Phloem exudation

Protocol

AUTHOR

Claudia Keitel

OVERVIEW

Phloem exudation allows the extraction of soluble compounds from bark tissue of species that do not bleed phloem sap when cut in the bark. Examples for bleeding tree species are European beech (Gessler et al. 2001, Keitel et al. 2003) and certain Eucalyptus species (Pate et al. 1998).

BACKGROUND

Three methods are presented, exudation with deionised water, with EDTA and polyphosphate buffer.

EDTA exudation was used first to retrieve soluble compounds out of phloem tissue, as it prevents the sieve tubes from clogging up (Schneider et al. 1996). Polyphosphate buffer has the advantage of not containing C and N compounds – as EDTA and chloramphenicol do – but it contains O.

Water exudation gives similar results to EDTA exudation as well as bled phloem sap and is the best choice for isotope analysis. Using water, there are no C, O and N compounds introduced into the exudation solution, which you have to correct for. A comparison of phloem samples collected with 4 different methods (water, EDTA and polyphosphate exudation and bled phloem sap), as well as the correction for introduced C, O and N compounds has been published in Gessler, Rennenberg & Keitel (2004).

MATERIALS/EQUIPMENT

5 to 10ml vials for exudation

2ml Eppendorf tubes

Disposable pipettes

Deionised water

Petri dish

Paper hand towels

Liquid nitrogen dewar and/or ice

Corer for bark pieces

Razor blade

Secateurs

10mM EDTA (ethylene diamine tetra-acetic acid)

0.015mM chloramphenicol (CAP)

15mM sodium hexametaphosphate (polyphosphate buffer, “phosphate glass”, Sigma)

HCl

PROCEDURE

  • Prepare exudation solution – either double demineralised water, 10mM EDTA and 0.015mM CAP or 15mM polyphosphate buffer. If necessary, adjust the pH to 7 with HCl. Pipette 2ml of solution into exudation vials.
  • Cut small pieces of bark from the stem or branch using a corer for thick bark pieces and a scalpel for branch pieces (ca. 150mg fresh weight, ca. 1cm in diameter if cored or 1 x 2 cm if from a branch).
  • Wash with double demineralised water in a petri dish to remove contaminants from xylem sap. Pat dry.
  • Immerse in exudation containers containing 2ml of exudation solution and leave for 5h at room temperature. The contamination of the phloem exudate with cellular constituents is minimal under these experimental conditions (Schneider et al. 1996). Please note: This is the standard technique that has been used for beech, spruce (Schneider et al. 1996) and Nothofagus (Keitel unpublished); a preliminary experiment of different incubation times should be performed to determine the ideal incubation time for different species.
  • Decant the supernatant after 5h and freeze in liquid N until further analysis or keep on ice until back in the laboratory. Store at -20∘C until analysis.

LITERATURE REFERENCES

Gessler A, Rennenberg H, Keitel C (2004). Stable isotope composition of organic compounds transported in the phloem of European beech – evaluation of different methods of phloem sap collection and assessment of gradients in carbon isotope composition during leaf-to-stem transport. Plant Biology 6: 721 – 729.

Gessler A, Schrempp S, Matzarakis A, Mayer H, Rennenberg H, Adams MA (2001). Radiation modifies the effect of water availability on the carbon isotope composition of beech (Fagus sylvatica L.). New Phytologist 50: 653 – 664.

Keitel C, Adams MA, Holst T, Matzarakis A, Mayer H, Rennenberg H, Gessler A (2003). Carbon and oxygen isotope composition of organic compounds in the phloem sap provides a short-term measure for stomatal conductance of European beech (Fagus sylvatica L.). Plant, Cell & Environment 26: 1157 – 1168.

Pate J, Shedley E, Arthur D, Adams M (1998). Spatial and temporal variations in phloem sap composition of plantation grown Eucalyptus globulus. Oecologia 117: 312 – 322.

Schneider S, Geßler A, Weber PV, Sengbusch D, Hanemann U, Rennenberg H ( 1996). Soluble N compounds in trees exposed to high loads of N: a comparison of spruce (Picea abies) and beech (Fagus sylvatica) grown under field conditions. New Phytologist 134: 103-114.

HEALTH, SAFETY & HAZARDOUS WASTE DISPOSAL CONSIDERATIONS

Handling cutting utensils, e.g. razor blade/scalpel, secateurs and bark corer

Use of corrosive (HCl) and irritant (EDTA, phosphate glass) substances

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