The overall goal of isolating intact and pure plant mitochondria is to study respiration, mitochondrial composition, biogenesis, and metabolism. This method can help answer key questions in plant energy biology, such as the importance of mitochondria and stress tolerance, under changing environmental conditions. The main advantage of this technique is that sufficient quantities of pure, intact, and functional mitochondria could be isolated from relatively small quantities of plant tissue.
Demonstrating the procedure will be Wenhui Lyu, and Jennifer Selinski, From my laboratory. On the day of mitochondrial isolation, wash the gradient pourer and PVC peristaltic tubing with deionized water to prepare the gradients. Incline to position two 50 mL centrifuge tubes on ice, such that the peristaltic tubing outlet touches the wall of the tubes.
Then turn the black lever down to close the connection between the inner and outer chambers. Place the gradient pourer with the magnetic stir bar inside the inner chamber on a magnetic stirrer. Then, pour 35 mL of heavy gradient solution into the inner chamber.
Dispense half of the heavy gradient solution through the peristaltic pump, at 300 mL per hour, into two centrifuge tubes, kept on ice. Then pour 35 mL of light gradient solution into the outer chamber. Immediately, turn the black lever up halfway to resume connection between the inner and the outer chambers, for the solutions to mix gently.
Expel the entire gradient mixture from the chambers, at a rate of 60 mL per hour, to form two continuous 0%to 4.4%PVP gradients. Always keep the gradients on ice before use. It is important not to overground the plant tissue, otherwise the mitochondria will break down.
For a single preparation, cut the whole rosette tissue from at least 80 four week old Arabidopsis plants, grown on soil. Then add half of the cut plant tissues to a cooled large mortar and pestle, containing 75 mL of grinding medium. Then use grinding medium to moisten four layers of filtration material, with a pore size of 20 to 25 m.
Use a plastic funnel to filter the homogeny through all four layers of filtration material, and collect it in a 500 mL conical flask. Use an additional 75 mL of grinding medium to grind the other half of the tissue. Then combine the entire homogenate in the filtration material, to filter out as much as possible.
Use 150 mL of grinding medium to grind all of the remaining tissue on the filtration material, and filter into a 500 mL conical flask. Distribute the filtered homogenate in eight pre-chilled 50mL plastic centrifuge tubes. And centrifuge at 2, 500 g at four degrees celsius, in a fixed angle rotor, for five minutes.
After pouring the supernatant into clean centrifuge tubes, centrifuge again at 17, 500 g, at four degrees celsius, for 20 minutes, in a fixed angle rotor. Leave 3 mL of supernatant in the tube. Then use a fine paintbrush to gently re-suspend the pellet in the remaining supernatant.
Add 10 mL of single strength wash buffer to each of the tubes, and pool all four of the tubes into one centrifuge tube, resulting in two tubes total. Adjust the volume to 50mL with single strength wash buffer, and repeat the centrifugation steps. Using a very small quantity of wash buffer, disperse the pellet with the fine paintbrush, and then use a dropper to pool the crude mitochondrial pellet into a tube.
Then carefully layer the mitochondrial suspension with a dropper, on two continuous 0%to 4%PVP gradients. Use single strength wash buffer to balance the weight of the tubes, and conduct a centrifugation run at 40, 000 g at four degrees celsius, for 40 minutes. Remember to turn off the brake.
Cautiously aspirate the upper five centimeters of solution above the mitochondrial band. Then distribute the mitochondrial solution into two clean tubes, and fill with single strength wash buffer. Cover the mouth of the tube with plastic paraffin film, and invert it several times to evenly distribute the colloidal density gradient, and mitochondria.
Then, centrifuge at 31, 000 g for 15 minutes at four degrees celsius, with slow brake. Aspirate the supernatant and adjust the tube volume up to 50mL with single strength wash buffer. Invert several times for proper mixing.
Centrifuge the solution at 31, 000 g for 15 minutes at four degrees celsius with slow brake. Aspirate the supernatant, and subsequently collect the mitochondrial pellet in 500 l of single strength wash buffer, using a pipette with modified 200 l tip. With the plunger open, add 970 l of air saturated respiration medium to the reaction chamber.
Then add 150 g of total mitochondrial protein, using a modified pipette, and 500 M Adenosine Triphosphate to the reaction chamber, to determine mitochondrial respiration. Use a magnetic stir bar to continuously stir the mitochondrial suspension. After closing the plunger, click Start Recording to record oxygen consumption.
Wait for a minute or two, to check for a linear oxygen consumption trace, indicating stable oxygen consumption. Then, add 5 mM of succinate, and record the oxygen consumption for approximately two minutes. Add 1 mM Adenosine Diphosphate, and keep recording the oxygen consumption rate for another two minutes.
Next, add 1 mM Nicotinamide Adenine Dinucleotide Hydrogen, and continue recording oxygen consumption for around four to eight minutes. Then add reagents to inhibit the cytochrome C pathway, and keep recording the oxygen consumption via alternative oxidase, for about two minutes. KCN, myxothiazol, and antimycin A are all respiratory poisons.
It is important to follow the safety instructions in the MSDS data sheets. Please note that different countries will have different legislations for the use of these inhibitors in our experiments. Next, add 5 mM dithiothreitol, and 10 mM pyruvate to activate the alternative oxidase.
And keep recording for another five to seven minutes. Finally, add 500 mM N-propyl gallate, and record for two minutes. A representative gradient, showing the presence of greenish-white mitochondrial fraction near the bottom of the gradient solution.
Thylakoids and other contaminants present on top of the gradient solution, are indicated in dark green bands. Representative traces of oxygen consumption by mitochondria, to show the outer membrane integrity before oxygen consumption measurement. The outer membrane integrity was well preserved and about 90%intact.
Representative traces of oxygen consumption by mitochondria, after adding substrates, inhibitors, and effectors. The results show a compromised cytochrome c, and alternative oxidase pathway. After watching this video, you should have a good understanding of how to isolate and analyze plant mitochondria.
