Materials

1. AT3 medium: Murashige and Skoog medium supplemented with 3% sucrose, 0.5 mg/L NAA and 0.05 mg/L kinetin, pH 5.5.

2. Murashige and Skoog media (Sigma, UK, cat. no. M5515).

3. Extraction buffer: 50 mM Tris-HCl, pH 7.5, with complete protease inhibitor cocktail.

4. Equilibration solution: 50 mM Tris-HCl, pH 8.8; 6 M urea; 30% glycerol; 2% sodium dodecyl sulfate (SDS); 0.025% bromophenol blue.

5. Protease inhibitor cocktail tablets (Roche).

6. 5'-iodoacetamide fluorescein (5'-IAF) made as a stock solution fresh (for example, 10 mM stock in dimethyl sulfoxide).

7. Two-dimensional electrophoresis solutions and reagents: all PlusOne reagents (GE Healthcare, UK).

8. H2O2 solution (30% v/v; Sigma-Aldrich Co., Ltd., Dorset UK).

9. Stomatal bioassay buffer: 10 mM MES; 5 mM KCl; 100 pM CaCl2, pH 6.15; with 1 M Tris-HCl, pH 7.5.

10. Levington's F2 compost with sand.

11. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assay buffer: 100 mM Tris-HCl, pH 8; 10 mM MgCl2; 0.2 mM NADH; 5 mM ATP; 2 mM phosphoglyceric acid.

12. Isolated GAPDH enzyme, for example, from rabbit muscle (Sigma).

13. MES/KCl buffer: 5 mM KCl, 10 mM MES, 50 pM CaCl2, pH 6.15.

14. Seed sterilizing solution: 10% bleach, 0.1% SDS.

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