Materials and Reagents

10-cm-diameter Petri dishes
Arabidopsis thaliana ecotype Columbia seeds (originally obtained from NASC, Nottingham Arabidopsis Stock Center)
Sweet green pepper fruits were provided by Syngenta Seeds S.A. (El Ejido, Spain)
70% (v/v) ethanol
0.1% (w/v) SDS
Commercial Bleach
Murashige and Skoog medium (Sigma-Aldrich, catalog number: M5524 )
Phyto-agar (Duchefa Biochemie, catalog number: P1001 )
Sucrose (Sigma-Aldrich, catalog number: 84097 )
Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 5000006 )
Bovine serum albumin (BSA) Fraction V (Roche Diagnostics, catalog number: 10735078001 )
Tris (AMRESCO, catalog number: 0497 )
Ethylenediaminetetraacetic acid, disodium salt, dihydrate (Na₂-EDTA) (Sigma-Aldrich, catalog number: E5134 )
Triton X-100 (AMRESCO, catalog number: 0694 )
Glycerol (AMRESCO, catalog number: E520 )
Dithiothreitol (DTT) (Roche Diagnostics, catalog number: 10708984001 )
30% acrylamide/Bis solution, 19:1 (Bio-Rad Laboratories, catalog number: 1610154 )
4x gel buffer
Glycine (AMRESCO, catalog number: 0167 )
Ammonium persulfate
TEMED
β-Nicotinamide adenine dinucleotide phosphate, sodium salt, hydrate (NADP) (Sigma-Aldrich, catalog number: N0505 )
Magnesium chloride, hexahydrate (MgCl₂·7H₂O) (EMD Millipore, catalog number: 442615 )
Nitroblue tetrazolium (NBT) (AMRESCO, catalog number: 0329-1G )
Phenazine methosulfate (PMS) (Sigma-Aldrich, catalog number: P9625 )
6-Phosphogluconic acid, trisodium salt (6PG) (Sigma-Aldrich, catalog number: P7877 )
Glacial acetic acid (Fisher Scientific)
Grinding buffer (see Recipes)
Non-denaturing polyacrylamide gel electrophoresis (PAGE) on 6% acrylamide gel (see Recipes)
Staining solution (see Recipes)
Stop solution (see Recipes)

Equipment

Plant Growth cabinet (Panasonic Biomedical, model: MLR-352-PE )
Porcelain mortar and pestle
Refrigerated centrifuge
Vertical Slab gels Electrophoresis System (Bio-Rad Laboratories, model: Mini-PROTEAN^® Electrophoresis Cell )

Software

ImageJ program (https://imagej.nih.gov/ij/)

Procedure

Plant extracts
1. Surface-sterilize Arabidopsis thaliana ecotype Columbia seeds for 5 min in 70% (v/v) ethanol containing 0.1% (w/v) SDS at room temperature and with a slight shaking. Then, place for 20 min in sterile water containing 20% (w/v) commercial Bleach and 0.1% (w/v) SDS and then wash four times in sterile water.
2. Grow seeds on Petri plates over commercial Murashige and Skoog medium (Sigma-Aldrich) with a pH of 5.5, containing 1% (w/v) sucrose and 0.8% (w/v) phyto-agar (Corpas and Barroso, 2014). Then, keep the seedlings for 14 days with a photoperiod of 16 h (light intensity of 100 μE m^-2 sec^-1) and 8 h dark, with a temperature of 22 °C and 18 °C, respectively.
3. Sweet green pepper fruits were provided by Syngenta Seeds S.A. (El Ejido, Spain) from plants grown in experimental greenhouses.
4. Homogenize plant samples in a mortar and pestle with grinding buffer (see Recipes) in a ratio 1:3 (w/v) for Arabidopsis and ratio 1:1 for pepper fruits. Perform these operations at 0-4 °C (on ice).
5. Centrifuge extracts at 27,000 x g at 4 °C for 20 min.
6. Use the supernatants for the enzymatic assays. Determine protein concentration with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as standard.
6PGDH activity staining
1. Separate plant proteins by non-denaturing polyacrylamide gel electrophoresis (PAGE) on 6% acrylamide gels at 4-7 °C. Usually, it takes around 2 h and 30 min.
2. After electrophoresis, incubate gels in staining solution (see Recipes) at room temperature (RT). Keep in the dark with shaking until blue-purple bands appear over a colorless background. It takes around 15 min (Figure 1).
3. Remove the staining solution and add stop solution (see Recipes) to cover gels.
  
  Figure 1. Analysis of 6-phosphogluconate dehydrogenase (6PGDH) isoforms by non-denaturing gel electrophoresis. A. 6PGDH isoforms activity were separated by native PAGE (6%) and identified by activity staining. Lane 1, Arabidopsis thaliana 14-day-old seedlings. Lane 2, sweet green pepper fruits. B. Densitometric scans of 6PGDH isoforms and its relative quantification (%) made by the ImageJ program.
Note: It is recommended to use fresh vegetable extracts because they provide better defined bands of activity.

Data analysis

Quantification of the activity of each 6PGDH isoform can be done by densitometric analysis (Figure 1B), for example, using the ImageJ program.

Recipes

Grinding buffer
50 mM Tris-HCl, pH 7.8 containing 0.1 mM EDTA
0.2% (v/v) Triton X-100
10% (v/v) glycerol
2 mM DTT
Non-denaturing polyacrylamide gel electrophoresis (PAGE) on 6% acrylamide gel
30% acrylamide/Bis solution, 19:1
4x gel buffer (1.5 M Tris-HCl, pH 8.9)
Electrode buffer (38 mM glycine plus 5 mM Tris, pH 8.2)
10% (w/v) ammonium persulfate (AP)
TEMED
The follow table shows the needed volume of each reagent to prepare two 1.5 mm thick gels to be used in a Bio-Rad Mini-PROTEAN Electrophoresis System

*Note: Added right before each use. The mix of the reagents should be done at 4 °C (on ice).

The standard running conditions are 15 mA per gel for 30 min and then increase to 25 mA per gel for approximately 90 min. The electrophoresis should be run in a cold room (5-7 °C)
Staining solution
50 mM Tris-HCl, pH 7.6 containing 0.8 mM NADP
5 mM EDTA
2 mM MgCl₂
0.24 mM NBT
65 mM PMS
10 mM 6PG
Note: Freshly prepared before use and protected from the light.
Stop solution
7% (v/v) acetic acid

Acknowledgments

This work has been supported by the ERDF-cofinanced grant AGL2015-65104-P from the Ministry of Economy and Competitiveness, Spain.

References

Corpas, F. J. and Barroso, J. B. (2014). Peroxynitrite (ONOO^-) is endogenously produced in Arabidopsis peroxisomes and is overproduced under cadmium stress. Ann Bot 113(1): 87-96.
Corpas, F. J., Barroso, J. B., Sandalio, L. M., Distefano, S., Palma, J. M., Lupiáñez, J. A. and del Río, L. A. (1998). A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes. Biochem J 330 (Pt 2): 777-784.
Fernández-Fernández, A. D. and Corpas, F. J. (2016). In silico analysis of Arabidopsis thaliana peroxisomal 6-phosphogluconate dehydrogenase. Scientifica (Cairo) 2016: 3482760.
Hölscher, C., Lutterbey, M. C., Lansing, H., Meyer, T., Fischer, K. and von Schaewen, A. (2016). Defects in peroxisomal 6-phosphogluconate dehydrogenase isoform PGD2 prevent gametophytic interaction in Arabidopsis thaliana. Plant Physiol 171(1): 192-205.
Krepinsky, K., Plaumann, M., Martin, W. and Schnarrenberger, C. (2001). Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach. Cyanobacterial genes for chloroplast and cytosolic isoenzymes encoded in eukaryotic chromosomes. Eur J Biochem 268(9): 2678-2686.
Mateos, R. M., Bonilla-Valverde, D., del Río, L. A., Palma, J. M. and Corpas, F. J. (2009). NADP-dehydrogenases from pepper fruits: effect of maturation. Physiol Plant 135(2): 130-139.