Materials and reagents

1.5 mL microcentrifuge tube (Sigma-Aldrich, catalog number: HS4323)
15 mL centrifuge tube (Thermo Fisher Scientific, catalog number: 339650)
Amicon ultra-0.5 centrifugal filter unit (EMD Millipore, catalog number: UFC500324)
10.4 mL polycarbonate bottle with cap assembly (Beckman Coulter, catalog number: 355603)
50 mL open-top thick-wall polycarbonate tube (Beckman Coulter, catalog number: 363647)
10 μL pipette tip (Thermo Fisher Scientific, catalog number: 3521-HR)
200 μL pipette tip (Thermo Fisher Scientific, catalog number: 3551-HR)
1,000 μL pipette tip (Thermo Fisher Scientific, catalog number: 3101-HR)
Disposable glass Pasteur pipettes 230 mm (VWR, catalog number: 612-1702)
1,000 mL plastic bag (e.g., Tingstad, catalog number: 398301-1)
Glass funnel (e.g., Sagitta, catalog number: 87807)
250 mL flask (e.g., Sagitta, catalog number: 86425)
25 mL beaker (e.g., VWR, catalog number: 213-0192)
Cuvette for chlorophyll quantification [e.g., Hellma, catalog number: HL104-002-10-40 (quartz, preferred) or Sarstedt, catalog number: 67.742 (plastic; ensure to measure right away so acetone does not degrade plastic and affect spectrophotometer reading)]
Paintbrush 6 mm (Ahlsell, catalog number: 384364)
Miracloth 22–25 μm pore size (Calbiochem, catalog number: 475855)
Arabidopsis thaliana plants (wild type and soq1-1 mutant, ecotype: Columbia-0)
Milli-Q water
2-Mercaptoethanol (Thermo Fisher Scientific, catalog number: 21985023)
Glycerol (Sigma-Aldrich, catalog number: G5516)
Bromophenol blue (Sigma-Aldrich, catalog number: B0126)
Ethanol (Sigma-Aldrich, catalog number: EX0290)
Acetic acid (Sigma-Aldrich, catalog number: 695092)
Coomassie blue R-250 (Sigma-Aldrich, catalog number: 1.12553)
Tris(hydroxymethyl)aminomethane (Tris base) (Sigma-Aldrich, catalog number: 648310-M)
Glycine (Sigma-Aldrich, catalog number: G8898)
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771)
Tween 20 (Sigma-Aldrich, catalog number: P9416)
Non-fat dried milk (e.g., Semper)
Tricine (Sigma-Aldrich, catalog number: T0377)
D-sorbitol (Sigma-Aldrich, catalog number: S1876)
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884)
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 529552)
Sodium L-ascorbate (Sigma-Aldrich, catalog number: A4034)
L-cysteine (Sigma-Aldrich, catalog number: C7352)
Sodium fluoride (Sigma-Aldrich, catalog number: 215309)
MgCl₂ (Sigma-Aldrich, catalog number: M2670)
Concentrated HCl (37%) (Sigma-Aldrich, catalog number: 320331)
Disodium hydrogen phosphate (Na2HPO4) (Sigma-Aldrich, catalog number: 567550)
Sodium dihydrogen phosphate (NaH₂PO₄) (Sigma-Aldrich, catalog number: 1.06370)
Benzamidine (Sigma-Aldrich, catalog number: 12072)
ϵ-Aminocaproic acid (Sigma-Aldrich, catalog number: A2504)
Phenylmethanesulfonyl fluoride solution (PMSF) (Sigma-Aldrich, catalog number: 93482)
cOmplete^TM, EDTA-free protease inhibitor cocktail (Roche, catalog number: 4693132001)
Quick Start^TM Bradford Protein Assay kit (Bio-Rad, catalog number: 5000201)
PageRuler prestained protein ladder (Thermo Fisher Scientific, catalog number: 26616)
Immobilon-P PVDF Membrane (Millipore Sigma, catalog number: IPVH00005)
ECL bright kit for immunodetection (Agrisera, catalog number: AS16 ECL-N)
Anti-PsaD, 1:1,000 dilution (Agrisera, catalog number: AS09 461)
Anti-Lhcb4, 1:7,500 dilution (Agrisera, catalog number: AS04 045)
Anti-RbcL, 1:7,500 dilution (Agrisera, catalog number: AS03 037)
Anti-PC, 1:2,000 dilution (Agrisera, catalog number: AS06 141)
Anti-ATPb, 1:5,000 dilution (Agrisera, catalog number: AS05 085)
Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody, 1:10,000 dilution (Sigma-Aldrich, catalog number: A6154)
1 M Tris-HCl stock solution (6.8 and 7.5) (see Recipes)
100 mM Sodium phosphate stock solutions (pH 7.8) (see Recipes)
Extraction buffer (see Recipes)
Resuspension buffer (see Recipes)
Lysis buffer (see Recipes)
Washing buffer (see Recipes)
4× sample loading buffer for SDS-PAGE (see Recipes)
Running buffer for SDS-PAGE (see Recipes)
Transfer buffer for immunodetection (see Recipes)
TBST buffer (see Recipes)
Blocking buffer (see Recipes)
Coomassie blue stain (see Recipes)
Coomassie blue destain (see Recipes)

Equipment

10 μL pipette (Thermo Fisher Scientific, catalog number: 4641040N)
20 μL pipette (Thermo Fisher Scientific, catalog number: 4641060N)
200 μL pipette (Thermo Fisher Scientific, catalog number: 4641080N)
1,000 μL pipette (Thermo Fisher Scientific, catalog number: 4641100N)
Plant growth cabinet (CLF plant Climatics, model: E-41L2)
Custom-designed LED panel, built by JBeamBio with cool white LEDs BXRA-56C1100-B-00 (Farnell)
Laboratory balance (e.g., Fisher Scientific, catalog number: 14-557-421)
Blender (e.g., Coline, 300 mL mixer cup with 2-bladed knife)
High-speed centrifuge (Beckman Coulter, model: Avanti J-20XP) with JA-25.50 fixed angle aluminum rotor (Beckman Coulter, catalog number: 363055)
Ultracentrifuge (Beckman Coulter, model: LE-70) with type 70.1 Ti fixed-angle titanium rotor (Beckman Coulter, catalog number: 342184)
Benchtop centrifuge (Beckman Coulter, catalog number: B06322)
UV-visible spectrophotometer (Hitachi, catalog number: U-5100)
Sonicator (Sonics, model: VCX130) with 2 mm microtip (Sonics, catalog number: 630-0417)
Dry block heater (MRC, catalog number: DBSC-001)
Immunodetection imaging system (Azure, model: c600)

Procedure

Plant material and growth conditions
Wild-type Arabidopsis thaliana and soq1-1 mutant were grown in a growth chamber with 120 μmol photons m^-2·s^-1 light intensity and 60% humidity at 20 °C during the day for 8 h and 18 °C during the night for 16 h. For cold and high light conditions (Cold HL), plants are illuminated in the cold room (4 °C) for 6 h at 1,600 μmol photons m^-2·s^-1 light intensity.
Thylakoid lumen isolation by sonication
1. Prepare extraction buffer [see Recipe 1 (Fristedt et al., 2009)], resuspension buffer (see Recipe 2), lysis buffer (see Recipe 3), and washing buffer (see Recipe 4). Keep at 4 °C.
2. Harvest the Arabidopsis rosettes from intended conditions (e.g., growth light and cold and high light conditions) into plastic bags. After removing the air, seal the bags and put them in the dark in the cold room. We usually use four rosettes from plants grown under short-day (8:16 h day/night) conditions for seven weeks (~10 g of fresh weight).
3. Blend the plant samples in 90 mL of extraction buffer three times for 5 s using a blender.
4. Filtrate the homogenate through a glass funnel with four layers of Miracloth to a 250 mL flask on ice.
5. Transfer the filtrated homogenate to 50 mL open-top thick-wall polycarbonate tubes (two tubes) and centrifuge at 1,000× g for 5 min.
6. Resuspend the chloroplast pellet in 35–40 mL of resuspension buffer using a soft paintbrush and centrifuge at 1,000× g for 5 min.
  Note: We initially use a small volume (~3 mL) to resuspend the chloroplast using a paintbrush, flash freeze one or two aliquots (100 μL) in liquid nitrogen, and store them at -70 °C until immunoblot analysis. Then, we add resuspension buffer to 35–40 mL.
7. Resuspend the chloroplast in lysis buffer at a final chlorophyll concentration of 0.2 mg/mL and centrifuge at 6,000× g for 5 min.
  Note: We usually keep the chloroplast in lysis buffer at 4 °C for 10 min to break the chloroplast envelopes by osmotic shock before centrifugation.
8. Concentrate approximately 2 mL of supernatant using Amicon ultra-0.5 centrifugal filters. Flash freeze the stromal aliquots (50 μL) in liquid nitrogen and store them at -70 °C until immunoblot analysis.
9. Wash the thylakoid pellet with 35–40 mL of washing buffer and centrifuge at 6,000× g for 5 min.
  Note: We usually use a small volume (~3 mL) to resuspend the pellet using a paintbrush and then add washing buffer to 35–40 mL.
10. Resuspend the thylakoids in washing buffer, usually ~4 mL, so that the chlorophyll concentration is approximately 0.5 mg/mL. The method to measure the chlorophyll concentration is from Porra et al. (1989). Flash freeze the thylakoid aliquots (100 μL) at -70 °C until immunoblot analysis.
11. Transfer the thylakoid mixture to a 25 mL pre-cooled beaker and sonicate 10 times for 30 s ON (power: 130 watt) and 10 s OFF at 4 °C.
  Note: We usually put half of the beaker into ice to keep the samples cold during the sonication operation.
12. Transfer the samples from the beaker to a 10.4 mL polycarbonate bottle, balance the samples with the washing buffer carefully, and ultracentrifuge at 200,000× g for 2 h at 4 °C to separate the thylakoid membranes in the pellet and the soluble lumenal proteins in the supernatant.
13. Transfer the supernatant (~3 mL) carefully to a new 15 mL centrifuge tube using a disposable glass Pasteur pipette and concentrate the soluble lumen sample (final volume is ~150 μL) at 14,000× g for 10 min at 4 °C using Amicon ultra-0.5 centrifugal filters. Flash freeze the lumen aliquots (25 μL) and store them at -70 °C until immunoblot analysis.
14. Resuspend the thylakoid membrane after isolation of lumen proteins in washing buffer (~4 mL) at a chlorophyll concentration of 0.5 mg/mL. Flash freeze the thylakoid membranes aliquots (100 μL) and store them at -70 °C until immunoblot analysis.
Sample preparation for SDS-PAGE
1. Take out samples (thylakoids, thylakoid membrane after isolation of lumen proteins, and lumen fraction) from -70 °C on ice and thaw rapidly at 37 °C on a heat block (less than a minute).
2. Wash the thylakoid and thylakoid membrane samples with 1 mL of 120 mM Tris-HCl (pH 6.8) and centrifuge at 20,000× g for 5 min at 4 °C.
3. Resuspend the pellets gently by adding 200 μL of 120 mM Tris-HCl (pH 6.8).
Measure the samples concentration using the Bradford assay (Kruger, 1994)
SDS-PAGE and immunoblot
1. Prepare the polyacrylamide gels for SDS-PAGE.
  Note: The optimal concentration of separating gel depends on the protein of interest. We usually use a 15% separating gel for lumenal plastocyanin protein (PC; PC1 runs closer to 14 kDa while PC2 runs closer to 19 kDa) and 12% separating gel for thylakoid membrane light-harvesting complex b4 protein (Lhcb4, 29 kDa), core subunit A of photosystem I (PsaA, 55–60 kDa), stromal ribulose bisphosphate carboxylase oxygenase large subunit (RbcL, 53 kDa), beta subunit of ATP synthase (ATPb, 54 kDa), and suppressor of quenching 1 (SOQ1, 108 kDa).
2. Load the samples (5 μg of protein and 5 µL PageRuler prestained protein ladder) and run the polyacrylamide gels.
  Note: We run the gels at a lower voltage (80 V) for 20 min and then increase to a higher voltage (120 V) until the dye front has reached the bottom of the gel.
3. After running the gel, transfer the proteins to a PVDF membrane in transfer buffer (see Recipe 7) by wet transfer at 200 mA for 90 min at 4 °C.
4. After transferring, incubate the PVDF membrane with blocking buffer (see Recipe 9) for 1 h at room temperature (20–25 °C).
5. Incubate the PVDF membrane with the primary antibody diluted with blocking buffer for 1 h at room temperature (20–25 °C).
  Note: Here, we used anti-PsaD, anti-Lhcb4, anti-RbcL, anti-PC, rabbit-specific antibodies against a C-terminal peptide of SOQ1 (TVTPRAPDAGGLQLQGTR) (1:200 dilution, produced and purified by peptide affinity by ThermoFisher), and anti-ATPb.
6. Wash the PVDF membrane with TBST buffer (see Recipe 8) (10 min × three times)
  Note: We used the horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody.
7. Incubate the PVDF membrane with the secondary antibody diluted with blocking buffer for 1 h at room temperature (20–25 °C).
8. Wash the PVDF membrane with TBST buffer (10 min × three times).
9. Incubate the PVDF membrane with the ECL bright kit for immunodetection.

Data analysis

We used sonication to break the thylakoids and collected thylakoid lumen by ultracentrifugation. The purity of the different fractions was assessed by immunoblot analysis using various antibodies against stromal (RbcL), thylakoid membrane (PsaA, Lhcb4), and thylakoid lumen (PC) proteins (Figure 1). The results in Figure 1 confirmed the purity of thylakoids, thylakoid membrane after isolation of lumen proteins (named membrane), and lumen. There are some lumen contaminants in the stroma fractions, possibly due to some thylakoids being broken when we lysed chloroplasts by osmotic shock. Of note, the lumen fraction is not completely pure. We observed a small amount of the stromal subunit of ATP synthase (ATPb) in the lumen, suggesting that there are some thylakoid membrane contaminants in the isolated lumen fraction (Figure 2).

Figure 1. Immunoblot analysis of marker proteins in chloroplast sub-fractions. The protein samples were prepared from 7-week-old Arabidopsis plants (Col-0) under growth light (GL, 120 μmol photons m^-2·s^-1) and 6 h cold and high light (Cold HL, 4 °C, 1,600 μmol photons m^-2·s^-1) conditions. Samples were loaded at the same quantity of total protein (5 μg). Four chloroplast proteins (RbcL, PsaA, Lhcb4, and PC) are shown. From Yu et al. (2022).
By applying this method, we can investigate the localization and accumulation of chloroplast proteins under different conditions (e.g., Cold HL) (Yu et al., 2022). Using either the Yeda press or sonication methods, we found that SOQ1, a thylakoid membrane-anchored protein involved in negative regulation of photoprotection qH (Brooks et al., 2013; Malnoë et al., 2018), accumulated as full length and also as three distinct truncated lumenal forms [comprising three domains thioredoxin-like (T) NHL (N) C-terminal domain (C) named TNC, two domains NC, or the C-terminal domain only (CTD)] in growth light and Cold HL conditions (Figure 2). Because of the large volumes of buffers used during the preparation with the Yeda press, no protease inhibitors were added except EDTA for metalloproteinases inhibition; together with the long isolation time, a concern was that the truncated SOQ1 forms observed in the lumen fraction were an artifact of the preparation. In comparison, the small volumes of buffers used in the sonication method allow to add inhibitors of different proteases (e.g., Roche cOmplete^TM protease inhibitor cocktail tablet, one tablet for 50 mL buffer). In the presence of protease inhibitors and a shorter isolation time, we confirmed the existence of SOQ1 truncated forms in the lumen fraction. The full-length SOQ1 present in the lumen fraction is likely due to contamination from the thylakoid membrane. These results were published in Yu et al. (2022).

Figure 2. The accumulation of SOQ1 in chloroplast sub-fractions. The protein samples were prepared from 7-week-old wild type (Col-0) and soq1 mutant under growth light (GL, 120 μmol photons m^-2·s^-1) and 6 h cold and high light (Cold HL, 4 °C, 1,600 μmol photons m^-2·s^-1) conditions. Samples were loaded at the same quantity of total protein (5 μg). Four chloroplast proteins (SOQ1, Lhcb4, PC, and ATPb) are shown. Three distinct truncated forms (TNC, NC, and CTD) of SOQ1 are accumulated in the lumen in GL and Cold HL conditions. TNC: thioredoxin-like, NHL and C-terminal domains (calculated molecular weight is 73 kDa); NC: NHL and C-terminal domains (calculated molecular weight is 53 kDa); CTD: C-terminal domain (calculated molecular weight is 17 kDa). From Yu et al. (2022).

Notes

All steps for thylakoid lumen isolation are performed in the cold room (4 °C). The centrifuge equipment with the rotor is pre-cooled to 4 °C. The paintbrushes should be washed after each isolation step. All buffers for thylakoid lumen isolation are prepared one day before and stored in the cold room. BSA, sodium ascorbate, and L-cysteine should be added in the extraction buffer right before use [see also notes from Hall et al. (2011)]. The protease inhibitors can be prepared with the stock concentration of 100 mM benzamidine (stored at -20 °C), 500 mM ϵ-aminocaproic acid, and 100 mM PMSF stored at 4 °C for several months [see also notes from Bouchnak et al. (2018)]. The protease inhibitors with final concentration of 1 mM benzamidine, 5 mM ϵ-aminocaproic acid, and 0.2 mM PMSF and/or 1× Roche cOmplete^TM protease inhibitor cocktail tablet must be added in all buffers right before use.

Recipes

(*) Added right before use

Extraction buffer
50 mM tricine-NaOH (pH 7.8)
330 mM sorbitol
1 mM EDTA
10 mM KCl
*0.15% (w/v) bovine serum albumin (traps fatty acids)
*4 mM sodium ascorbate (limits protein oxidation)
*7 mM L-cysteine (limits protein oxidation)
*Protease inhibitors
Resuspension buffer
50 mM sodium phosphate (pH 7.8)
330 mM sorbitol
10 mM sodium fluoride (NaF) (inhibits phosphatase)
*Protease inhibitors
Lysis buffer
10 mM sodium phosphate (pH 7.8)
5 mM MgCl₂
10 mM NaF
*Protease inhibitors
Washing buffer
50 mM sodium phosphate (pH 7.8)
100 mM sorbitol
10 mM NaF
5 mM MgCl₂
*Protease inhibitors
4× sample loading buffer for SDS-PAGE
8% (w/v) SDS
20% (w/v) 2-mercaptoethanol
40% (v/v) glycerol
0.008% (w/v) bromophenol blue
25 mM Tris-HCl (pH 6.8)
Running buffer for SDS-PAGE
25 mM Tris base
192 mM glycine
0.1% (w/v) SDS
Transfer buffer for immunodetection
25 mM Tris base
192 mM glycine
20% (v/v) ethanol
0.0375% (w/v) SDS
Tris-buffered saline with 0.1% Tween 20 (TBST) buffer
20 mM Tris-HCl (pH 7.5)
150 mM NaCl
0.1% (v/v) Tween 20
Blocking buffer
5% (w/v) non-fat dried milk in TBST
Coomassie blue stain
0.1% (w/v) Coomassie blue R-250
40% (v/v) ethanol
10% (v/v) acetic acid
50% (v/v) Milli Q water
Coomassie blue destain
30% (v/v) ethanol
10% (v/v) acetic acid
60% (v/v) Milli Q water
1 M Tris-HCl stock solution (6.8 and 7.5, 500 mL)
1. Mix 60.57 g of Tris base with 400 mL of Milli Q water
2. Adjust the pH to 6.8 or 7.5 by adding concentrated HCl
3. Add Milli Q water until final volume is 500 mL
  Note: The stock solution is used to prepare 25 mM and 120 mM Tris-HCl (pH 6.8) or 20 mM Tris-HCl (pH 7.5)
100 mM Sodium phosphate stock solution (pH 7.8)
10.4 mM NaH₂PO₄
89.6 mM Na₂HPO₄
Note: The stock solution is used to prepare 10 mM and 50 mM sodium phosphate (pH 7.8).

Acknowledgments

This protocol was adapted from Peltier et al. (2000) and Levesque-Tremblay et al. (2009). We thank W.P. Schroder for help with lumen preparation using the Yeda press method and critical reading of this protocol. This work was supported by a starting grant to A.M. from the Swedish Research Council Vetenskapsrådet (2018-04150), European Commission Marie Skłodowska-Curie Actions Individual Fellowship Reintegration Panel (845687) to A.M., by a consortium grant from the Swedish Foundation for Strategic Research (ARC19-0051) and by grants to UPSC from the Knut and Alice Wallenberg Foundation (2016.0341 and 2016.0352), and the Swedish Governmental Agency for Innovation Systems (2016-00504).

Competing interests

The authors declare that there is no conflict of interest.

Ethics considerations

The material used in this protocol is Arabidopsis thaliana.

References

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