# Summary

John Boyer

### Definition

Thermocouple psychrometers measure the water potential and its components by determining the vapour pressure in a sealed atmosphere in equilibrium with the sample containing liquid water. The liquid does not need to be continuous, making the method more versatile than others requiring liquid continuity. The sample is enclosed in a small, air-tight chamber and allowed to equilibrate. A thermocouple in the chamber above the sample determines the vapour pressure in the atmosphere and thus in the sample (Boyer, 1995).

### Terminology and equations

Leaf water potential is the sum of the forces acting on water in the liquid. The water potential is

where is the chemical potential (energy/mol), V is the partial molal volume of water (m3/mol), and the subscripts w and o are water in the sample and in the reference, respectively. V is considered constant over most of the biological range of conditions. ℼw is thus proportional to the difference in chemical potentials. ℼw has units of energy/m3, i.e., force/m2 = pressure (Kramer and Boyer, 1995).

The energy of liquid water is affected by several components that in turn affect the vapour pressure in equilibrium with the liquid. The components are solute, pressure, matrix (solid) and gravity, and their algebraic sum determines the water potential

where subscripts s, p, m, and g indicate solute, pressure, matrix (solid) and gravity. The reference is defined to be pure water (no solute), at atmospheric pressure, free (no solids), a defined gravitational position, and the same temperature as the sample. The water potential of the reference is zero and by comparison, solute and solid components always decrease the energy (always have less energy than the reference, i.e., negative) while pressure and gravitational components can be positive or negative. Units of the water potential and its components are usually megapascals (MPa) or kilopascals (KPa) where 1 MPa = 1000 KPa = 10 bars = 9.87 atmospheres.

The vapour pressure is related to the water potential by the equation:

where w is the water potential of the sample, ew is the vapour pressure of the sample, and eo is the vapour pressure of the reference (which is equivalent to 100% humidity at T). The additional terms are constants, specifically, R (gas constant), T (Kelvin temperature) and Vw. Although
varies with T, it is constant at a particular temperature and has a value of 137.2 MPa at 298 K. The water potential thus is proportional to the logarithm of the ratio of the two vapour pressures. Because of the large size of the proportionality constant, the ratio need not be much different from 1 to have a large effect. This indicates that thermocouple psychrometers work in nearly saturated atmospheres (ratio slightly less than 1, i.e. slightly less than 100% relative humidity). Because the vapor pressure of the sample is usually less than that of the reference, the water potential is negative.

### Measurement approaches

All thermocouple psychrometers rely on wetting a thermocouple in the atmosphere above the sample. The water on the thermocouple evaporates to the atmosphere, and the temperature of the junction is measured electrically and indicates how much water vapour is in the atmosphere. Because the vapour pressures are not far below saturation, temperatures around the sample chamber must be uniform to within 0.001C to avoid uncontrolled condensation from the atmosphere. The evaporation from the thermocouple is slow in this atmosphere, making the electrical output of the thermocouple small and needing to be amplified. Although there are three basic psychrometer designs – isopiestic, dewpoint, and Peltier types – all have these thermal and amplification requirements.

Instruments

Isopiestic – the thermocouple junction is designed to hold a droplet of solution that can be changed by the investigator (Boyer and Kipling, 1965). A solution (usually sucrose) having a known vapour pressure (known water potential) is placed on the junction until a steady reading is obtained. From the reading, an approximate water potential is predicted and followed by a second solution having that potential. The steady reading for the second solution is close to zero and can be extrapolated a short distance to zero, which is the isopiestic value. The isopiestic value is very accurate (Boyer, 1966) because it is in equilibrium with the liquid in the sample and unaffected by the rate of vapour transfer (which is zero) or the temperature (sample chamber is isothermal). Because the water potential of the solution is known, the water potential of the sample is known. No calibration is required and temperature affects the isopiestic value according to
.

Dewpoint – the vapour pressure in the atmosphere above the sample is obtained by cooling the thermocouple junction to the dewpoint (Neumann and Thurtell, 1972; Campbell et al., 1973; 1974). An electrical current cools the junction (Peltier effect) until condensation just begins. The condensate increases the heat capacity of the junction, which is detected electronically. The current is then removed and the temperature of the junction is recorded. By measuring the dewpoint of solutions of known vapour pressure (known water potential), the system is calibrated so that the dewpoint indicates the water potential of the sample. The measurement system is not isothermal (thermocouple is cooler than the rest of the chamber), so the calibration must be at the temperatures expected in the sample. The vapour pressure of the dewpoint is close to that of the sample, which minimizes vapour transfer between the thermocouple and sample and thus decreases some of the variability caused by varying rates of transfer from the sample to the atmosphere.

Peltier – the vapour pressure in the atmosphere above the sample is obtained by cooling the junction until condensate accumulates, then removing the cooling and determining the rate of evaporation to the atmosphere (Brown and van Haveren, 1972; Brown et al., 1980; Brown and Oosterhuis, 1992). An electric current is first used to cool the junction (Peltier effect). After removing the cooling, the accumulated condensate evaporates for long enough to give a steady reading, usually a minute or so. By measuring the evaporation above solutions of known vapour pressure (known water potential), the system is calibrated so that the evaporation rate indicates the water potential of the sample. The measurement system is not isothermal (thermocouple is cooler than the rest of the chamber), so the calibration must be at the temperatures expected in the sample. Also, the measurements tend to be wetter than the sample because the rate of evaporation from the thermocouple is impeded by the epidermis of the sample (Boyer and Knipling, 1965). Keeping the junction small minimizes the amount of condensate and thus the disturbance of the atmosphere.

Components of water potential

Conditions in the psychrometer can be arranged to eliminate certain components of the water potential or cancel their effects, allowing each component to be measured in terms of its vapour pressure. In tissues, the vapour emanates from the evaporating surface that for plants is at the outer surface of the cell walls. The liquid in the wall (apoplast) is in equilibrium with that in the cytoplasm (symplast), but the components of the water potential can be different in the two compartments, e.g., low solute concentrations in the apoplast and large concentrations in the symplast, or negative pressures (tensions) in the apoplast but positive ones in the symplast. This compartmentation needs to be considered when measuring the component potentials.

Solute – the sample of plant tissue can be placed in the vapour chamber, frozen in liquid nitrogen while in the covered chamber, then thawed, and the vapour pressure reading taken. Freezing and thawing breaks the plasma membrane and eliminates pressure in the symplasm (Ehlig, 1962). If different samples do not differ in position in a gravitational field, and solid matrices are flooded by the water released from the symplast, gravity and matrix effects can be ignored and the solute component is considered to be the only one remaining,

.

Alternately, the solution in the sample can be extracted with a press after freezing and thawing to break the cell membranes. The osmotic potential is measured in the extract using the psychrometer. The isopiestic method is especially useful for this method because the thermocouple can hold small quantities of the extract, then be exposed to known vapour pressures in the chamber.

When measuring the osmotic potential with these methods, the symplast solution is inevitably mixed with the apoplast solution and can be diluted by it. If the apoplast is a substantial portion of the tissue volume, corrections for the dilution can be made.

Pressure – in tissues, the water potential of the sample is measured followed by freezing and thawing, then repeating the vapour measurement as above to obtain the solute component. The freeze/thaw disrupts cell membranes and eliminates the turgor pressure in the symplast (Ehlig, 1962; Nonami et al., 1987). The turgor pressure component is determined from

Matrix (solid) – in plant tissues, psychrometers directly measure the matric component in the cell walls in equilibrium with potential in the symplast. Porous solids such as cell walls contain surfaces that attract water (Boyer, 1967a). The attraction holds water in the interstices of the wall matrix, which generates tension on the water and decreases its vapour pressure. In addition, the wall solution may contain solute so that the apoplast water potential is

,

where the subscript a denotes the apoplast compartment (Boyer, 1967b). When the wall solution is dilute, the solute component may be ignored (often in leaves) and

.

Psychrometers thus indicate mostly matric tension with which the symplast is in equilibrium. The symplasm normally does not have significant matric potentials (too hydrated). It is worth noting that the xylem of transpiring plants is under tension that is transmitted to the apoplast and greatly affects the tension in the wall matrix.

In soils, the soil solution is extracted and its osmotic potential is determined as above for solute. The water potential of the soil is then determined. The matric potential is calculated according to

.

Gravity – the mass of water becomes important in columns such as the vascular system of trees where water at the bottom is under more pressure than at the top. For example, a tree 10 meters high will have a gravitational potential 0.1 MPa higher at the bottom than at the top in the xylem solution. To measure the gravitational potential, samples of leaves are collected from various heights at the same time in a tree with hydrated roots but not transpiring (pre-dawn), and the gravitational potential is assessed from

because gravitational differences are the only component altering the water potential in the apoplast (note that the apoplast is in hydraulic contact with the xylem solution, and thermocouple psychrometers detect potentials in the apoplast). This measurement assumes that solute is negligible in the apoplast.

### Ranges of values

The high accuracy of the isopiestic method is achieved by having no net vapour transfer between the thermocouple and the sample at the isopiestic value. When working with leaves, this is important because the epidermis impedes vapour transfer and can cause error if the method depends on transfer of vapour (Peltier) or heat (dewpoint). Avoiding this problem decreases the variability between replicate samples and eliminates the need of calibration, making the isopiestic method quite simple. Standard solutions need to be manipulated during the measurement, so the method is most suited for small sample numbers. By contrast, the dewpoint and Peltier methods are calibrated beforehand, which minimizes the manual effort of the sample measurement itself. This makes these methods more suited for screening large numbers of samples where high accuracy may be less important. Tissues such as leaves tend to appear wetter with dewpoint and especially Peltier methods than with the isopiestic one.

The walls of the psychrometer chamber can sorb water from the atmosphere. This is minimized by coating the walls with melted and re-solidified petrolatum, giving a smooth hydrophobic surface. Coated in this fashion, the vapour pressure reflects that of the sample rather than the walls.

Samples that were previously in contact with a dry atmosphere have dry sorption sites on the surface, especially on the epidermis of leaves and stems (Boyer, 1995). In order for the vapour pressure in the psychrometer to accurately equilibrate with liquid in the tissue, the surface sorption sites must hydrate, which can take 1-3 h and delay the approach to a steady reading with psychrometers. Roots and enclosed shoot tissues are not normally exposed to the dry atmosphere and equilibrate in 10-30 min.

Thermocouple psychrometers typically use excised plant parts. Care must be taken to prevent dehydration of the sample after excision (Boyer, 1995). Experience indicates that the sample must be sealed in the psychrometer chamber within 10 s of excision. If additional time is required, a simple glove box should be constructed to allow the chamber to be loaded in an atmosphere nearly saturated with water.

There also are effects of metabolic heat, gradients in water potential, changes in water potential due to tissue growth, and oxygen requirements that need to be considered and are treated more fully by Boyer (1995).

Boyer JS. 1995. Measuring the Water Status of Plants and Soils. Academic Press: San Diego. 178 pp. https://udspace.udel.edu/handle/19716/2828

Kramer PJ, Boyer JS. 1995. Water Relations of Plants and Soils. Academic Press: San Diego. 495 pp. https://udspace.udel.edu/handle/19716/2830

Boyer JS. 1969. Measurement of the water status of plants. Annual Review of Plant Physiology 20, 351-364.

#### Literature references

Boyer JS. 1966. Isopiestic technique: measurement of accurate leaf water potentials. Science 154, 1459-1460.

Boyer JS. 1967a. Matric potentials of leaves. Plant Physiology 42, 213-217.

Boyer JS. 1967b. Leaf water potentials measured with a pressure chamber. Plant Physiology 42, 133-137.

Boyer JS. 1995. Measuring the Water Status of Plants and Soils. Academic Press: San Diego. 178 pp. https://udspace.udel.edu/handle/19716/2828

Boyer JS, Knipling EB. 1965. Isopiestic technique for measuring leaf water potentials with a thermocouple psychrometer. Proceedings of the National Academy of Sciences USA 54, 1044-1051.

Brown RW, Collins JM. 1980. A screen-caged thermocouple psychrometer and calibration chamber for measurements of plant and soil water potential. Agronomy Journal 72, 851-854.

Brown RW, Oosterhuis DM. 1992. Measuring plant and soil water potential with thermocouple psychrometers: some concerns. Agronomy Journal 84, 78-86.

Brown RW, van Haveren BP (eds). 1972. Psychrometery in Water Relations Research. Utah Agricultural Experiment Station, Utah State University, Logan, UT.

Campbell EC, Campbell GS, Barlow WK. 1973. A dewpoint hygrometer for water potential measurement. Agricultural Meteorology 12, 113-121.

Campbell GS, Campbell MD. 1974. Evaluation of a thermocouple hygrometer for measuring leaf water potential in situ. Agronomy Journal 60, 24-27.

Ehlig CF. 1962. Measurement of energy status of water in plants with a thermocouple psychrometer. Plant Physiology 37, 288-290.

Kramer PJ, Boyer JS. 1995. Water Relations of Plants and Soils. Academic Press: San Diego. 495 pp. https://udspace.udel.edu/handle/19716/2830

Neumann HH, Thurtell GW. 1972. A Peltier cooled thermocouple dewpoint hydrometer for in situ measurements of water potentials. In :Psychrometry in Water Relations Research” (Brown RW, van Haveren BP, eds). PP 103-112. Utah Agricultural Experiment Station, Utah State University, Logan, Ut.

Nonami H, Boyer JS, Steudle ES. 1987. Pressure probe and isopiestic psychrometer measure similar turgor. Plant Physiology 83, 592-595.