Notes

1. Sterilize flasks by baking them at 200°C for 2 h.

2. The time of H2 O2 treatment is determined empirically for different tissue types. The time mentioned here has been tested for cell cultures.

3. Volume of buffer to sample: 1.5-mL buffer: 3 g of fresh weight tissue, calculated by aspirating all media from cell cultures and weighing wet weight of cells in Falcon tube.

4. If for some reason the protein does not digest into peptides easily using trypsin, then alternative enzymes can be used (e.g., chymotrypsin).

5. For TCA/acetone precipitation, add ice cold 100% TCA to make a final concentration of10% (v/v). Chill on ice for 15 min and centrifuge the sample for 20 min to remove the supernatant. Wash the pellet with 1 ml of 100% acetone, vortex well and re-centrifuge for 10 min. Repeat this process twice to wash the pellet. Air-dry the pellet and resuspend in the required buffer.

6. Soluble protein extract is obtained by homogenising the cells, followed by centrifugation at 12,000x g for 10 min to obtain soluble proteins.

7. The activity of GAPDH can be followed by the oxidation of NADH as it is a stoichiometric reaction.

8. To confirm that the inhibition of GAPDH is by affects on the enzyme itself, rather than on the phosphoglyceric phosphokinase (PPK), the assay can be carried out in the reverse direction, using glyceraldehyde 3-phosphate as the substrate and following the reduction of NADH instead of its oxidation.

9. Over-wetting should be avoided.

10. It is very important to have the lights of the appropriate intensity as this vastly affects the growth of the plants.

11. Overwatering of plants must be avoided.

12. If the plants start flowering do not use them for the stomatal assays.

13. For getting accurate concentrations of H2O2 in each plate, make a 1,000x stock of the required concentration of H2O2, and dilute this in 50 ml of medium in Falcon tubes, enough to pour two plates. Repeat with fresh tubes for each dose.

14. The amount of seeds in a microfuge tube which comes up to the 0.1 ml mark is approximately 300-500 seeds.

References

1. Cooper, C., Patel, R. P., Brookes, P. S., and Darley-Usmar, V. M. (2002) Nanotransduc-ers in cellular redox signalling: modification of thiols by reactive oxygen and reactive nitrogen species. Trends Biochem Sci 27, 489-492.

2. Hancock, J. T., Henson, D., Nyirenda, M., Desikan, R., Harrison, J., Lewis, M., Hughes, J., and Neill, S. J. (2005) Proteomic identification of glyceraldehyde 3-phos-phate dehydrogenase as an inhibitory target of hydrogen peroxide in Arabidopsis. Plant Physiol Biochem. 43, 828-835.

3. Desikan, R., Hancock, J. T., Bright, J., Harrison, J., Weir, I., Hooley, R., and Neill, S. J. (2005) A novel role for ETR1: hydrogen peroxide signalling in stomatal guard cells. Plant Physiol 137, 831-834.

4. Pei, Z.-M., Murata, Y., Benning, G., Thom-ine, S., Klusener, B., Allen, G. J., Grill, E., and Schroeder, J. I. (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406, 731-734.

5. Foreman, J., Denidchik, V., Bothwell, J. H.F., Mylona, P., Miedema, H., Torres, M. A., Instead, P., Costa, S., Brownlee, C., Jones, J. D.G., Davies, J. M., and Dolan, L. (2003) Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422, 442-446.

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