Cyclohexane Diamine Tetraacetic Acid (CDTA) Extraction of Plant Cell Wall Pectin   

Materials and Reagents

1. Plant material or cell wall preparation of choice (see Note 1)
   
2. Cyclohexane diamine tetraacetic acid (CDTA) (Fluka, catalog number: 34588 )
   
3. Tris-base (Sigma-Aldrich, catalog number: T6066 )
   
4. Sodium hydroxide (Thermo Fisher Scientific, catalog number: S318 )
   
5. Liquid nitrogen
   
6. CDTA extraction buffer (see Recipes)
   

Equipment

1. Liquid nitrogen container
   
2. Paint shaker (e.g. Harbil, model: 5G-HD) and ball bearings (e.g. 3 mm diameter steel beads) (mortar and pestle or bead beater)
   
3. Media bottle
   
4. Single-channel pipettor
   
5. Microfuge tubes appropriate for use at 95 °C and for freezing in liquid N₂ (Thermo Fisher Scientific, catalog number: 02-707-355 )
   Note: We use 2 ml screw cap tubes.
   
6. Pipette tips
   
7. Water bath or incubator
   
8. Vortex mixer
   
9. Microcentrifuge capable of spinning at 10,000 x g
   
10. Optional: Screw cap tubes or lid locks
    

Procedure

1. Pre-heat water bath or incubator to 95 °C and pre-heat CDTA extraction buffer.
   
2. Collect plant tissue in tubes - we use leaf material from approx. 4 week old Arabidopsis thaliana plants. Three fully expanded Arabidopsis leaves weigh about 250 mg.
   
3. Freeze tubes containing the tissue in liquid nitrogen (tissue can be stored at -80 °C).
   
4. Pulverize frozen tissue using ball bearings and a paint shaker, mortar and pestle or a bead beater. The method used to pulverize tissue is not critical. Keep tissue frozen. If paint shaker is used three cycles of three minutes each are generally sufficient to pulverize tissue.
   
5. Add 1 ml of CDTA extraction buffer to 250 mg of frozen pulverized Arabidopsis tissue.
   
6. Place tubes at 95 °C. Use caution, the liquid will become very hot and lids may open; therefore use screw cap tubes or lid locks to secure lids.
   
7. Incubate for 15 min, vortex every 5 min.
   
8. Centrifuge at 10,000 x g for 10 min at room temperature.
   
9. The supernatant contains the pectin.
   
10. Supernatant can be frozen and freeze dried for downstream applications.
    

Notes

1. We directly use pulverized Arabidopsis leaf material (Bethke et al., 2014). Alternatively crude cell wall preparations like alcohol insoluble residue (Gille et al., 2009) preparations etc. can be used (pre-heat the water bath and extraction buffer and proceed to step 5).
   
2. Different groups have reported different extraction times and temperatures e.g. 15 min at 95 °C (Siedlecka et al., 2008) or 4 h at room temperature (Moller et al., 2008). In our experience about twice the amount of pectin could be extracted when extraction was performed for 15 min at 95 °C as compared to the longer extraction at room temperature.
   
3. Different groups have utilized similar protocols for the extraction of pectin from various monocotyledonous and dicotyledonous plants e.g. cucumber, tomato, celery (Jarvis et al., 1982), poplar (Siedlecka et al., 2008), soybean (Huisman et al., 2001) or wheat (Wiethölter et al., 2003).
   
4. The extraction buffer can be stored at room temperature for several weeks.
   
5. Pectin extracted using this procedure can be used for various downstream application including dot-blot analysis with pectin specific antibodies, sugar analysis, ion-exchange chromatography or determination of degree of esterification of pectin (Bethke et al., 2014; MacDougall et al., 1997).
   

Recipes

1. CDTA extraction buffer
   50 mM CDTA
   50 mM Tris-base
   Adjust pH to 7.2 using sodium hydroxide
   

Acknowledgments

This work was funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-FG02-05ER15670 to J.G.
Many labs have used similar protocols in the past. We have adapted this protocol from Siedlecka et al. (2008).

References

1. Bethke, G., Grundman, R. E., Sreekanta, S., Truman, W., Katagiri, F. and Glazebrook, J. (2014). Arabidopsis PECTIN METHYLESTERASEs contribute to immunity against Pseudomonas syringae. Plant Physiol 164(2): 1093-1107.
   
2. Gille, S., Hansel, U., Ziemann, M. and Pauly, M. (2009). Identification of plant cell wall mutants by means of a forward chemical genetic approach using hydrolases. Proc Natl Acad Sci U S A 106(34): 14699-14704.
   
3. Harholt, J., Suttangkakul, A. and Vibe Scheller, H. (2010). Biosynthesis of pectin. Plant Physiol 153(2): 384-395.
   
4. Huisman, M. M. H., Fransen, C. T. M., Kamerling, J. P., Vliegenthart, J. F. G., Schols, H. A. and Voragen, A. G. J. (2001). The CDTA-soluble pectic substances from soybean meal are composed of rhamnogalacturonan and xylogalacturonan but not homogalacturonan. Biopolymers 58(3): 279-294.
   
5. Jarvis, M. C. (1982). The proportion of calcium-bound pectin in plant cell walls. Planta 154(4): 344-346.
   
6. MacDougall, A. J., Rigby, N. M., Ring, S. G. (1997). Phase separation of plant cell wall polysaccharides and its implications for cell wall assembly. Plant Physiol 114(1): 353-362.
   
7. Mohnen, D. (2008). Pectin structure and biosynthesis. Curr Opin Plant Biol 11(3): 266-277.
   
8. Moller, I., Marcus, S. E., Haeger, A., Verhertbruggen, Y., Verhoef, R., Schols, H., Ulvskov, P., Mikkelsen, J. D., Knox, J. P. and Willats, W. (2008). High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles. Glycoconj J 25(1): 37-48.
   
9. Siedlecka, A., Wiklund, S., Peronne, M. A., Micheli, F., Lesniewska, J., Sethson, I., Edlund, U., Richard, L., Sundberg, B. and Mellerowicz, E. J. (2008). Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus. Plant Physiol 146(2): 554-565.
   
10. Wiethölter, N., Graeβner, B., Mierau, M., Willats, W. G. T., Knox, J. P., Moerschbacher, B. M. (2003). Isolation and characterisation of the homogalacturonan from type II cell walls of the commelinoid monocot wheat using HF-solvolysis. Carbohydrate Res 338(5):423-431.
    

1. Bethke, G. and Glazebrook, J. (2014). Cyclohexane Diamine Tetraacetic Acid (CDTA) Extraction of Plant Cell Wall Pectin. Bio-protocol 4(24): e1357. DOI: 10.21769/BioProtoc.1357.
   
2. Bethke, G., Grundman, R. E., Sreekanta, S., Truman, W., Katagiri, F. and Glazebrook, J. (2014). Arabidopsis PECTIN METHYLESTERASEs contribute to immunity against Pseudomonas syringae. Plant Physiol 164(2): 1093-1107.