Plant mitochondria1

BIOLOGY 369 PLANT PHYSIOLOGY

Plant Mitochondria

Plant mitochondria perform many functions. They supply most of the ATP and NADPH used by non-photosynthetic tissues at all times and by photosynthetic tissues at night. They also make many necessary biochemicals and help recycle phosphoglycolate formed by photorespiration. Consequently, plant mitochondria are very active in all plant tissues, even in source leaves during the day.

Plant mitochondria have many differences with animal mitochondria. Their genomes are much larger and some encode proteins which poison pollen development, resulting in cytoplasmically-inherited male sterility. They also perform many more reactions than animal mitochondria. In particular, electron transport has additional components in many plants. Most plants have extra NADH dehydrogenases in addition to the “Complex I” NADH dehydrogenase which starts the textbook version of mitochondrial electron transport. These alternative NADH dehydrogenases oxidize NADH and reduce ubiquinone but don’t transport protons across the membrane. Some are associated with the matrix side, and some are associated with the intermembrane space, enabling cells to oxidize NADH from either the cytoplasm or the matrix in this manner. Since no protons are transported all of the energy released is lost as heat. Complex I is very sensitive to rotenone whereas these alternative NADH dehydrogenases are insensitive, allowing them to be easily assayed by measuring oxygen consumption before and after adding rotenone.

Many plants also have an alternative oxidase which takes electrons directly from ubiquinone (Q) and uses them to reduce oxygen to water. This enzyme is insensitive to cyanide, azide or CO, thus allowing cells treated with these poisons to continue respiring. The alternative oxidase also does not pump any protons when it oxidizes Q, so the energy is released as heat. Some plants, such as the Voodoo Lily, use the alternative oxidase to raise the temperature of their flowers as much as 25˚ C above ambient to help attract pollinators. Most plant mitochondria have some alternative oxidase activity, which frequently increases in wounded tissues. It has also recently been discovered that many plants contain an uncoupler protein which acts as a proton ionophore, allowing protons pumped into the intermembrane space by electron transport to reenter without making ATP (or doing other work).

It seems odd that plants have so many ways to oxidize NADH to release heat rather than make ATP, yet these mechanisms must serve a vital function since they are so prevalent. One hypothesis is that this serves as yet another safety valve to help vent excess energy captured by photosynthesis, alternatives include the hypothesis that this allows the safe disposal of NADH created by the synthesis of necessary biochemicals, or that the burst of heat may help fight an attack by pathogens.

In this lab we will study respiration by mitochondria prepared from various plant tissues including freshly-prepared and aged beet and potato slices, fresh broccoli and fresh cauliflower. We will test the hypothesis that stress induces the alternative oxidase by comparing the initial respiration rates of freshly-prepared and aged beet and potato slices and the amount that is resistant to cyanide (or azide). We will also measure total and cyanide-resistant respiration in mitochondria from freshly-prepared broccoli and cauliflower to establish a baseline for comparison.

Preparing Mitochondria

We will prepare mitochondria from beets, broccoli, cauliflower, mung bean sprouts, corn seedlings, pea seedlings and potatoes. We will disrupt the tissue by grinding in a buffered, isosmotic solution so that cellular contents will be released into a solution similar to the intracellular environment. At this point, the brei contains soluble macromolecules, along with membrane fragments and organelles (nuclei, mitochondria, peroxisomes, plastids, etc.).

Next we will separate mitochondria from other cellular contents by differential centrifugation. This relies on the fact that larger particles sediment more rapidly than smaller particles upon centrifugation, therefore repeated centrifugation of a brei at successively higher speeds sediments progressively smaller particles. We can thus separate mitochondria from larger and smaller particles by choosing an appropriate series of centrifugations. We will first give our brei a short spin at low speed (1000 x g for 5’) to sediment most large particles, such as nuclei or intact cells. Most smaller particles such as mitochondria will remain in the supernatant after this centrifugation, therefore we will discard the pellet (called the nuclear fraction) and save the supernatant. Next we will sediment mitochondria (and other particles, unfortunately) by centrifuging the post-nuclear supernatant at 10,000 x g for 10 min.

We will next separate the mitochondria from other constituents by sedimentation through a Percoll step gradient. We will recover the mitochondria which band at the interface between 25% and 40% Percoll, and then use these for our experiments.

Measuring Mitochondrial Activity

We will assay mitochondrial electron transport by measuring the oxidation of succinate. Succinate dehydrogenase catalyzes oxidation of succinate to fumarate, transferring 2 electrons from succinate to the coenzyme flavin adenine dinucleotide (FAD):

succinate + FAD <=> fumarate + FADH2

In active mitochondria, the electrons from FADH2 are transferred to ubiquinone, then are passed down the respiratory electron transport chain to either cytochrome C oxidase or the alternative oxidase which both use them to reduce O2 to H2O. Succinate dehydrogenase is the best enzyme for this purpose because it is the only Krebs cycle enzyme that is embedded in the inner mitochondrial membrane and is therefore most likely to be present even if the mitochondria are somewhat damaged during isolation.

We will use an oxygen electrode to measure O2consumption dependent on succinate oxidation. To do so, we will compare the rates with and without added succinate. We will then compare the rates before and after adding cyanide or azide to assay the amount of alternative oxidase activity in the different tissues.

Procedures

Preparing mitochondria

1. Obtain 20 g of your plant tissue and chop it into small pieces with a razor blade

2. Place 5 g tissue in a chilled mortar with 10 ml of ice-cold mannitol grinding medium. Grind the tissue into a brei with a chilled pestle, then filter the brei through four layers of cheesecloth into a chilled 50 ml centrifuge tube.

3. Repeat three times (grinding 5 g each time), then wring out the juice into the tube.

4. Centrifuge the brei at 1000 x g for 5 min at 2˚ C. This gets rid of the large chunks, but leaves the mitochondria in the supernatant.

5. Decant the supernatant (which contains mitochondria and other goodies) into a clean, chilled centrifuge tube and discard the pellet (which contains intact cells, nuclei, and other debris).

6. Centrifuge the supernatant at 10,000 x g for 10 min at 2˚ C. While our mitochondria are spinning prepare a tube containing 10 mls each of 40% Percoll, 25% Percoll and 15% Percoll ( prepared like we did last week).

7. Decant and discard the supernatant.

8. Gently resuspend the pellet (which contains mitochondria and other goodies) in 1 ml cold mannitol assay buffer with a small paint brush or with a pasteur pipet. It is important that the clumps be completely dispersed. Once completely resuspended, add 5 more mls mannitol assay buffer.

9. Carefully layer them on top of the step gradient, then centrifuge 15 minutes at 15,000 x g, 2˚ C.

10. Recover the band that collects at the 25%-40% Percoll interface. Transfer to a fresh tube, dilute with as much assay buffer as possible, then centrifuge 10 minutes at 10,000 x g, 2˚ C.

11. Gently resuspend the pellet (which contains mitochondria and other goodies) in 1 ml cold mannitol assay buffer with a small paint brush or with a pasteur pipet. It is important that the clumps be completely dispersed. Once completely resuspended, add 5 more mls mannitol assay buffer. Be sure to keep the tube on ice for the remainder of the experiment!

12. Transfer 0.6 ml of the mitochondrial suspension to a microcentrifuge tube. Place this in a boiling water bath for 5 min to denature mitochondrial enzymes.

Measuring succinate dehydrogenase activity

Each group will be provided with two oxygen electrodes attached to a single sensing unit.

1. Add 5 mls distilled water and a stir bar to your cuvette.

2.Turn on the stirrer and once the display has settled down adjust it to read 100% oxygen saturation.

3.Remove the water, then rinse with assay buffer.

4. Add the contents of tube 1 prepared as described below. This is our baseline

5.Turn on the stirrer and once the display has settled down inject 0.5 mls ADP, thenread the % oxygen saturation at 30” intervals for 5 minutes, entering the data in Table I.

6.Remove the contents of the cuvette and rinse with assay buffer.

7.Add the contents of tube 2.This tests the effects of azide

8.Turn on the stirrer and once the display has settled down inject 0.5 mls ADP, thenread the % oxygen saturation at 30” intervals for 5 minutes, entering the data in Table I.

9. Remove the contents of the cuvette and rinse with assay buffer.

10.Add the contents of tube 3. This tests how quickly azide acts

11.Turn on the stirrer and once the display has settled down inject 0.5 mls ADP, thenread the % oxygen saturation at 30” intervals for 2 minutes, entering the data in Table I.

12. Inject 100 µl of 50 mM sodium azide, Once the display has settled down read the % oxygen saturation at 30” intervals for 3 minutes, entering the data in Table I.

13.Remove the contents of the cuvette and rinse with assay buffer.

14.Add the contents of tube 4.This tests the effects of cyanide

15.Turn on the stirrer and once the display has settled down inject 0.5 mls ADP, thenread the % oxygen saturation at 30” intervals for 3 minutes, entering the data in Table I.

16.Remove the contents of the cuvette and rinse with assay buffer.

17.Add the contents of tube 5. This tests how quickly cyanide acts

18.Turn on the stirrer and once the display has settled down inject 0.5 mls ADP, thenread the % oxygen saturation at 30” intervals for 2 minutes, entering the data in Table I.

19.Inject 100 µl of 50 mM cyanide, Once the display has settled down read the % oxygen saturation at 30” intervals for 3 minutes, entering the data in Table I.

20.Remove the contents of the cuvette and rinse with assay buffer.

21.Add the contents of tube 6. This tests the effects of leaving out ADP

22.Turn on the stirrer and once the needle has settled down read the % oxygen saturation at 30” intervals for 5 minutes, entering the data in Table I. Do not inject ADP!

  1. Remove the contents of the cuvette and rinse with assay buffer.
  2. Add the contents of tube 7. This tests the effects of leaving out succinate
  3. Turn on the stirrer and once the display has settled down inject 0.5 mls ADP, thenread the % oxygen saturation at 30” intervals for 5 minutes, entering the data in Table I.
  4. Remove the contents of the cuvette and rinse with assay buffer.
  5. Add the contents of tube 8. This tests the effects of leaving out ATP
  6. Turn on the stirrer and once the display has settled down inject 0.5 mls ADP, thenread the % oxygen saturation at 30” intervals for 5 minutes, entering the data in Table I.
  7. Remove the contents of the cuvette and rinse with assay buffer.
  8. Add the contents of tube 9. This tests for oxygen consumption that is not due to mitochondria
  9. Turn on the stirrer and once the display has settled down inject 0.5 mls ADP, thenread the % oxygen saturation at 30” intervals for 5 minutes, entering the data in Table I.
  10. Mix 100 µl of mitochondria with 4.9 mls Bradford’s and measure the A595. Dilute as necessary to get onto the standard curve.
  11. Calculate the mg protein in 0.5 mls of your mitochondrial preparation.
  12. Calculate the rate of oxygen production per mg protein as follows. First determine the rate of oxygen consumption for each tube as % change in oxygen saturation. Next convert this to ngs O2/mL using the conversion factor that at 25˚ C and today’s atmospheric pressure 1% saturation is 82 ng/mL. Finally, convert this to ngs O2/ mg protein remembering that you have 5 mls of solution in each cuvette, and that you added the amount of protein calculated in step 33.

Tube / Assay
Buffer / Azide / Cyanide / ATP / Succinate / mitochondria
1 / 3.5 ml / --- / --- / 0.5 ml / 0.5 ml / 0.5 ml
2 / 3.4 ml / 0.1 ml / --- / 0.5 ml / 0.5 ml / 0.5 ml
3 / 3.5 ml / --- / --- / 0.5 ml / 0.5 ml / 0.5 ml
4 / 3.4 ml / --- / 0.1 ml / 0.5 ml / 0.5 ml / 0.5 ml
5 / 3.5 ml / --- / --- / 0.5 ml / 0.5 ml / 0.5 ml
6 / 3.5 ml / --- / --- / 0.5 ml / 0.5 ml / 0.5 ml
7 / 4.0 ml / --- / --- / 0.5 ml / --- / 0.5 ml
8 / 4.0 ml / --- / --- / --- / 0.5 ml / 0.5 ml
9 / 3.5 ml / --- / --- / 0.5 ml / 0.5 ml / 0.5 ml*

*- This is the boiled mitochondrial suspension

Your introduction should explain why plant mitochondria are important, that they have several alternative mechanisms for oxidizing substrates not found in animal cells, that some of these alternative mechanisms may be induced by wounding, and that the purpose of this experiment was to see whether any of the tissues studied had alternative oxidase activity.

Your materials and methods should succinctly describe the procedure followed. Your results should state the purpose of the experiment, state that the raw data are presented in Table I and the processed class data are presented in Table II, and should point out the key features. Did any tissues have alternative oxidase activity? Were there differences between tissues?

Your discussion should explain the results/ Why were there differences between tissues (if any)? Why did leaving out ADP, ATP or succinate make a difference?