Protocol

Authors

Frank W. Ewers¹, Maria A. Lo Gullo², Jorge López-Portillo³, Rodrigo Méndez Alonzo⁴, Andrea Nardini⁵, Elisabetta Oddo⁶, Fabio Raimondo², Tadeja Savi⁷, Patrizia Trifilò²

Authors contributed equally to this work

Author affiliations

¹Biological Sciences Department, California State Polytechnic University, Pomona, 3801 West Temple Avenue, Pomona, California 91768 USA

²Dipartimento di Scienze Biologiche e Ambientali, Università di Messina, Salita F. Stagno D’Alcontres 31, 98166 Messina, Italy

³Red de Ecología Funcional, Instituto de Ecología, A. C., Carretera antigua a Coatepec 351, El Haya, 91070  Xalapa, Veracruz, México

⁴Departamento de Biología de la Conservación, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Carretera Ensenada-Tijuana No. 3918, 22860 Ensenada, B.C., México

⁵Dipartimento di Scienze della Vita, Università di Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy

⁶Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Università di Palermo, via Archirafi 38, 90123, Palermo, Italy

⁷University of Natural Resources and Life Sciences, Vienna, Department of Integrative Biology and Biodiversity Research, Institute of Botany, Gregor-Mendel-Straße 33, 1180 Vienna, Austria

Overview

This protocol describes how to extract xylem sap from branch samples using a vacuum chamber. The analysis of extracted sap solute content, chemical composition, and physicochemical properties

(i.e. pH, osmotic potential, ion and carbohydrate concentration) is relevant to investigate the osmotic balance between symplast and apoplast within the xylem.

Background

Xylem sap extraction by the vacuum chamber technique is an easy, rapid, and low-cost method that permits the extraction of greater volumes of sap in comparison with the pressure chamber method, allowing detailed analytical determination. However it has the caveat that it cannot be used with all species, so the technique should be tested in each case. Sap extraction is obtained by applying a gentle aspiration to xylem vessels. Vacuum force allows sap extraction due to the lower axial resistance in the xylem vessels than in the other compartments of the sample. Because the applied pressure is not enough to overcome the resistance of the air to pass through the pit membrane, it is necessary to consecutively cut small segments of the samples to progressively open vessels and facilitate sap collection.

Materials/Equipment

• Black plastic bags, up to 1m long
• ice
• osmometer (if sap osmolality measurements are desired)
• plastic tubes or microcentrifuge save-lock tubes
• razor blade
• sealing film
• shears
• superglue (optional to seal secondary stubborn branches that do not fit in the flask-vacuum chamber)
• adhesive putty (optional to seal stem in the rubber stopper)
• vacuum chamber such as a Büchner or Kitasato flask
• vacuum/pressure pump (many options)
• wet tissue paper

Units, terms, definitions

MPa: megapascal, a measure of pressure. 1 MPa= 10 bars

Osmolality, mmol kg^-1

Procedure

Step 1

Field sampling: A minimum of 5-6 terminal branches or stems between 50-80 cm long and 1- 3 cm wide should be defoliated to avoid loss of water in functional vessels due to evaporation, and then cut using shears. Samples should be enclosed rapidly in a sufficiently large black plastic bag with a piece of wet filter paper inside to prevent desiccation and transported to the laboratory.

Step 2

Stem preparation: After removing the sample from the plastic bag, about 5 cm of bark at the approximately 1-cm diameter distal end is removed with a sharp razor blade and the exposed wood is shaved with a knife until a clean surface is obtained. Rinse directly with deionized water and blot dry with clean filter paper. This step prevents contamination of the xylem sap sample with phloem sap on the distal end. If contamination with phloem sap, gums or latex is feared, all bark should be removed from the rest of the stem with the back of a field knife or any other blunt object, removing up to the cambium but taking care not to damage the stem. This will take care of any contamination from the bark. In most species this will be enough.

Step 3

Insertion of the stem in the vacuum chamber: The clean and shaved apical end of the branch is inserted in the plastic tube, sealed with sealing film, and placed inside the vacuum chamber or Buchner flask (in this case the stem must be inserted in a rubber stopper and sealed with adhesive putty to avoid the escape of vacuum and then proceed to insert the apical end of the branch in the plastic tube). The plastic tube may be immersed in an ice bath to avoid evaporation of the xylem sap during extraction.

Step 4

Sequential stem cuts and sap extraction: After connecting the chamber to a vacuum pump and applying a pressure of at least -0.08 MPa, about 1-5 cm long segments (depending on vessel length) are consecutively cut at 20-30 s intervals with the shears starting from the stem end protruding outside the chamber. This procedure allows the xylem sap of open vessels to be sucked out of the sample and collected in the plastic tube such as an ultracentrifuge tube. At the end of the extraction, after obtaining the desired amount vessel sap, the tube is immediately collected and sealed with its integrated cap. If more sap needs to be collected, another tube may be inserted as before and more segments may be cut. The tubes containing the xylem sap can be directly analysed or stored in a freezer (which is normally at –20 °C) to prevent evaporation and microorganism growth until further analysis.

Step 5

Measurement of sap osmolality: Sap solute concentration (i.e. osmolality, in mmol/kg) can be measured with a vapour pressure osmometer (such as, for example the VAPRO 5560 or 5520, Wescor Inc, Logan, Utah, US). To conduct sap osmolality measurements, extracted sap should be collected in solute-free paper discs. The required amount to saturate the disc is approximately 10 ml, which is the ideal amount for the measurement in the osmometer. Each disc must be kept in the sealed tube to eliminate evaporation until used. The osmolality of extracted sap can also be measured with a freezing point osmometer (for example the Osmomat 3000, Gonotec GmbH, Berlin, Germany).

Sap ionic composition or sugar composition can be further analysed using high-performance liquid chromatography (HPLC). Ion concentration may be also measured directly with ion-specific electrodes.

 

 

Figure 1. Stem placed in a Büchner flask for vacuum sap extraction. Sequential cuts are made at the distal end to open vessels and the sap is collected in plastic tubes such as microcentrifuge tubes with integrated cap at the distal end.

 

 

Figure 2. Lab setting of two branches connected in series to the vacuum pump (right) and ready for sap extraction. The vacuum is released with the three way valve (left) at the end of tubing.

Notes and trouble shooting tips

• If possible, use only straight branches, with few or no secondary branches. If needed, use superglue to seal the wounds and then wrap the branch in parafilm.
• Sap extraction should be performed within 1 h after branch excision to prevent desiccation of the sample or the branch should be kept inside a plastic bag in a cooler or an icebox.
• Scholander et al. (1966) used “an automobile tire pump with reversed piston valve” to extract xylem sap in the field, which is handy if no electricity is available.
• In stems with short vessels, several cuts should be made before the first sap from the open vessels appears in the plastic tube.
• If a 10 µL sample is desired, place the paper disc directly on the surface of the shaved stem distal end before it is inserted in the plastic tube, apply the vacuum and check every time a cut is made until the disc is saturated with the sap. Several sequential cuts may be made if more sap is needed.
• If there are canals or laticifers in the xylem, every time a cut is made on the distal part of the stem, some gum or latex will be extruded (at positive pressure) on the cut surface and the amount will depend on the pressure and length of the cut canals, so eventually the “bleeding” will stop. Dry such extrusion until it stops, clean with a damped cotton or paper and then connect the vacuum again to extract the xylem sap. The sap extraction process will be slower than in other species, but contamination will be minimized.
• The osmometer should be calibrated and cleaned according to the manufacturer’s manual, normally after 100 measurements depending on the type of sample: those with higher osmolality require more frequent calibrations (every 50 samples). Be careful to place the filter disc in the correct location within the sample holder and clean and dry the sample holder after each measurement and before placing the next disc.

Links to resources and suppliers

http://www.sigmaaldrich.com/labware/labware-products.html?TablePage=9580325

http://www.camlab.co.uk/buchner-flasks-glass-p17457.aspx

http://welchvacuum.com/products/wob-l-pump-2522

http://www.wescor.com/biomedical/osmometer/vapro5600.html

Literature references

Améglio T, Decourteix M, Alves G, Valentin V, Sakr S, Julien JL, Petel G, Guilliot A, Lacointe A (2004) Temperature effects on xylem sap osmolarity in walnut trees: evidence for a vitalistic model of winter embolism repair. Tree Physiology 24,785–793. doi: 10.1093/treephys/24.7.785

Bollard EG (1953) The use of tracheal sap in the study of apple-tree nutrition. Journal of Experimental Botany 4, 363–368. doi: 10.1093/jxb/4.3.363

Charrier G, Cochard H, Améglio T (2013) Evaluation of the impact of frost resistances on potential altitudinal limit of trees. Tree Physiology 33, 891–902. doi: 10.1093/treephys/tpt062

Ewers FW, Améglio T, Cochard H, Beaujard F, Martignac M, Vandame M, Bodet C, Cruiziat P (2001) Seasonal variation in xylem pressure of walnut trees: root and stem pressures. Tree Physiology 21, 1123-1132. doi: 10.1093/treephys/21.15.1123

López-Portillo J, Ewers FW, Méndez-Alonzo R, Paredes-López C, Angeles G, Alarcón Jiménez AL, Lara-Domínguez AL, Torres Barrera MC (2014) Dynamic control of osmolality and ionic composition of the xylem sap in two mangrove species. American Journal of Botany 101, 1013–1022. doi:10.3732/ajb.1300435

Milburn JA, Ranasinghe MS (1996) A comparison of methods for studying pressure and solute potentials in xylem and also in phloem laticifers of Hevea brasiliensis. Journal of Experimental Botany 47, 135–143. doi: 10.1093/jxb/47.1.135

Scholander  PF, Bradstreet ED, Hammel HT, Hemmingsen EA (1966) Sap concentrations in halophytes and some other plants. Plant Physiology 41, 529–532. doi: http:/​/​dx.​doi.​org/​10.​1104/​pp.​41.​3.​529

Schurr, U. 1998. Xylem sap sampling – new approaches to an old topic. Trends in Plant Science 3, 293-298. doi: 10.1016/S1360-1385(98)01275-8.

Secchi F, Zwieniecki MA (2012) Analysis of xylem sap from functional (nonembolized) and nonfunctional (embolized) vessels of Populus nigra: chemistry of refilling. Plant Physiology 160, 955–964. doi: http:/​/​dx.​doi.​org/​10.​1104/​pp.​112.​200824

Health, safety & hazardous waste disposal considerations

Dispose safety blades properly covering them with masking tape

Search terms and classification

This protocol has been used to extract xylem sap of numerous and different species.

Examples are:

• Acanthaceae, Avicennia germinans L., woody tree by López-Portillo J, Ewers FW, Méndez-Alonzo R, Paredes López CL, Angeles G, Alarcón Jiménez AL, Lara-Domínguez AL, Torres Barrera MD (2014) American Journal of Botany 101(6),1013-1022.
• Actinidiaceae, Actinidia chinensis Planch, woody vine by Ferguson AR, Eiseman JA, Leonard JA (1983) Annals of  Botany 51 (6), 823-833
• Combretaceae, Laguncularia racemosa L. (Gaertn.), woody tree by López-Portillo J, Ewers FW, Méndez-Alonzo R, Paredes López CL, Angeles G, Alarcón Jiménez AL, Lara-Domínguez AL, Torres Barrera MD (2014) American Journal of Botany 101(6),1013-1022.
• Fabaceae, Ceratonia siliqua L., woody tree by Trifilò P, Barbera PM, Raimondo F, Nardini A, Lo Gullo MA (2014) Tree Physiology 34, 109–122.
• Fabaceae, Glycine max (L.) Merrill, annual herb by Herridge DF, Roughley RJ, Brockwell J  (1984) Australian Journal of Agricultural research 35(2), 149-161.
• Solanaceae, Solanum lycopersicum L., annual herb by Trifilò P, Lo Gullo MA,  Raimondo F,  Salleo S, Nardini A (2013) Functional Plant Biology 40(5), 459-465.