Materials and Reagents

Rosette leaves of Arabidopsis (Columbia, 3 weeks)
AlCl₃ (Sigma-Aldrich, catalog number: 237051 )
CaCl₂ (Sigma-Aldrich, catalog number: C7902 )
HCl (Sigma-Aldrich, catalog number: 258148 )
FDA (Sigma-Aldrich, catalog number: 596-09-8 )
TRI reagent (Sigma-Aldrich, catalog number: 93289 )
Cellulase R10 (Yakult Honsha, catalog number: C6260 )
Macerozyme R10 (Yakult Honsha, catalog number: 8032-75-1 )
Mannitol (Sigma-Aldrich, catalog number: M4125 )
MES (Sigma-Aldrich, catalog number: M8250 )
KCl (Sigma-Aldrich, catalog number: P3911 )
Bovine serum albumin (Sigma-Aldrich, catalog number: A-6793 )
NaCl (Sigma-Aldrich, catalog number: S6150 )
Glucose (Sigma-Aldrich, catalog number: G7528 )
Trition X-100 (Sigma-Aldrich, catalog number: T-8787 )
Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: P7626 )
Tris (Sigma-Aldrich, catalog number: T1378 )
SDS (Sigma-Aldrich, catalog number: L6026 )
(NH₄)₂S₂O₈ (Sigma-Aldrich, catalog number: A3426 )
TEMED (Sigma-Aldrich, catalog number: T9281 )
Tween-20 (Sigma-Aldrich, catalog number: P9416 )
SuperScript II first-strand synthesis system (Life Technologies, Invitrogen^TM, catalog number: 11904-018 )
SYBR Premix Ex Taq (Takara, catalog number: RR420A )
Bio-Rad protein assay kit (Bio-Rad Laboratories, catalog number: 500-0001 )
Anti-AOX antibody (Agrisera, catalog number: AS10699 )
Anti-Rubisco antibody (Agrisera, catalog number: AS03037 )
Anti-rabbit IgG (Dylight^TM 800 4x PEG Conjugate) secondary antibody (CST, catalog number: 5151 )
Anti-mouse IgG (Dylight^TM 800 Conjugate) secondary antibody (CST, catalog number: 5257 )
Enzyme solution (see Recipes)
W5 solution (see Recipes)
5 mM AlCl₃ solution in Ca medium
Real time PCR reaction solution (see Recipes)
Lysis buffer (see Recipes)
10 ml separating gel (see Recipes)
6 ml stacking gels (see Recipes)
TBST (see Recipes)

Equipment

pH meter
Thermal Cycler (Roche, model: Light cycler 2.0 )
Confocal laser-scanning microscope (Carl-Zeiss, model: LSM510/ConfoCor2 )
Auto microplate reader (Tecan Trading AG, model: infinite M200 )
Two-color infrared imaging system (Odyssey, model: 9120 )
Centrifuge
Orbital shaker
PVDF membranes (Bio-Rad Laboratories, catalog number: 162-0177 )
Nylon mesh
96-well plates
1.5 ml microcentrifuge tube
Generic razor blade
Vacuum pump (Vacuubrand, model: MZ 2C )
BioPhotometer (Eppendorf, model: AG 22331 )

Software

Image J 1.43 software

Procedure

Prepare for Arabidopsis protoplasts
1. Healthy rosette leaves from Arabidopsis thaliana (3 weeks) were sliced with a razor blade into small leaf strips (0.5-1 mm).
2. The leaves were covered with enzyme solution in a petri dish and placed in a vacuum chamber connected to a vacuum pump, and approx. 100 to 400 mmHg of vacuum was applied for 30 min. The leaves were then incubated in the dark for 3 h without shaking at room temperature.
3. The digested sample was filtrated through a 75 µm nylon mesh, the crude protoplast filtrates were sedimented by centrifugation for 3 min at 100 x g at room temperature.
4. The purified protoplasts were suspended in W5 solution.
Al treatment for Arabidopsis protoplasts
1. 10 μl AlCl₃ solution in Ca medium was added to 90 µl of protoplast solution in 96-well plates.
2. The protoplasts were incubated for 1 h at room temperature in darkness.
3. The protoplasts were incubated with 50 µM FDA for 5 min and Al-induced protoplast death was observed by confocal microscopy (Figure 1).
  
  Figure 1. Al-induced protoplast death. Viability of wild-type (WT) Arabidopsis protoplasts after 0.5 mM Al treatment. Protoplasts (2 x 10⁵/ml) were incubated with 50 µM FDA and observed with a confocal laser-scanning microscope.
RNA extraction and quantitative RT-PCR
1. Total RNA was extracted from Arabidopsis protoplasts according to the manufacturer’s instructions using TRI reagent.
2. The concentration of RNA was determined by measuring A₂₆₀ using BioPhotometer. 4 μg RNA was used for reverse transcription PCR.
3. First strand cDNA was synthesized with SuperScript II First strand synthesis system for qRT-PCR. Eppendorf BioPhotometer was used to determine the concentrations of cDNA.
4. The transcript of AOX genes were analyzed by quantitative RT-PCR using ACTIN2 as an endogenous control. The AOX1a gene was amplified with the primers 5’-ATGATGATAACTCGCGGTGGAGC-3’ and 5’-GCAACATTCAAAGAAAG CCGAATC-3'. PCR was carried out using 50 ng of cDNA and SYBR PCR Master Mix following the manufacturer’s protocol. For quantitative RT-PCR analyses, the Light Cycler 2.0 instrument was used to run the two-step program. PCR cycling conditions for amplification were 95 °C for 30 sec followed by 40 cycles of 95 °C for 5 sec, 55 °C for 30 sec.
5. Relative expression levels were calculated using the 2^(-∆∆Ct) analysis method.
  
  Figure 2. qRT-PCR analysis of the gene expression levels of AOX1a gene in Al-treated WT protoplasts
Protein extraction and western blot
1. The treated protoplasts were re-suspended in lysis buffer and incubated on ice with gentle shaking on a level shaker for 30 min.
2. The samples were centrifuged for 5 min at 1,2000 x g at 4 °C, and the supernatants were transferred to new 1.5 ml tubes.
3. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard (Bio-Rad protein assay kit).
4. Proteins extracts were separated by 12% sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE).
5. The gel was transferred to PVDF membranes and then electrophoresis for 45 min.
6. The membrane was blocked with TBST containing 5% non-fat milk for 1 h.
7. The membrane was incubated with anti-AOX antibody or anti-Rubisco antibody at 4 °C overnight.
8. The membrane was washed with TBST three times (about 10 min each time), and then incubated with secondary antibody [AOX: Anti-rabbit IgG (Dylight^TM800 4x PEG Conjugate) secondary antibody; Rubisco: Anti-mouse IgG (Dylight^TM800 Conjugate) secondary antibody] at room temperature for 2 h.
9. When the membrane was dried, detected by using Odyssey two-color infrared imaging system.
  
  Figure 3. Western blot analysis of the level of total AOX proteins in Al-treated WT protoplasts. Blots were probed with monoclonal anti-AOX antibody.
  
  Quantitative analysis was carried out using Image J 1.43 software (Figure 4). A step-by-step guide on how to use Image J for this purpose is shown in Supplementary data 1.
  
  Figure 4. Quantitative analysis of western blot. Quantitative analysis was carried out using Image J software.

Recipes

Enzyme solution
1%-1.5% (w/v) cellulose R10
0.2%-0.4% (w/v) macerozyme R10
0.4 M mannitol
20 mM MES
20 mM KCl
10 mM CaCl₂
0.1% (w/v) bovine serum albumin
pH 5.7
W5 solution
154 mM NaCl
125 mM CaCl₂
5 mM KCl
5 mM Glc
15 mM MES-KOH (pH 5.6)
5 mM AlCl₃ solution in Ca medium
5 mM AlCl₃
30 mM CaCl₂
pH 4.5
Real time PCR reaction solution
SYBR premix Ex Taq
10.0 µl
PCR forward primer (10 µM)
0.4 µl
PCR reverse primer (10 µM)
0.4 µl
cDNA
2.0 µl
ddH₂O
7.2 µl
Lysis buffer
15 mM NaCl
1% Triton X-100
100 mg/ml phenylmethylsulfonyl fluoride
50 mM Tris-HCl (pH 8.0)
10 ml separating gel
30% polyacrylamide
4.0 ml
1.5 M Tris-HCl (pH 8.8)
2.5 ml
10% SDS
100 µl
10% (NH₄)₂S₂O₈
100 µl
TEMED
10 µl
ddH₂O
3.29 ml
6 ml stacking gels
30% polyacrylamide
1.0 ml
1 M Tris-HCl (pH 6.8)
0.75 ml
10% SDS
60 µl
10% (NH₄)₂S₂O₈
60 µl
TEMED
6 µl
ddH₂O
4.124 ml
TBST
10 mM Tris-HCl (pH 7.4)
150 mM NaCl
0.1% Tween-20

Acknowledgments

This protocol was supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT0829), the Key Program of NSFC-Guangdong Joint Funds of China (U0931005) and the National High Technology Research and Development Program of China (863 Program) (2007AA10Z204). This protocol was adapted from previous work (Yamamoto et al., 2002; Ye et al., 2013; Sun et al., 2012).

References

Liu, J., Li, Z., Wang, Y. and Xing, D. (2014). Overexpression of ALTERNATIVE OXIDASE1a alleviates mitochondria-dependent programmed cell death induced by aluminium phytotoxicity in Arabidopsis. J Exp Bot 65(15): 4465-4478.
Li, Z. and Xing, D. (2011). Mechanistic study of mitochondria-dependent programmed cell death induced by aluminium phytotoxicity using fluorescence techniques. J Exp Bot 62(1): 331-343.
Sun, A., Nie, S. and Xing, D. (2012). Nitric oxide-mediated maintenance of redox homeostasis contributes to NPR1-dependent plant innate immunity triggered by lipopolysaccharides. Plant Physiol 160(2): 1081-1096.
Yamamoto, Y., Kobayashi, Y., Devi, S. R., Rikiishi, S. and Matsumoto, H. (2002). Aluminum toxicity is associated with mitochondrial dysfunction and the production of reactive oxygen species in plant cells. Plant Physiol 128(1): 63-72.
Ye, Y., Li, Z. and Xing, D. (2013). Nitric oxide promotes MPK6-mediated caspase-3-like activation in cadmium-induced Arabidopsis thaliana programmed cell death. Plant Cell Environ 36(1): 1-15.