Determination of Cell wall mass and nitrogen concentration from plant leaf tissue.

Protocol

Authors

Stephanie McCaffery, John Evans

Background

This protocol has been adapted from: Harrison M.T., Edwards E.J., Farquhar, G.D., Nicotra, A.B. and Evans J.R. (2009)in Plant, Cell and Environment 32, 259-270

Materials/Equipment

  • finely ground freeze-dried leaf material
  • buffer containing 50 mM Tricine, pH 8.1, 1% PVP40.
  • 2ml round bottom Eppendorf tube
  • ball bearing (type 316 stainless steel 5/32″ #BNMX-21/2)
  • vortex
  • centrifuge
  • buffer (50 mM Tricine, pH 8.1, 1% SDS)
  • drying oven at 90∘C and 80∘C
  • 0.2 M KOH
  • deionised water
  • ethanol
  • forceps
  • elemental analyser

Units, terms, definitions

supernatant

eppendorf tube

deionised water – DI

elemental analyser

Procedure

  1. Extract approximately 10 mg of finely ground freeze-dried leaf material in 1.5 mL of a buffer containing 50 mM Tricine, pH 8.1, 1% PVP40.
  2. Carry out the extraction in a preweighed 2ml round bottom Eppendorf tube, containing a ball bearing (type 316 stainless steel 5/32″ #BNMX-21/2).
  3. Thoroughly vortex the sample, then centrifuge it at 12 000xg for 5 min (Eppendorf AG 5424, Hamburg, Germany) before carefully removing the supernatant.
  4. Resuspend the pellet in buffer (50 mM Tricine, pH 8.1, 1% SDS), incubate at 90∘C for 5 min, then centrifuge at 12 000 g for 5 min.
  5. Following centrifugation remove the supernatant and repeat step (4).
  6. Wash the pellet (as step 3) twice with 0.2 M KOH, twice with deionised water and finally twice with ethanol. The ball bearing should remain in the tube throughout the washing steps.
  7. During the final ethanol wash, hold the ball bearing with forceps and thoroughly wash any plant material into the tube. Repeat this step if any material remains attached to the ball bearing.
  8. The tube containing the final pellet is then oven-dried at 80∘C and the pellet mass determined by weighing and subtracting the preweighed tube mass.
  9. The remaining dry mass of pellet is assumed to represent the leaf structural biomass, and the N content is determined on 2-5 mg of material using an elemental analyser.

Eppendorf, Hamburg, Germany

Literature references

Harrison M.T., Edwards E.J., Farquhar, G.D., Nicotra, A.B. and Evans J.R. (2009) Determination of Cell wall mass and nitrogen concentration from plant leaf tissue. Plant, Cell and Environment 32, 259-270

Health, safety & hazardous waste disposal considerations

RISK CATEGORY B1

Safety equipment required:

Eye protection, lab coat, enclosed shoes and appropriate gloves must be worn at all times.

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