José M. Torres-Ruiz1,2, Annika E Huber3 and Taryn Bauerle3.
1 Université de Bordeaux, BIOGECO, Unité Mixte de Recherche 1202, F-33615 Pessac, France.
2 Institut National de la Recherche Agronomique, Unité Mixte de Recherche 1202 BIOGECO, F-33610 Cestas, France.
3Cornell University, School of Integrative Plant Science, Ithaca, NY 14853 USA
Maximum xylem vessel length is an important trait used in wood anatomical studies for xylem tissue characterization, as well as in functional studies to evaluate hydraulic functionality of diverse plant species.
In general, xylem vessel length distribution has typically been used in wood anatomical studies both to establish evolutionary traits, as well as to evaluate the resistivity of water flow in xylem (Zimmerman and Jeje 1981, Ewers et al. 1990, Sperry et al. 2007). Although less common, maximum xylem vessel length is an important trait and must be considered for, evaluating plant hydraulic efficiency (e.g. Comstock and Sperry 2000) or the vulnerability to cavitation of the species (e.g. Torres-Ruiz et al. 2014). The air-injection technique (Greenidge 1952, Ewers and Fisher 1989) is based on the premise that air cannot pass through wet pit membranes at low pressures (ca. 100 kPa) but can pass through the vessels when they are cut open at both ends. This technique is a very useful and a simple way to determine the maximum vessel length of plant material (i.e. roots, branches, shoots, etc.). When possible, double checking the values obtained by air injection is recommended using a different method (e.g. silicon injection, Wheeler et al. 2005, or by determining the “hydraulically-weighted vessel length”, Cochard et al. 1994) to detect possible overestimation due to alternative paths for the air (e.g. pith). In any case, air injection is the most used method for a fast estimation of the approximate max. vessel length.
- Air tank
- Shears or razor blade
- PVC flexible tubes
- Plastic clamps
- Lab tray full of water.
- Magnifying glass (optional).
Units, terms, definitions
The meter (m) is the standard unit of the maximum vessels length (Lmax).
- A very long organ sample that is at least equal to the maximum vessel length (see ‘Notes and trouble shooting tips’ section below) is collected from the plant with shears or a razor blade. As the max. vessel length can be variable, a minimum of five different samples are required for proper determination.
- Samples should be perfused with water at high pressure (ca. 0.15MPa) for 30 minutes to re-establish the air/water meniscus in the pores of pit membranes and vessels that were cavitated under native conditions (Fig. 1, Step i). This will avoid air passage through these cells during measurement and, therefore, the overestimation of the Lmax.
- The PVC flexible tube should be attached to the basal end of the sample and to the air tank by using plastic clamps to avoid leaks.
- The sample basal end is injected with air at low pressure (50-100 kPa) while its apical end is immersed in water (Fig. 1, Step ii)
- The sample should be successively shortened by short portions (10-20 mm) using the shears/razor blade. After removing each portion, the apical end should be immersed again in water to check for air bubbles continuously exiting the sample (Fig. 1, Step iii).
- The emergence of air bubbles at the apical end after cutting indicates that at least one vessel is opened at both ends, so the remaining sample length can then be considered equal to the maximum vessel length of the sample (Fig. 1, Step iv).
Fig 1. Diagram showing the protocol to measure the maximum xylem vessel length (Lmax) by air-injection
Notes and trouble shooting tips
– If there is no previous information about the maximum vessel length of our plant material, we recommend collecting a reasonably long sample (e.g. 1.5m from branches) for the first measurements. Once initial experimentation on vessel length indicates the range of Lmax values for a particular species, we recommend reducing future sample lengths to samples 20-30cm longer than the Lmax.
– The air should never be injected at pressures higher than 100 kPa due to likelihood of breaking the air/water meniscus of the pores in the pit membranes and, therefore, overestimating Lmax.
– This technique could be problematic for those species that show a large and/or hollow pith structure since the air could circulate easily through it.
– Using a magnifying glass can facilitate the detection of the emerging air bubbles at the distal end.
– Some herbaceous plants like sunflowers produce slimy mucilage sealing the whole vascular tissue at the cut surfaces which prevents the air from flowing into the organ. Re-cutting (5mm-10 mm) the ends of the organ after a few minutes removes the seal and prevents the plant from producing new mucilage.
Cochard H, Ewers FW, Tyree MT (1994) Water relations of a tropical vine-like bamboo (Rhipidocladum racemiflorum). Root pressures, vulnerability to cavitation and seasonal changes in embolism. Journal of Experimental Botany 45, 1085-1089.
Comstock JP, Sperry JS (2000) Theoretical considerations of optimal conduit length for water transport in vascular plants. New Phytologist 148, 195–218
Ewers FW, Fisher JB (1989) Techniques for measuring vessel lengths and diameters in stems of woody plants. American Journal of Botany 76, 645–656.
Ewers F.W., Fisher J.B., Chiu S. (1990) A survey of vessel dimensions in stems of tropical lianas and other growth forms. Oecologia 84, 544–552.
Greenidge KNH (1952) An approach to the study of vessel length in hardwood species. American Journal of Botany 39, 570-574.
Sperry JS, Hacke U, Feild TS, Sano Sikkema E (2007) Hydraulic consequences of vessel evolution in angiosperms. International Journal of Plant Sciences 168, 1127–1139.
Scholz A, Klepsch M, Karimi Z, Jansen S (2013) How to quantify conduits in wood? Front Plant Science 4, 56. doi: 10.3389/fpls.2013.00056
Torres-Ruiz JM, Cochard H, Mayr S, Beikircher B, Diaz-Espejo A, Rodriguez- Dominguez CM, Badel E, Fernández JE (2014) Vulnerability to cavitation in Olea europaea current-year shoots: further evidence of an open-vessel artifact associated with centrifuge and air-injection techniques. Physiologia Plantarum 152: 465–474. doi: 10.1111/ppl.12185
Wheeler JK, Sperry JS, Hacke UG, Hoang N (2005) Inter-vessel pitting and cavitation in woody Rosaceae and other vesselled plants: a basis for a safety versus efficiency trade-off in xylem transport. Plant Cell Environ 28: 800–812.
Zimmermann M H and Jeje AA (1981). Vessel-length distribution in stems of some American woody plants. Canadian Journal of Botany 59, 1882–1892.
Scholz A, Klepsch M, Karimi Z and Jansen S (2013) How to quantify conduits in wood? Frontiers in Plant Science. 4, 56. doi: 10.3389/fpls.2013.00056
Health, safety & hazardous waste disposal considerations
There are no health, safety or hazardous waste disposal considerations for this technique.
Search terms and classification
Xylem anatomy, maximum vessel length, air-injection.