Materials and Reagents

Corex^® centrifuge tubes (30 ml) (Corning, USA)
Note: This product has been discontinued. Replaceable item: 30 ml glass tubes from Krackeler Scientific, catalog number: 6-45500-30 .
Miracloth (pore size: 22-25 µm) (EMD Millipore, catalog number: 475855 )
Pipette tips (10 µl, 200 µl, 1 ml) and ‘cut-off’ pipette tips (1 ml) with the top (~2 mm) cut with a scissors
Falcon tubes (50 ml) (Greiner Bio One, catalog number: 227261 )
2 ml tubes
Aluminum foil
Scintillation vials (SARSTEDT, catalog number: 58.536 ) and push caps (SARSTEDT, catalog number: 65.816 )
Arabidopsis plants, approximately 4 weeks old (see Notes)
Percoll (GE Healthcare, catalog number: 17-0891-01 )
H₂O_bidest
80% (v/v) acetone (Carl Roth, catalog number: 9372.2 )
⁴⁵Ca stock solution (1 mCi [37 MBq], CaCl₂ > 10 Ci/g) (PerkinElmer, catalog number NEZ013001MC )
⁵⁴Mn stock solution (200 µCi [7.4 MBq], MnCl₂ in 0.5 N HCl > 20 Ci/g) (PerkinElmer, catalog number: NEZ040200UC )
Calciumchlorid (CaCl₂) (Carl Roth, catalog number: A119.1 )
Mangan(II)-sulfat monohydrate (MnSO₄·H₂O) (Carl Roth, catalog number: 4487.1 )
0.1% (w/v) SDS (Carl Roth, catalog number: CN30.4 )
Rotiszint^® Eco Plus scintillation cocktail (Carl Roth, catalog number: 0016.3 )
Sorbitol (Carl Roth, catalog number: 6213.2 )
Tricine (Carl Roth, catalog number: 6977.5 )
Natriumhydroxid (NaOH) (Carl Roth, catalog number: P031.2 )
EDTA (Carl Roth, catalog number: 8040.2 )
Magnesiumchlorid (MgCl₂) (Carl Roth, catalog number: KK36.1 )
Sodium bicarbonate (NaHCO₃) (Sigma-Aldrich, catalog number: S5761 )
BSA (Carl Roth, catalog number: 8076.3 )
Manganese(II) chloride dehydrate (MnCl₂) (EMD Millipore, catalog number: 105934 )
EGTA (Carl Roth, catalog number: 3054.2 )
HEPES
⁴⁵Ca working solution (see Recipes)
⁵⁴Mn working solution (see Recipes)
5x resuspension buffer, pH 8.4 (see Recipes)
Homogenization buffer, pH 8.4 (see Recipes)
Import buffer, pH 8.0 (see Recipes)
Washing buffer, pH 8.0 (see Recipes)
Lysis buffer, pH 7.6 (see Recipes)

Equipment

Cold room equipped with green light (500-530 nm)
Automatic pipette controller (BrandTech Scientific, model: Accu-Jet^® Pro ) and a 10 ml glass pipette
Waring blender (Waring Laboratory Science, model: 7011S) with a steel container and four razor blades
Beckman-Avanti^TM J-25 centrifuge with rotors JA-14 and JS-13.1 (Beckman Coulter, model: AvantiTM J-25 )
Note: This product has been discontinued. Replaceable item: Beckman Avanti J-E series centrifuge (Beckman Coulter, model: Avanti J-E).
Centrifuge Type 4K15C for Falcon tubes (Sigma Laborzentrifugen, model: 4K15C) or any other centrifuge, which is adequate for Falcon tubes
UV/Vis spectrophotometer (Biochrom, model: Ultrospec^TM 2100 ) with quartz cuvettes
Light source Faser-Illuminator (Heinz Walz, model: FL-460 ) set at 90 µmol photons m^-2 sec^-1
Vortex shaker (Scientific Industries, model: Genie2 )
Centrifuge 5418 R with rotor FA 45-18-11 for 2 ml tubes (Eppendorf, model: 5418 R)
Liquid scintillation counter (PerkinElmer, model: Tri-Carb^® 2910TR )
Isotope lab with permission for ⁵⁴Mn handling, lead coat and lead shields (approximately 5 cm)

Procedure

Preparation of a Percoll gradient
1. Place all materials, reagents and buffers in the cold room for temperature equilibration at 4 °C.
2. Prepare 10 ml Percoll solution (40%) by mixing 4 ml Percoll, 2 ml 5x resuspension buffer, pH 8.4 and 4 ml H₂O_bidest.
3. Prepare 10 ml Percoll solution (80%) by mixing 8 ml Percoll and 2 ml 5x resuspension buffer, pH 8.4.
4. Fill into a Corex tube 9 ml of 40% Percoll solution and carefully underlay 9 ml of 80% Percoll solution using a 10 ml glass pipette and an automatic pipette controller, keep the gradient on ice until further use (Figure 1).
  
  Figure 1. Preparation of Percoll gradient and isolation of intact chloroplasts (Procedures A and B)
Isolation of intact chloroplasts
1. Harvest 12 g fresh leaf material of overnight dark adapted Arabidopsis plants (see Notes). Perform all steps in a cold room equipped with green light (500-530 nm).
2. Add 180 ml of homogenization buffer, pH 8.4 to the leaf material and homogenize in a Warring blender for 4 x 3 sec at 18,000 rpm (corresponding to button ‘Hi’).
3. Filter the homogenate through two layers of Miracloth and centrifuge at 1,500 x g for 5 min at 4 °C in a JA-14 rotor.
4. Remove supernatant and resuspend the pellet in 1 ml of 1x resuspension buffer pH 8.4 by carefully inverting the tube.
5. Transfer the resuspended pellet (= chloroplast suspension) by using a cut-off pipette tip carefully on top of the Percoll gradient (Figure 1).
6. The density gradient centrifugation is carried out in a JS-13.1 rotor for 15 min at 4 °C and 7,000 x g, use a slow acceleration and turn deceleration off. After centrifugation broken chloroplasts reside in the top fraction, intact chloroplasts reside in the interface of the two Percoll layers (Figure 1).
7. Remove broken chloroplasts and the upper solution and carefully transfer intact chloroplasts using a cut-off pipette tip into a Falcon tube.
8. Wash intact chloroplasts with 25 ml of 1x resuspension buffer pH 8.4. This step removes residual Percoll. Centrifuge at 1,500 x g for 5 min at 4 °C.
9. Resuspend pellet (= Intact chloroplasts) in 500 µl import buffer, pH 8.0 (Ettinger et al., 1999), transfer to a 2 ml tube, wrap in aluminum foil and keep on ice.
Determination of chlorophyll concentration
1. Dilute 10 µl of intact chloroplast solution with 990 µl 80% acetone and incubate for 10 min on ice and in the dark.
2. Centrifuge at 16,000 x g (centrifuge 5418 R) for 2 min at room temperature and immediately transfer supernatant to a quartz cuvette for measurement at 750 nm (for baseline correction), 663 nm and 646 nm in a UV/Vis spectrophotometer. Use 80% acetone as a blank sample.
3. Calculate the chlorophyll content with following formulae (Porra, 2002):
  Chl_a = 12.25 x A₆₆₃ - 2.55 x A₆₄₆
  Chl_b = 20.31 x A₆₄₆ - 4.91 x A₆₆₃
  (Chl_a + Chl_b) x 100 = xxx µg Chl/ml
  Adjust chlorophyll concentration to 0.3 mg/ml with import buffer, pH 8.0 and divide sample into 45 µl aliquots (= Intact chloroplasts), wrap in aluminum foil and keep on ice. The whole procedure requires 15 aliquots per isotope experiment.
Preparation of ⁴⁵Ca or ⁵⁴Mn working solution
1. Perform all steps in the isotope lab. For handling ⁵⁴Mn it is necessary to wear a lead coat and to shield radioactive samples with a lead wall (5 cm). Perform ⁴⁵Ca assays and ⁵⁴Mn assays separately, e.g., on two following days, but use freshly prepared chloroplasts.
2. Take 4 µl of ⁴⁵Ca stock solution and dilute it (1:20) with 76 µl of CaCl₂ (15 µM). Mix well, this mixture corresponds to the ⁴⁵Ca working solution and it is suitable for 15 (+1) reactions.
3. Take 4 µl of ⁵⁴Mn stock solution and dilute it (1:20) with 76 µl of MnSO₄ (1.5 µM). Mix well, this mixture corresponds to the ⁵⁴Mn working solution and it is suitable for 15 (+1) reactions.
Uptake of ⁴⁵Ca or 54Mn into chloroplasts (Figure 2)

Figure 2. Schema of experimental set-up. Incubation of intact chloroplasts with ⁴⁵Ca (A and B) or ⁵⁴Mn (C and D) is done for 20 min in 90 µmol photons m^-2 sec^-1. After light incubation samples are incubated in sorbitol, without vortexing to keep chloroplasts intact (A and C) or samples are incubated without sorbitol, but with vortexing to release thylakoids (B and D). EGTA is used as chelating agent to complex divalent cations, attached to the outside of the membranes.
1. To start the import reaction, mix a 45 µl aliquot (= Intact chloroplasts, step C3) with 5 µl of ⁴⁵Ca working solution (step D2) or with ⁵⁴Mn working solution (step D3), remove aluminum foil and immediately expose samples for 20 min to 90 µmol photons m^-2 sec^-1.
  Set up the reaction with three aliquots corresponding to three replicates.
2. Add 1 ml import buffer, pH 8.0 to each aliquot and incubate on ice, in the dark for 10 min (the total incubation time is 30 min). The import buffer contains sorbitol therefore the chloroplasts are kept intact during this incubation step.
3. Centrifuge at 16,000 x g (centrifuge 5418 R) for 1 min at 4 °C and remove supernatant. Resuspend pellet in 1 ml washing buffer, pH 8.0 and vortex for 2 sec. This step removes ⁴⁵Ca or ⁵⁴Mn bound at the surface of the membrane, because the washing buffer contains EGTA as chelating agent.
4. Centrifuge at 16,000 x g (centrifuge 5418 R) for 1 min at 4 °C and remove supernatant. Resuspend pellet in 50 µl of 0.1% SDS and immediately transfer to 4 ml scintillation cocktail. Mix well and keep samples at room temperature until counting.
Uptake of ⁴⁵Ca or ⁵⁴Mn into chloroplasts and subsequent release of thylakoids (Figure 2)
1. To start the import reaction, mix a 45 µl aliquot (= Intact chloroplasts, step C3) with 5 µl of ⁴⁵Ca working solution (step D2) or with ⁵⁴Mn working solution (step D3), remove aluminum foil and immediately expose samples for 20 min to 90 µmol photons m^-2 sec^-1.
  Set up the reaction with three aliquots corresponding to three replicates.
2. Add 1 ml import buffer, pH 8.0 to each aliquot and incubate on ice, in the dark for 10 min (the total incubation time is 30 min). The import buffer contains sorbitol therefore the chloroplasts are kept intact during this incubation step.
3. Centrifuge at 16,000 x g (centrifuge 5418 R) for 1 min at 4 °C and remove supernatant. Resuspend pellet in 1 ml washing buffer, pH 8.0 and vortex for 2 sec. This step removes ⁴⁵Ca or ⁵⁴Mn bound at the surface of the membrane, because the washing buffer contains EGTA as chelating agent.
4. Centrifuge at 16,000 x g (centrifuge 5418 R) for 1 min at 4 °C and remove supernatant. Resuspend pellet in 50 µl of 0.1% SDS and immediately transfer to 4 ml scintillation cocktail. Mix well and keep samples at room temperature until counting.
Additional reaction for estimating uptake of ⁴⁵Ca or ⁵⁴Mn during 10 min incubation step on ice
To start the import reaction, mix a 45 µl aliquot (= Intact chloroplasts, step C3) with 5 µl of ⁴⁵Ca working solution (step D2) or with ⁵⁴Mn working solution (step D3), remove aluminum foil and immediately perform steps E2 to E4 and F2 to F4. The total incubation time is therefore 10 min on ice in the dark. Set up the reactions with three aliquots each corresponding to three replicates.
Additional reaction for estimating background radiation
To estimate background radiation, mix a 45 µl aliquot (= Intact chloroplasts, step C3) with 5 µl of ⁴⁵Ca working solution (step D2) or with ⁵⁴Mn working solution (step D3), remove aluminum foil and immediately perform steps E3 to E4. Set up the reaction with three aliquots each corresponding to three replicates.
Determination of incorporated radioactivity
Collect scintillation vials form steps E4, F4, G and H and determine counts per minutes (cpm) in a liquid scintillation counter Tri-Carb^® 2910 TR. Determine ⁴⁵Ca values using program #5 and determine ⁵⁴Mn values using program #8 with 5 min count time per individual scintillation vial.

Data analysis

Collect cpm values from step I and insert values into an Excel sheet.
Subtract cpm values for mean background radiation (step H) from all other cpm values (steps E4, F4 and G). Although the value derived after 10 min ice-incubation (step G) is not used for subsequent calculations, it should be clearly lower than the value obtained after 30 min incubation. If this is not the case, results can not be interpreted as ‘light dependent uptake’.
Convert cpm values into relative units, e.g., take the mean cpm value after 30 min incubation time from step E4 and set it as a relative unit to ‘one’. Experiments performed with independent grown plants and isotopes can thus easily be compared.
The thylakoid to stroma distribution in chloroplasts can be estimated from the amount of radioactivity separately detected in thylakoid fractions (step F4) and in intact chloroplast fractions (step E4). Set the cpm values obtained from intact chloroplast fraction (step E4) to 100% and calculate the percentage of cpm values from thylakoid fractions (step F4).
The whole procedure (steps A to I) is described for three technical replicates (see step F1). It should be performed two to three times with independently grown plants (= independent experiments).

Notes

Arabidopsis growth can be performed as described in Steinberger et al., 2015. This protocol is also suitable for two-week old pea plants. For isolation of intact chloroplasts, it is important to avoid any starch granules, which accumulate during the light period. Starch granules inside chloroplasts might prevent accumulation of intact chloroplasts at the interface of the two Percoll layers. Therefore, dark adapt plants for at least 16 h; during the night starch degradation takes place.

Recipes

⁴⁵Ca working solution (use a fresh preparation)
4 µl of ⁴⁵Ca stock solution
76 µl of CaCl₂ (15 µM)
⁵⁴Mn working solution (use a fresh preparation)
4 µl of ⁵⁴Mn stock solution
76 µl of MnSO₄ (1.5 µM)
5x resuspension buffer, pH 8.4 (can be stored at 4 °C for a few days)
0.4 M sorbitol
20 mM Tricine/NaOH, pH 8.4
2.5 mM EDTA
5 mM MgCl₂
Homogenization buffer, pH 8.4 (can be stored at 4 °C for a few days)
0.4 M sorbitol
20 mM Tricine/NaOH, pH 8.4
10 mM EDTA
5 mM NaHCO₃
0.1% (w/v) BSA
1 mM MgCl₂
1 mM MnCl₂
Import buffer, pH 8.0 (can be stored at 4 °C for a few days)
0.33 M sorbitol
50 mM Tricine/NaOH, pH 8.0
5 mM MgCl₂
Washing buffer, pH 8.0 (can be stored at 4 °C for a few days)
0.33 M sorbitol
50 mM Tricine/NaOH, pH 8.0
50 mM EGTA
Lysis buffer, pH 7.6 (can be stored at 4 °C for a few weeks)
10 mM HEPES/NaOH, pH 7.6
5 mM MgCl₂

Acknowledgments

We thank Gabi Burkhard for excellent technical assistance. This work was carried out in the laboratory of Prof. Dario Leister (Biozentrum der LMU München, Department Biologie I, Munich, Germany) and supported by funds from the Deutsche Forschungsgesellschaft. This protocol is adapted from Schneider et al., 2016.

References

Ettinger, W. F., Clear A. M., Fanning, K. J., and Peck, M. L. (1999). Identification of a Ca²⁺/H⁺ antiport in the plant chloroplast thylakoid membrane. Plant Physiol 119: 1379-1386.
Porra, R. J. (2002). The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res 73(1-3): 149-156.
Schneider, A., Steinberger, I., Herdean, A., Gandini, C., Eisenhut, M., Kurz, S., Morper, A., Hoecker, N., Ruhle, T., Labs, M., Flugge, U. I., Geimer, S., Schmidt, S. B., Husted, S., Weber, A. P., Spetea, C. and Leister, D. (2016). The evolutionarily conserved protein photosynthesis affected mutant71 is required for efficient manganese uptake at the thylakoid membrane in Arabidopsis. Plant Cell 28(4): 892-910.
Steinberger, I., Egidi, F., and Schneider, A. (2015). Chlorophyll fluorescence measurements in Arabidopsis wild-type and photosystem II mutant leaves. Bio-Protocol 5: e1532.