophotometer, adjust the cell density to an optical density of 2.0 at 750 nanometers. To remove the polysaccharides, centrifuge 50 milliliters of culture and remove the supernatant. Repeat washing of the cells in 50 milliliters of buffer A two times.
Resuspend the washed pellet in 25 milliliters of buffer A and break the cells using a French pressure cell at 1, 100 pound square inches. Repeat the process three times in cold conditions until the lysed color changes from green to red, blue, green under white light. Remove the aliquot of the cell homogenate and set it aside to be stored as a representative total cell sample.
Distribute the resulting homogenate in eight three-milliliter ultracentrifuge tubes filled to a maximum of 2.5 milliliters in each tube. Overlay this homogenate with 400 microliters of medium R, producing a step gradient. Carefully balance the tubes by adding additional medium R as necessary before centrifuging the tubes.
The thylakoid and other heavier organelles, including any polyhydroxyalkanoate bodies, show pellet formation, while plastoglobules form a yellow oily pad on or near the top of the sucrose gradient. Harvest the resulting floating pad of crude plastoglobules with a syringe, as demonstrated earlier, and deposit the harvest into a two-milliliter tube. Scrape plastoglobules off the side of the ultracentrifuge tube wall with a needle tip if necessary.
Either continue with cyanobacteria processing or store crude plastoglobules at minus 80 degrees Celsius and purify later. To harvest the pure plastoglobules using the plant tissue processing method, prepare the sucrose gradient described earlier using 500 microliters of crude plastoglobules, 400 microliters of medium R 0.2, and 400 microliters of medium R.After centrifugation, harvest the resulting floating pad of pure plastoglobules into a two-milliliter tube. After aliquoting the pure plastoglobules and flash freezing the aliquots in liquid nitrogen, either store them at minus 80 degrees Celsius or lyophilize them to a dry powder.
To harvest pure plastoglobules from cyanobacteria, create a sucrose gradient, as mentioned earlier, using 500 microliters of the crude plastoglobules, 750 microliters of medium R 0.7, and 750 microliters of medium R 0.2. After centrifugation, collect the pure plastoglobules in a 1.5-milliliter tube and aliquot the pure plastoglobules. Once done, flash freeze the pure plastoglobules aliquots in liquid nitrogen and store them directly at minus 80 degrees Celsius or lyophilize to a dry powder.
In this method, a significant amount of plastoglobule or lipid droplet material was seen floating on the top layer of the sucrose cushion. After subsequent centrifugation, purified plastoglobules were obtained at or near the surface of the sucrose gradient. After the successful isolation of plastoglobules, immunoblots developed for purity assessment using antibodies raised against arabidopsis thaliana fibrillin 1a and arabidopsis thaliana photosystem II subunit D1 showed that the fibrillin homologues were primarily associated with thylakoids than the plastoglobules.
To ensure the efficient and concentrated extraction of the floating pad, place the syringe needle orifice just below the liquid surface of the buffer while completely submerged, then gently pull up. Isolated plastoglobule samples are amenable to subsequent proteomic or lipidomic studies and have been used to demonstrate the changes in the targeted protein-lipid composition under differing environmental conditions. This technique has facilitated new studies of the plastoglobule, including the discovery of associated protein kinase activity and the presence of oligomeric protein complexes through subsequent biochemical investigation.
