Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

1. Growth of Arabidopsis Plants and the Stress Treatment

1. Prepare 1 L of Murashige and Skoog (MS) medium by adding 4.3 g of MS Basal Salt Mixture, 10 g sucrose, 0.5 g 2-(N-morpholino) ethane sulfonic acid (MES) to deionized water up to 1 L, and adjust the pH to 5.7 with potassium hydroxide (KOH). Add 6 g of phytoagar and autoclave for 20 min at 120 °C.
   1. Before solidification, pour the medium into round Petri plates (diameter 9 cm, height 1.5 cm, with 20 – 25 mL MS medium per plate). Allow the plates to dry for ~1 h in a laminar flow hood before closing the lids.
2. Place Arabidopsis thaliana seeds into a 1.5 mL test tube for surface sterilization by adding 1 mL of 70% (v/v) ethanol containing 0.02% (v/v) Triton X-100 and continuously shaking for 5-10 min. For each genotype/condition, prepare 10 Petri plates each carrying ~100 – 150 seedlings.
3. Wait for about 10 s until the seeds settle to the bottom of the tube, and then discard the supernatant by pipetting. Add 1 mL of 100% ethanol, and shake the tube again for 10 min.
4. Prepare the laminar flow hood while the seeds are sterilizing. Take one piece of filter paper per sample, fold it in half to facilitate sowing, and soak it in 100% ethanol in the hood. Wait until it dries out completely. Note that 100% ethanol is used as it evaporates more quickly than 70% ethanol.
5. Transfer the seeds onto the filter paper by pipetting using a 1 mL pipette tip cut ~5 mm from the fine end. Allow them to dry, which should take about 10 – 15 min.
6. Sow ~100 – 150 sterilized seeds evenly onto each MS medium Petri plate, and seal each plate with surgical tape.
7. Store the plates upside down at 4 °C for 2 – 4 d to encourage and synchronize germination. Old seeds may need to stay longer (up to a week) at 4 °C.
8. Transfer the plates to a plant tissue culture chamber and leave them upside up. Grow the plants for 8 d under a long-day cycle (16 h 100 µmol·m⁻²·s⁻¹ light, 8 h dark) at 20 °C.
9. For stress treatment, when the plants are 8 d old, in the flow hood, transfer them from the agar medium, by gently scraping them off by hand wearing ethanol-sterilized gloves, into a sterilized flask of liquid MS medium containing the stressor (e.g., 200 mM mannitol). Avoid carrying over agar medium. Cover the flask mouth with sterilized foil and allow the plants to grow under the same conditions as in step 1.8 for an additional 2 d on an orbital shaker with gentle shaking (~100 rpm).

2. Making a Precursor Protein by In Vitro Transcription/Translation

Note: This protocol assumes the use of Arabidopsis photosystem I subunit D precursor (pPsaD) as the template/preprotein, but the method is compatible with others.

1. Clone the coding sequence (CDS) of pPsaD into the multiple cloning site (MCS) of the pBlueScript II SK vector (or any other similar vector with an upstream T7 promoter) ¹³. Purify the plasmid (pBSK-pPsaD) using a DNA isolation kit and verify the sequence by DNA sequencing.
   NOTE: All these steps employ standard molecular cloning techniques.
2. Determine the pBSK-pPsaD plasmid DNA concentration using a spectrophotometer set to measure absorbance at 260 nm. Dilute the plasmid to a final concentration of 10 ng/µL.
3. Run a 20 µL PCR (for 35 cycles) using the diluted plasmid as template. Prepare the reaction as follows: 2 µL 10× polymerase buffer, 2 µL 2 mM dNTPs, 1 µL 5 mM M13 forward primer (5'-TGT AAA ACG ACG GCC AGT-3'), 1 µL 5 mM M13 reverse primer (5'-CAG GAA ACA GCT ATG ACC-3'), 1 µL diluted pBSK-pPsaD plasmid, 1 U Taq polymerase, and distilled water to make the total reaction volume up to 20 µL. Use a standard PCR program, as follows: 95 °C, 5 min; 35 cycles of [95 °C, 30 sec; 56 °C, 30 sec; 72 °C, 30 sec); and 72 °C, 5 min.
4. Run 5 µL of the PCR product on a 1% (w/v) agarose gel in TAE buffer (Tris-HCl, pH 7.6, 20 mM acetic acid, 1 mM EDTA) to verify correct amplification of the cDNA, and quantify the relevant band relative to standards to confirm the concentration. Store the rest of the product at −20 °C for further applications.
5. Prepare a 50 µL reaction using a rabbit reticulocyte lysate based cell-free transcription/translation system compatible with PCR DNA, as follows: 40 µL reticulocyte lysate from the transcription/translation system, 2.5 µL radiolabeled [³⁵S] methionine, 11 µCi/mL (specific activity: >1,000 Ci/mmol), 2.5 µL sterile distilled water, and 5 µL pPsaD PCR product (100 – 800 ng).
   Caution: Wear gloves, laboratory clothing and safety glasses when handling radioactive material. Monitor and decontaminate the working surface and equipment. Dispose of all radioactive waste in an approved waste container.
6. Incubate the reaction for 90 min in a 30 °C water bath. Stop the reaction by placing the sample on ice.
7. Remove 1 µL of the reaction as a test sample (the same volume can also serve as an input control for step 5.7) for verification on standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, fluorography or phosphor imaging. A good result is indicated by the observation of a distinct and strong band corresponding to the molecular weight of pPsaD (~23 kD). Store the rest of the reaction at −80 °C for further applications.

3. Chloroplast Isolation

1. Prepare the following stock solutions:
   1. Prepare Chloroplast Isolation Buffer (CIB, 2x): 0.6 M sorbitol, 10 mM magnesium chloride (MgCl₂), 10 mM Ethylene Glycol Tetraacetic Acid (EGTA), 10 mM ethylenediaminetetraacetic acid (EDTA), 20 mM sodium bicarbonate (NaHCO₃), 40 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES); adjust to pH 8.0 with KOH. Prepare 2 L of 2x CIB, make aliquots and keep them at -20 °C for long-term storage, or at 4 °C for short-term storage. To make 1x CIB, dilute the 2x CIB 1:1 with distilled water; this can be prepared freshly on the day of chloroplast isolation experiment.
   2. Prepare HEPES-MgSO₄-Sorbitol buffer (HMS, 1x): 50 mM HEPES, 3 mM magnesium sulfate (MgSO₄), 0.3 M sorbitol; adjust to pH 8.0 with sodium hydroxide (NaOH). Prepare 400 mL of buffer, make aliquots and keep them at -20 °C for long-term storage, or at 4 °C for short-term storage.
2. Conduct all of the following procedures in the cold room or on ice. Precool all of the solutions, devices, equipment, and rotors before starting.
3. Prepare a continuous density gradient by mixing 13 mL Percoll, 13 mL 2x CIB buffer, and 5 mg glutathione in a 50 mL centrifuge tube, and centrifuging the mixture at 43,000 × g for 30 min (brake off) at 4 °C. After centrifugation, handle the tube with care to avoid disturbing the gradient and keep it on ice for later use.
4. Transfer 100 mL of 1x CIB per genotype/condition to a 1 L beaker.
   1. Remove the seedlings from the liquid medium by tipping them into a sieve, and transfer them into another CIB-containing beaker; then, rinse the tissue with the CIB to remove any residual liquid medium before replacing it with 100 mL of fresh CIB.
5. For homogenization, use a total of 100 mL of 1x CIB per sample in five consecutive rounds of homogenization, each round using 20 mL of fresh CIB held in a 50 mL beaker.
   1. For the first round of homogenization, transfer the plant tissue into the 50 mL beaker by hand, allowing the previous holding buffer to drain through your fingers. Try to use most of the tissue in the first round, but make sure it is immersed with buffer; if this is not possible, introduce the remaining unused tissue at the second round.
6. Place probe of the tissue homogenizer into the tissue and homogenize with two pulses of 1 – 2 s each. Filter the homogenate through two layers of filtration cloth into a 250 mL centrifuge tube by gentle squeezing. Retain the filtrate, and transfer the tissue back to the 50 mL beaker.
7. Place a second 20 mL CIB aliquot into the 50 mL beaker, adding any remaining tissue not used in the first round of homogenization, and repeat the homogenization and filtration steps. Thus, repeat steps 3.5 and 3.6 until all of the 100 mL of CIB has been used up (i.e., 5 aliquots of 20 mL), and combine the resulting filtrates.
8. Centrifuge the pooled homogenate at 1,000 × g for 5 min (brake on) at 4 °C, and pour off the supernatant immediately following completion of the centrifugation, taking care not to disturb the pellet. Resuspend the pellet in the residual supernatant left in the tube by gently agitating the tube on ice. Do not resuspend vigorously by pipetting or vortexing.
9. Gently transfer the homogenate onto the top of the premade continuous density gradient, using a Pasteur pipette to apply it via the wall of the receiving tube. Avoid disturbing the gradient. Centrifuge in a swinging-bucket rotor at 7,800 × g for 10 min (brake off) at 4 °C.
   NOTE: Two green bands can be seen in the gradient after centrifugation: the lower band contains intact chloroplasts, and the upper band contains broken chloroplasts.
10. Discard the top band by pipetting, and then transfer the lower band with a Pasteur pipette into a fresh 50 mL centrifuge tube, retaining a volume of up to 8 mL per gradient.
11. Add ~25 mL of 1x HMS buffer into the tube and invert the tube twice to wash the Percoll from the chloroplasts. Place the tube again in the swinging-bucket rotor and centrifuge at 1,000 × g for 5 min (brake on) at 4 °C.
12. Pour off the supernatant and carefully resuspend the chloroplast pellet in the residual HMS left in the tube by gently agitating the tube on ice. Add an additional 100 – 300 µL of HMS if necessary (based on the size of the pellet and the counting in Section 4), but try not to dilute the sample too much.
    1. Keep the chloroplasts on ice. Each time before the chloroplasts are used for downstream applications, shake the tube to resuspend them. Always use a cut pipette tip (with an enlarged pore) to transfer the chloroplasts.

4. Analysis of the Yield and Intactness of Chloroplasts

1. Add 5 µL of isolated chloroplasts to 495 µL of 1x HMS buffer in a 1.5 mL tube, and then mix gently by inverting the tube to obtain a 1:100 dilution.
2. Place a cover glass on top of the counting chamber of the haemocytometer. Slowly pipette ~40 – 60 µL of the diluted chloroplast suspension into the gap between the cover glass and the counting chamber. Using a phase-contrast microscope with a 10X or 20X objective, intact chloroplasts look round and bright and are surrounded by a halo of light.
   NOTE: Under the microscope, there is a 1 mm² counting area with 25 large squares, each containing 16 small squares in the center of the counting chamber.
3. Count 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 (step 4.1 above) accordingly, and repeat the procedure.
4. Calculate the number of chloroplasts per mL (concentration) as follows: n (the average number of chloroplasts per large square calculated in step 4.3) × 25 (total number of large squares) × 100 (the dilution factor) × 10⁴ (scaling factor to express data per 1 mL, since the volume above the 25 squares is 0.1 mm³).
5. Calculate the actual yield of chloroplasts by multiplying the concentration by the volume of chloroplast suspension obtained in step 3.12. Normally, more than 50 × 10⁶ chloroplasts can be obtained.

5. Chloroplast Protein Import

1. Prepare the following stock solutions:
   1. Prepare 10x HMS buffer: 500 mM HEPES, 30 mM MgSO₄, and 3.0 M sorbitol; adjust to pH 8.0 with NaOH. Prepare 50 mL of this buffer, aliquot, and store at −20 °C for long-term storage and at 4 °C for short-term storage.
   2. Prepare Import stop buffer: 50 mM EDTA dissolved in 1x HMS buffer. Prepare 50 mL of this buffer, aliquot, and store at −20 °C.
   3. Prepare 2x Protein loading buffer: 60 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 0.005% (w/v) bromophenol blue. Make 50 mL, and store at 4 °C. Just before use, add 100 µL of 1 M 1,4-dithiothreitol (DTT) to 900 µL of buffer.
2. To run a time-course with 3 time-points, prepare 3 tubes each containing a 130 µL aliquot of import stop buffer, and leave them on ice. Prepare a 450 µL import reaction in a 2 mL tube (per genotype/condition). Thaw all ingredients just prior to use.
   1. For one import reaction, typically use 10 × 10⁶ chloroplasts in A µL volume; for example, if the chloroplast concentration is 2.5 × 10⁸ /mL, use 40 µL chloroplast suspension. Three time-points will need 3 × A µL (i.e., 120 µL in our example).
   2. Mix the reactions components on ice in the following order: distilled water (to make the total volume 600 µL), B µL 10x HMS buffer (where B = [600-3×A]/10; i.e., 48 in our example), 12 µL 1 M gluconic acid (potassium salt), 6 µL 1 M NaHCO₃, 6 µL 20% (w/v) BSA, 30 µL 100 mM adenosine 5′-triphosphate magnesium salt (MgATP), 24 µL 250 mM methionine (not radiolabeled), and 30 µL precursor protein. Immediately before starting the import reaction, add 3 × A µL of chloroplasts and mix by gently tapping the tube.
3. Incubate the reaction tube at 25 °C in a water bath under 100 µmol·m⁻²·s⁻¹ light. Occasionally flick the tubes to resuspend chloroplasts.
4. To conduct a time-course, withdraw 130 µL aliquots from the reaction at the required time-points within the linear range of import, which for pPsaD is up to ~12 min (4-, 8- and 12-min time points are suitable in this case). The linear range period can vary for different proteins ¹⁴. Thus, it is suggested to test each individual preprotein before to optimize the time points.
5. Immediately upon withdrawal, transfer each 130 µL aliquot to a tube of ice-cold import stop buffer, mix by gently tapping the tube, and retain all tubes on ice until the time-course has been completed.
6. Centrifuge all samples for 30 s at 12,000 × g in a microfuge, discard the supernatants by pipetting, and resuspend pellets in 15 µL of 2x Protein loading buffer by vortexing.
7. Analyze all the samples plus the pPsaD input control (containing pPsaD equivalent to 10% of the amount added to each import reaction, from step 2.7) by standard SDS-PAGE and autoradiography, fluorography or phosphor imaging ¹⁵. Use image analysis software to quantify and analyze the results. To provide an indication of import efficiency, the amount of imported protein in different genotypes/conditions can be assessed by measuring the radioactivity associated with each mature band.