Materials and Reagents

40 μm cell strainer (Corning, catalog number: 431750)
50 mL tubes (Sangon Biotech, catalog number: F602788)
Cellulase R10 (Yakult, catalog number: L0012)
Macerozyme R10 (Yakult, catalog number: L0021)
D-mannitol (Sigma-Aldrich, catalog number: M9647)
MES hydrate (Sigma-Aldrich, catalog number: M2933)
Tris-base (Thermo Fisher Scientific, catalog number: BP152-5)
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C5670)
Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266)
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333)
Percoll (GE Healthcare, catalog number: 17-0891-09)
HEPES (Sigma-Aldrich, catalog number: H3375)
EGTA (Sigma-Aldrich, catalog number: H3889)
Sucrose (Sigma-VETEC, catalog number: V900116)
K-gluconate (Sigma-Aldrich, catalog number: G4500)
D-sorbitol (Sigma-Aldrich, catalog number: S1876)
MnSO₄ (Sigma-Aldrich, catalog number: M7634)
HNO₃(GHTECH, catalog number: 1.14003.018)
H₂O₂(Sinopharm Chemical Reagent, catalog number: 10011208)
Murashige and Skoog (MS) base salts with vitamins (Phytotech Labs, catalog number: M519)
Agar (Sigma-Aldrich, catalog number: A1296)
KNO₃ (Sinopharm Chemical Reagent, catalog number: 10017218)
Ca(NO₃)₂·4H₂O (Sigma-Aldrich, catalog number: SC278601)
NH₄H₂PO₄ (Sinopharm Chemical Reagent, catalog number: 10002808)
MgSO₄·7H₂O (Sinopharm Chemical Reagent, catalog number: 10013018)
H₃BO₃ (Sinopharm Chemical Reagent, catalog number: 10004808)
MnCl₂·4H₂O (Sigma-Aldrich, catalog number: SM500501)
(NH₄)₆Mo₇O₂₄·4H₂O (Sinopharm Chemical Reagent, catalog number: 10002318)
ZnSO₄·7H₂O (Sinopharm Chemical Reagent, catalog number: 10024018)
CuSO₄·5H₂O (Sinopharm Chemical Reagent, catalog number: 10008218)
EDTA-Fe(III)Na (Biotopped, catalog number: Q0028-100g)
BSA (Sigma-Aldrich, catalog number: V900933)
β-mercaptoethanol (14.3 M) (Millipore, catalog number: 444203)
1/2 MS medium (see Recipes)
1/5 Hoagland solution (see Recipes)
Protoplasting solution (see Recipes)
Medium B (see Recipes)
Medium I (see Recipes)
Medium II (see Recipes)
Medium III (see Recipes)
Medium IV (see Recipes)
Medium V (see Recipes)
Medium VI (see Recipes)
Medium VII (see Recipes)
Medium VIII (see Recipes)

Equipment

Artificial illumination incubator (PERCIVAL, model: LT-36VL)
Horizontal centrifuge (Eppendorf, model: Centrifuge 5810R)
Dissolver (LabTech, model: DigiBlock ED54)
ICP-MS (Thermo Fisher Scientific, model: ICAP Qc)
Perfluoroalkoxy alkane (PFA) vessels (LabTech, model: GC-36-L)
Fuchs-Rosenthal hemacytometer (Marienfeld, model: Dark-Line 0650010)

Procedure

Protoplast preparation
1. Sterilize approximately 50 Arabidopsis seeds, plant on 1/2 MS medium, and then grow in an artificial illumination incubator under normal light conditions (100 µmol m^-2 s^-1) with a long-day cycle (16:8 h light/dark) at 22 °C. After seven days, clamp the seedlings with a small sponge and place them on a reticulated floating board; immerse the roots in 1/5 Hoagland solution and then grow in an artificial illumination incubator under normal light conditions (100 µmol m^-2 s^-1) with a short-day cycle (8:16 h light/dark) at 22 °C. Change the solution every three days. After seven weeks, transfer the seedlings to the same solution containing 240 μM MnSO₄ for another seven days.
2. Take 10–15 seedling roots and cut them into small pieces with a blade, depositing them into a 100 mL flask containing the protoplasting solution (see Recipes). Generally, 20 mL of protoplasting solution is used per 15 seedling roots.
3. Shake the flasks gently (75 rpm) at room temperature for 1 h. A longer incubation time may increase the protoplast yield.
4. Filter the protoplast solution with a 40 μm cell strainer and carefully pour into a 50 mL tube.

Vacuole extraction
1. In order to reduce the disruption of protoplasts, first add 5 mL of medium I to the bottom of the tube and then gently mix the filtered protoplasts. After centrifugation (200 × g, 10 min), discard the supernatant with a pipette to obtain sedimented protoplasts.
2. Slowly add 5 mL of medium I into the tube and gently mix with the sedimented protoplasts.
3. Using a pipette, add 5 mL of medium II to the tube against the wall, and then 5 mL of medium III. Each layer of liquid should be added slowly to ensure that the layers do not break apart. After adding medium III, the gradient is formed and can be clearly seen in the tube. After centrifugation (800 × g, 10 min), purified colorless protoplasts are obtained in the interfaces between medium I and II or between medium II and III. Discard the latter protoplasts (between medium II and III) with the pipette to avoid contamination of the vacuolar fractions with protoplasts.
4. Gently mix approximately 2 mL of the remaining purified protoplasts with 2 mL of medium B and incubate on ice for 5 min.
5. Gently mix the mixture of protoplasts, vacuoles, and lysate (from step B4) with 5 mL of medium IV first. Then, a gradient is formed by consecutive overlaying with 5 mL each of medium V, medium VI, medium VII, and medium VIII.
6. After centrifugation (800 × g, 10 min), discard medium VIII with a pipette. Carefully aspirate approximately 1 mL of liquid between medium VII and medium VIII as the purified vacuoles.

Elemental analysis
1. Determine the number of vacuoles using a Fuchs-Rosenthal hemacytometer. (Approximately 10 vacuoles per square millimeter of the Fuchs-Rosenthal hemacytometer, whose height is 0.1 mm through microscope observation; so, there are 100 vacuoles in each cubic millimeter of solution.) The total number of vacuoles in the solution is obtained by multiplying the number of vacuoles per cubic millimeter by the volume of the solution.
2. Digest the vacuoles in PFA vessels with 1.5 mL of HNO₃ (65%) and 0.6 mL of H₂O₂ (30%) for 15 min in the dissolver at 170 °C.
3. After cooling down to room temperature, transfer the digestion solution to a plastic volumetric flask and dilute to 5 mL with ultrapure water (18.25 MΩ cm⁻¹).
4. Determine Mn in the digestion solution using an inductively coupled plasma mass spectrometer (ICP-MS).

Data analysis

The vacuolar elemental concentration of each plant material requires three biological replicates to be measured. The mean value of three biological replicates was calculated and statistically analyzed (Student’s t-test). Detailed data and statistical methods could be found in the previously published articles (Figure 5B in Zhang et al., 2021 and Figure 4B in Ju et al., 2022).

Recipes

1/2 MS medium (adjust the pH to 5.7 using Tris)

Reagent Final concentration Amount
MS base salts with vitamins 0.222% 2.22 g
Sucrose 1% 10 g
Agar
ddH₂O
Total
1%
n/a
n/a
10 g
Up to 1 L
1 L

1/5 Hoagland Solution

Reagent

Final concentration

Amount

KNO₃

1 μM

0.51 g

Ca(NO₃)₂·4H₂O

1 μM

1.18 g

NH₄H₂PO₄

MgSO₄·7H₂O

H₃BO₃

MnCl₂·4H₂O

(NH₄)₆Mo₇O₂₄·4H₂O

ZnSO₄·7H₂O

CuSO₄·5H₂O

EDTA-Fe(III)Na

ddH₂O

Total

0.2 μM

0.4 μM

3 nM

0.5 nM

1 nM

0.4 nM

0.2 nM

20 nM

n/a

n/a

0.12 g

0.49 g

1.85 μg

0.99 μg

12.36 μg

1.15 μg

0.05 μg

8.42 μg

Up to 1 L

1 L

MES-Tris buffer

Reagent Final concentration Amount
MES hydrate 0.5 M 9.76 g
Tris-base
Adjust pH to 5.7
ddH₂O n/a Up to 100 mL
Total n/a 100 mL

Protoplasting solution

Reagent	Final concentration	Amount
Cellulase R10	1.25%	1.25 g
Macerozyme R10	0.3%	0.3 g
D-mannitol (0.8 M)	0.4 M	50 mL
MES-Tris buffer (0.5 M, pH 5.7)	20 mM	4 mL
KCl (1 M)	20 mM	2 mL
Heat the solution to 55 °C for 10 minutes and let it cool down to room temperature
BSA	0.1%	0.1 g
CaCl₂ (1 M)	10 mM	1 mL
β-mercaptoethanol (14.3 M)	5 mM	35 μL
ddH₂O	n/a	Up to 100 mL
Total	n/a	100 mL

Medium B

Reagent Final concentration Amount
HEPES-Tris (1 M, pH 7.2) 30 mM 3 mL
K-gluconate 30 mM 0.7 g
MgCI₂(1 M) 2 mM 0.2 mL
EGTA (0.5 M) 2 mM 0.4 mL
ddH₂O n/a Up to 100 mL
Total n/a 100 mL

Medium I

Reagent Final concentration Amount
Sucrose 400 mM 13.69 g
Percoll 50% 50 mL
Medium B n/a Up to 100 mL
Total n/a 100 mL

Medium II

Reagent Final concentration Amount
Sucrose 400 mM 13.69 g
Percoll 7.5% 7.5 mL
Medium B n/a Up to 100 mL
Total n/a 100 mL

Medium III

Reagent Final concentration Amount
D-sorbitol 400 mM 7.27 g
Medium B n/a Up to 100 mL
Total n/a 100 mL

Medium IV

Reagent Final concentration Amount
D-sorbitol 200 mM 3.64 g
Percoll 25% 25 mL
Medium B n/a Up to 100 mL
Total n/a 100 mL

Medium V

Reagent Final concentration Amount
Sucrose 200 mM 6.85 g
Percoll 7.5% 7.5 mL
Medium B n/a Up to 100 mL
Total n/a 100 mL

Medium VI

Reagent Final concentration Amount
Sucrose 200 mM 6.85 g
Percoll 5% 5 mL
Medium B n/a Up to 100 mL
Total n/a 100 mL

Medium VII

Reagent Final concentration Amount
Sucrose 200 mM 6.85 g
Percoll 2.5% 2.5 mL
Medium B n/a Up to 100 mL
Total n/a 100 mL

Medium VIII

Reagent Final concentration Amount
Sucrose 200 mM 6.85 g
Medium B n/a Up to 100 mL
Total n/a 100 mL

Acknowledgments

This research was funded by a grant from the National Natural Science Foundation of China (31770289 to C.W.), Northwest A&F University (Z111021604 to C.W.), and the National Natural Science Foundation of China (31900236 to Z.Z.).

The initial publication on preparation of the plant material is published in Eroglu et al. (2016); the initial publication on protoplast preparation of the plant material is published on Bargmann et al. (2010); the initial publication on vacuole extraction is published on Shimaoka et al. (2004).

Competing interests

Non-financial competing interests on behalf of all authors.

References

Bargmann, B. O. and Birnbaum, K. D. (2010). Fluorescence activated cell sorting of plant protoplasts. J vis exp (36).
Eroglu, S., Meier, B., von Wirén, N. and Peiter, E. (2016). The vacuolar manganese transporter MTP8 determines tolerance to iron deficiency-induced chlorosis in Arabidopsis. Plant Physiol 170(2): 1030-1045.
Ju, C., Zhang, Z., Deng, J., Miao, C., Wang, Z., Wallrad, L., Javed, L., Fu, D., Zhang, T., Kudla, J., Gong, Z. and Wang, C. (2022). Ca²⁺-dependent successive phosphorylation of vacuolar transporter MTP8 by CBL2/3-CIPK3/9/26 and CPK5 is critical for manganese homeostasis in Arabidopsis. Mol Plant 15(3): 419-437.
Martinoia, E., Schramm, M. J., Kaiser, G., Kaiser, W. M. and Heber, U. (1986). Transport of anions in isolated barley vacuoles : I. Permeability to anions and evidence for a cl-uptake system. Plant Physiol 80(4): 895-901.
Massonneau, A., Martinoia, E., Dietz, K. J. and Mimura, T. (2000). Phosphate uptake across the tonoplast of intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don. Planta 211(3): 390-395.
Shimaoka, T., Ohnishi, M., Sazuka, T., Mitsuhashi, N., Hara-Nishimura, I., Shimazaki, K., Maeshima, M., Yokota, A., Tomizawa, K. and Mimura, T. (2004). Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana. Plant Cell Physiol 45(6): 672-683.
Zhang, Z., Fu, D., Sun, Z., Ju, C., Miao, C., Wang, Z., Xie, D., Ma, L., Gong, Z. and Wang, C. (2021). Tonoplast-associated calcium signaling regulates manganese homeostasis in Arabidopsis. Mol Plant 14(5): 805-819.