The overall goal of this methodology is to study the import of nucleus encoded proteins into chloroplasts with a particular focus on the effects of stress. This method can help answer key questions in the chloroplast field, such as how chloroplast protein import is regulated. A particular advantage of this technique is that it allows for the effects of stress on chloroplast protein import to be assessed.

Though this method has been developed for use with the model plant Arabidopsis thaliana, it could be modified and adapted for use with other systems, such as crop plants. Demonstrating the procedure will be Qihua Ling, a postdoc from my laboratory. To begin, first grow the Arabidopsis plants as described in the accompanying text protocol.

Then when the plants are eight days old, perform a stress treatment. Gently scrape the plants from the agar plate by hand, and place them into a sterilized flask of liquid MS medium containing the stressor. Placing additional plants into a similar flask without stressor as a control might also be necessary, depending on the nature of the experiment.

Cover the mouth of the flasks with sterilized foil and allow the plants to grow for an additional two days on an orbital shaker with gentle shaking. Prepare a continuous density gradient by first mixing 13 milliliters of Percoll, 13 milliliters of two x chloroplast isolation buffer, and five milligrams of glutathione in a 50 milliliter centrifuge tube. Then centrifuge the mixture.

After centrifugation, handle the tube with care to avoid disturbing the gradient, and keep it on ice to use later. Next, transfer 100 milliliters of chloroplast isolation buffer per condition to a one liter beaker. Remove the seedlings from the liquid medium by tipping them into a sieve.

Then transfer them into the beaker containing chloroplast isolation buffer. Rinse the tissue with the buffer to remove any residual medium, and then replace the buffer with 100 milliliters of fresh isolation buffer. For the first round of homogenization, transfer the plant tissue into a 50 milliliter beaker by hand, allowing the previous buffer to drain through your fingers.

Then place the probe of the tissue homogenizer into the tissue and homogenize with two pulses of one to two seconds each. Filter the homogenate through two layers of filtration cloth into a 250 milliliter centrifuge tube. Finish by gently squeezing the cloth.

Retain the filtrate and transfer the plant tissue back to the 50 milliliter beaker. Then place a second 20 milliliters of chloroplast isolation buffer into the 50 milliliter beaker and add any remaining tissue that was not used in the first round of homogenization. Repeat the homogenization and filtration steps four more times.

Centrifuge the pooled homogenate at 1, 000 times g for five minutes at four degrees Celsius. Pour off the supernatant immediately following centrifugation, taking care not to disturb the pellet. Place the tube on ice and resuspend the pellet in the residual supernatant by gently agitating the tube.

Next, use a Pasteur pipette to gently transfer the homogenate onto the top of the premade continuous density gradient via the wall of the receiving tube. Then centrifuge the sample and gradient in a swinging bucket rotor at 7, 800 times g for 10 minutes at four degrees Celsius with the brake off. When finished, observe the two green bands that can be seen in the gradient.

Discard the upper band, which contains broken chloroplasts, and use a Pasteur pipette to transfer the lower band containing intact chloroplasts to a fresh 50 milliliter centrifuge tube. Next, add 25 milliliters of HMS buffer into the tube, and invert the tube twice to wash the Percoll from the chloroplasts. Then place the tube back into the swinging bucket rotor and repellet the chloroplasts.

Pour off the supernatant and resuspend the pellet in the residual buffer by gently agitating the tube on ice. Add an additional 100 to 300 microliters of the HMS buffer if necessary, but try not to dilute the sample too much. Store the resuspended chloroplasts on ice.

Start by adding five microliters of the isolated chloroplasts to 495 microliters of HMS buffer in a 1.5 milliliter tube. Invert the tube to gently mix the solution. Then place a cover glass on top of the counting chamber of a hemacytometer.

Slowly pipette 40 to 60 microliters of the diluted chloroplast suspension into the gap between the cover glass and the counting chamber. Using a phase contrast microscope, image the chloroplasts with a 20x objective. Intact chloroplasts look round and bright and are surrounded by a halo of light.

Then use the 10x objective to the number of chloroplasts in 10 large squares. The number of chloroplasts per large square should average between 10 and 30. If too few or too many chloroplasts are present, adjust the dilution factor and repeat the procedure.

Once within the proper range, calculate the number of chloroplasts per milliliter as described in the accompanying text protocol. To run a time course with three time points, prepare three tubes, each containing a 130 microliter aliquot of import stop buffer, and leave them on ice. In a two milliliter tube, prepare the import reaction containing the reaction buffer and radio-labeled precursor protein.

Note that there should be one reaction for each condition to be tested. Use approximately 10 million chloroplasts per time point. Incubate the reaction tube at 25 degrees Celsius in a water bath under a limited amount of light.

Occasionally flick the tubes to resuspend the chloroplasts. To conduct a time course, with 130 milliliter aliquots from the reaction at the required time points within the linear range of import. Immediately upon withdrawal, transfer each 130 microliter aliquot to a tube of ice-cold import stop buffer mixed by gently tapping the tube, and place the tubes on ice until the time course has been completed.

Once the last sample has been removed, centrifuge all of the samples for 30 seconds at 12, 000 times g in a Microfuge. Next, discard the supernatants and resuspend the pellets in 15 microliters of two x protein loading buffer. Then mix the samples by vortexing.

Finally, analyze all of the samples, plus a precursor input control, using standard SDS-PAGE and autoradiography, fluorography, or phosphorimaging. PsaD is an 18 kilodalton component of photosystem I that is synthesized as a larger precursor of 23 kilodaltons. Processing to the mature 18 kilodalton form indicates that import into the chloroplasts has occurred.

The sp1 mutant of Arabidopsis studied here carries a defect in an important regulator of the chloroplast protein import machinery, the sp1 protein. Under stress conditions, the amount of PsaD protein import into sp1 mutant chloroplasts is increased relative to the wild type, but not under normal non-stressful conditions. While the mature protein form of PsaD accumulated in a time-dependent manner with chloroplasts from both plant genotypes, the rate of import was significantly higher for sp1 mutant chloroplasts than for wild type chloroplasts.

These results imply an important role for the sp1 protein in regulating chloroplast protein import under stress conditions. Once mastered, this method from chloroplast isolation through to the completion of the import assay can be completed in five to six hours if it's performed properly. While attempting this procedure, it's important to remember to conduct all of the chloroplast isolation steps at four degrees Celsius to ensure that the chloroplasts retain maximal activity.

After its initial development several decades ago, the in vitro chloroplast protein import assay paved the way for researchers in the field to explore many different aspects of the chloroplast protein import mechanism. The refinement presented here enables investigation into the effects of stress conditions on protein import. Don't forget that working with radioactive material requires special care.

Precautions such as wearing gloves, a lab coat, and safety glasses should always be taken while performing this procedure.

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