Pectin Methylesterase Activity Assay for Plant Material

Materials and Reagents

1. Arabidopsis thaliana plant or seed materials
   
2. Liquid nitrogen
   
3. 100 mM sodium phosphate buffer (pH 7.5)
   
4. 0.5% (w/v) Pectin (in dH₂O) (Sigma-Aldrich, catalog number: P-9135 )
   
5. 0.1 U/μl Alcohol oxidase (in 100 mM phosphate buffer) (pH 7.5) (Sigma-Aldrich, catalog number: A2404 )
   
6. 0.5 U/μl Formaldehyde dehydrogenase (in 100 mM phosphate buffer) (pH 7.5) (Sigma-Aldrich, catalog number: F1879 )
   
7. 0.4 mM NAD+ (in 100 mM phosphate buffer) (pH 7.5) (Sigma-Aldrich, catalog number: N8410 )
   
8. PME from orange peel (Sigma-Aldrich, catalog number: P5400 )
   
9. Protease inhibitor cocktail (1x) (contains 100 mM PMSF, 2 mM Bestatin, 0.3 mM Pepstatin A, and 0.3 mM E-64) (abmGood, catalog number: G135 )
   
10. Protein extraction buffer (see Recipes)
    
11. Master mix (see Recipes)
    

Equipment

1. Eppendorf tubes
   
2. Mortar and pestle
   
3. Vortexer
   
4. Centrifuge with cooling function
   
5. 96 well microplates
   
6. Microplate reader
   

Procedure

1. Plant or seed tissue (Arabidopsis thaliana) is weighed. Use about 100 mg per extraction.
   
2. Tissues are ground to a fine powder in liquid nitrogen using a mortar and pestle. The tissue must be kept frozen during grinding.
   
3. Twice the fresh weight (w/v) of extraction buffer is added to the powder, and the powder allowed to thaw in the buffer.
   
4. Vortex for 10 sec.
   
5. Extracts are rotated at 4 °C for 30 min and centrifuged at 11,500 x g at 4 °C for 20 min.
   
6. The supernatant is the soluble protein extract. Use fresh supernatants immediately for the PME enzyme assay, as the activity can be affected by freezing.
   
7. Four replicates of each sample (10 μl each) are pipetted into microplate wells. For the negative control, use protein extraction buffer only. For a positive control, use a solution of commercially available PME in protein extraction buffer.
   
8. Master mix (180 μl) is added to each well and mixed by pipetting up and down. Avoid the formation of bubbles.
   
9. To start the reaction, add 10 μl of the pectin solution to the samples, the negative and the positive control, but not to the background controls. Mix well by pipetting up and down.
   
10. Immediately put the plate into the microplate reader. If bubbles have formed during the mixing process, shake plate for 5 sec. Record the changes in absorption at 340 nm over 15 min at room temperature.
    
11. The change in absorption per unit time over the linear part of the reaction is calculated for each well, and used to calculate the increase in concentration of NADH. The NADH concentration is calculated using Lambert-Beer's law with the extincion coefficient ε₃₄₀ for NADH (6,220 M^-1cm^-1). One nkat PME activity is defined as 1 nmol NADH formed per second.
    
12. The activities of the triplicates are averaged.
    

Recipes

1. Protein extraction buffer
   100 mM Tris-HCl (pH 7.5)
   500 mM NaCl
   1x protease inhibitor cocktail
   
2. Master Mix (per sample)
   20 μl pectin solution
   2 μl alcohol oxidase solution
   2 μl formaldehyde dehydrogenase solution
   156 μl NAD+ solution
   

Acknowledgments

This work was supported by the Swiss National Science Foundation (grant 31003A_127563; to TB) and by stipends to SB from the European Molecular Biology Organisation (EMBO: ALTF 61-2010) and the Leopoldina Fellowship Programme of the National Academy of Science Leopoldina (LPDS 2009-35).

References

1. Grsic-Rausch, S. and Rausch, T. (2004). A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors. Anal Biochem 333(1): 14-18.
   
2. Mueller, K., Levesque-Tremblay, G., Bartels, S., Weitbrecht, K., Wormit, A., Usadel, B., Haughn, G. and Kermode, A. R. (2013). Demethylesterification of cell wall pectins in Arabidopsis plays a role in seed germination. Plant Physiol 161(1): 305-316.
   
3. Wolf, S., Mouille, G. and Pelloux, J. (2009). Homogalacturonan methyl-esterification and plant development. Mol Plant 2(5): 851-860.