Protocol
Author
Louis Santiago
Overview
This protocol outlines how to obtain initial hydraulic conductivity and maximum hydraulic conductivity of un-branched stem segments using the pipette method. A pipette distal to the stem segment is used to measure the volume of water that has moved through the stem segment for a given amount of time, providing a measurement of mass flow of water. The mass flow of water (F) combined with measurement of the height of a water reservoir providing the driving force ( P) and the length of the stem (L) allow calculation of conductivity (Kh = F L / ( P), kg s-1 m-1 MPa-1).
Background
Hydraulic conductivity (Kh) provides a measure of the capacity of a plant stem segment to conduct water. Variation in rates of Kh among different species or plants in contrasting experimental treatments is the product of several xylem characteristics including the distribution of vessel diameters and lengths, vessel end plates, and sap ionic concentration.
Because vessel characteristics are an especially critical factor determining variation in Kh, knowledge of vessel diameter is an important consideration in deciding on an experimental measurement length for stem segments. The length of the longest vessel length is normally determined by the methods of Ewers and Fisher (1989) and involves pushing air through a long stem segment with one end under water and cutting sections until bubbles appear in the water. Once the length of the longest vessels is known, this information can be used to cut stem segments from plants that are longer than the longest vessel to avoid cavitating vessels in measurements stems, and to decide how long of a stem segment on which to measure Kh. Many researchers have measured Kh on relatively short stem segments and obtained ecologically meaningful data suggesting that even if a proportion of xylem vessels are completely open in the stem segment, that the diameter distribution of vessels still controls rates of mass flow. Other researchers argue that if a proportion of vessels are completely open, higher rates of Kh will be obtained.
Materials/Equipment
Sharp clippers
Parafilm
large plastic bags
paper towels
1 ml glass pipettes
tygon tubing (various sizes)
surgical tubing (for connecting pipette to stem)
filtered, de-gassed water
a syringe and needle
pressurized gas and regulator
captive air tank
safety glasses (used whenever pressure is involved)
hose clamps
stopcocks
Units, terms, definitions
(Kh = F L / ( P), kg s-1 m-1 MPa-1)
Procedure
- Select stems from the measurement plant of interest that are straight and approximately 0.5-1 cm in diameter (other sizes may be used depending on species)
- Cut a section of the stem from the plant that is longer than the longest xylem vessel, or a section that is shorter than the longest xylem vessel may be cut if it is cut from the plant under water
- Seal the cut ends with parafilm
- Seal the cut stem segment into a plastic bag with wet paper towels inside to maintain high humidity and transport the stem segment to the laboratory, keeping it out of direct sunlight (a large cooler can be helpful while transporting the stem in a vehicle)
- In the lab, label & mark which end is up
- Trim the stem to measurement size under water
- Prepare stem:
a. Trim off bark (down to sapwood) about 1cm long on each end
b. Cut a slice off each end
c. Wrap parafilm around edge of bark and sapwood - Connect stem to tygon tubing with hose clamp (end near main branch)
- Attach pipettes to other end of stem with surgical tubing (end near leaf area)
- Measure height of hydraulic head
- Run initial flush for 3 min. to be sure water is flowing through stem and there are no leaks and no bubbles in the line. There must be a constant column of water from the hydraulic head to the stem, and from the stem through the surgical tubing to the pipette
- Get initial hydraulic conductivity:
a. bring water level in pipette down to 0 by extracting water from the surgical tubing between the stem and pipette
Start timer for 3-5min. (depending on speed of water movement)
c. at end of 3 min. record water level for each stem
d. repeat up to 4 times as needed - Run high pressure flush for 20 min., be sure to clamp apparatus to stems
- Get maximum hydraulic conductivity (follow same steps as initial hydraulic conductivity)
- Disconnect from apparatus
- Measure xylem diameter, pith diameter, and stem length
- Find total leaf area. See alternate protocol: Measuring leaf perimeter and leaf area.
Example of hydraulic conductivity apparatus (photo by T.J. Jones)
Notes and troubleshooting tips
If there is no flow
- Check for leaks
- Is the stem clogged
- Is there debris in the line To prevent microbial growth, the line should be cleaned by running a dilute bleach solution through the tubing once or twice per week
- Are all of the stopcocks open
- Are there bubbles in the line somewhere
Links to resources and suppliers
hose clamps: http://www.coleparmer.com/catalog/product_view.asp sku=0683299
tygon tubing: http://www.coleparmer.com/catalog/product_index.asp search=tygon%20tubing&Request=brand
Literature references
Ewers, F. W., and J. B. Fisher. 1989. Techniques for measuring vessel lengths and diameters in stems of woody plants. American Journal of Botany 76:645-656.
Santiago, L. S., G. Goldstein, F. C. Meinzer, J. B. Fisher, K. Machado, D. Woodruff, and T. Jones. 2004. Leaf photosynthetic traits scale with hydraulic conductivity and wood density in Panamanian forest canopy trees. Oecologia 140:543-550.
Sperry, J. S., J. R. Donnelly, and M. T. Tyree. 1988. A method for measuring hydraulic conductivity and embolism in xylem. Plant, Cell and Environment 11:35-40.
Zwieniecki, M. A., P. J. Melcher, and N. M. Holbrook. 2001. Hydrogel control of xylem hydraulic resistance in plants. Science 291:1059-1062.
Health, safety & hazardous waste disposal considerations
When pressurized gas is used, safety goggles must be worn and a regulator must be used to temper the pressure leading to the captive air tank.