Agronomy Journal 92:538-541 (2000)
© 2000 American Society of Agronomy
NOTES & UNIQUE PHENOMENA
Leaf-Cutter Psychrometers
A Cautionary Note
Neil C. Turnera,
Kenneth A. Shackelb and
Ian F. Le Coultrea
a CSIRO Plant Industry, Private Bag No. 5, Wembley (Perth), W.A. 6913, Australia
b Dep. of Pomology, Univ. of California, Davis, CA 95616-8683 USA
n.turner{at}ccmar.csiro.au
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ABSTRACT
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Nontranspiring (covered) leaf water potential on greenhouse-grown chickpea (Cicer arietinum L.) leaves was measured with the pressure chamber and leaf-cutter psychrometer techniques over a range of water potentials. Potentials measured with the leaf-cutter psychrometer technique were highly variable and not well correlated with the values measured with the pressure chamber. The objectives of this study were to identify the basis for the discrepancy between the two techniques and to identify procedures to minimize the errors. Intentionally damaging 15% of the disc caused an increase (less negative) in the potentials measured in the psychrometers relative to those measured in the pressure chamber. When leaves were sampled with a sharp razor or a new biopsy punch, however, potential values similar to those measured by the pressure chamber were obtained. We conclude that unintentional damage caused by the psychrometer's cutter can give erroneous values of leaf water potential measured by leaf-cutter psychrometry.
Abbreviations: 
V/V, relative cell volume • 
, volumetric elastic modulus • 
, water potential(s) • p, turgor pressure • s, osmotic potential
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INTRODUCTION
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THERMOCOUPLE PSYCHROMETERS are widely used to measure water potentials () of plants and soils (Brown and Oosterhuis, 1992; Boyer, 1995). Leaf-cutter psychrometers have been developed to minimize evaporation and contamination when collecting leaf discs for the measurement of (Brown, 1976). Each psychrometer has a sharpened edge to allow a disc to be cut from the leaf directly into the psychrometer chamber, which is then quickly sealed with a tight-fitting cap. Leaf-cutter psychrometers are useful for analyzing leaf samples taken from the field or greenhouse, when associated with equilibration and measurement in the laboratory.
In a comparison of measured psychrometrically to that measured using the pressure chamber technique, we observed significant differences between the values observed on adjacent leaves. As leaf damage is thought to affect measurements of by the psychrometric technique (Oosterhuis and Wullschleger, 1987) and the cut edge to surface area ratio of the leaf-cutter psychrometer sample is high, damage from the psychrometer's cutter was suspected to have induced these differences. Our objectives were to identify the basis for the discrepancy between the two techniques, to evaluate whether damage was the cause, and to seek procedures to minimize the errors.
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Materials and methods
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Seeds of chickpea were inoculated with a commercial strain of Bradyrhizobium and planted in 4-L plastic pots containing a commercial potting soil. Several batches of plants, with each plant in a separate pot, were grown between July 1996 and May 1997 in a naturally lit, evaporatively cooled greenhouse at CSIRO, Floreat Park, Perth, Western Australia. Plants were generally irrigated daily, but water was withheld from selected pots to lower the leaf water potential as measured by the pressure chamber technique to -1.8 MPa.
In all experiments, near sunset on the day before measurement, three or four adjacent leaves per plant were individually inserted into close-fitting plastic bags covered with aluminum foil and sealed around the petioles with a paper clip. This technique eliminated transpiration by the covered leaf and is considered to allow the of that leaf to equilibrate with the of the stem at the point of leaf insertion (Begg and Turner, 1970). It has the advantage of eliminating any gradients in within the leaf that are associated with leaf transpiration (Shackel, 1987) and has been suggested as a more stable and reliable measure of plant water status than the of a transpiring leaf (McCutchan and Shackel, 1992). At about 0900 h (Australian Western Standard Time) the following day, adjacent covered leaves were cut from the plant. One leaf with the cover remaining attached was placed into a pressure chamber (Model 3005, Soilmoisture Equipment Corp., Santa Barbara, CA) located in the greenhouse, and the of this leaf measured using the precautions mentioned by Turner (1988). The other leaves were individually transferred to a humid chamber (Oosterhuis and Wullschleger, 1987) located close by. Each leaf was removed from its cover and, in the first series of measurements, two 0.24 cm2 leaflet discs per leaf were sampled into each leaf-cutter psychrometer (J.R.D. Merrill Specialty Equipment, Logan, UT). The measurement instructions supplied by the manufacturer were followed. The leaf was placed on a hard rubber stopper, a leaflet sample was cut with a slight twisting motion, and after both samples were collected, the chamber was quickly sealed. The psychrometers were relatively new and their cutting edges had no obvious wear. The central leaf was used in the pressure chamber and those immediately above and below it used for the psychrometric measurements. Two samples per chamber were used to reduce equilibration times. Further study tested whether the cutting edge of the psychrometers was affecting the measurement. Two leaflet segments of about the same area as cut by the psychrometers were cut in the humidified chamber with (i) a parallel set of new razor blades, positioned about 5 mm apart, or (ii) a 5-mm-diameter (0.20 cm2) Stiefel (Stiefel Laboratorium GmbH, Offenbach am Main, Germany) biopsy punch, and carefully lifted into the psychrometer chamber before sealing to minimize any damage. Both the new razor blades and the biopsy punch were disposable and were only used for one set of measurements with no noticeable damage (darkening) along the cut edge. Finally, to determine the influence of damage to the leaflet disc, four samples were cut with a biopsy punch from the same chickpea leaf and the center of two, randomly assigned, were deliberately damaged (crushed) with a 2-mm-diameter (0.03 cm2) nail punch before sealing into the chamber; the other two were left undamaged.
After sampling, the psychrometers were transferred to the laboratory, where they were placed in a water bath at 20°C in a temperature-controlled room (20 ± 1°C) for equilibration. After 4 h the total was measured with an HP-115 water potential data system (Wescor, Logan, UT) operated in the dew point mode. In some cases, osmotic potential (s) was measured on the same samples after freezing the tissue, still enclosed in the psychrometer chamber, in liquid N, and re-equilibrating in the 20°C water bath for 1 h. The psychrometers were calibrated using NaCl solutions before and after the series of measurements, and the psychrometer chambers were carefully cleaned and dried as per the manufacturer's instructions after each use.
For each leaf measured with the pressure chamber, two or three comparable psychrometer samples from the leaves immediately above and below the pressure chamber leaf were collected, and the mean and standard error of these samples was calculated. Results are presented as comparisons between the average measured by psychrometry and the measured by the pressure chamber technique.
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Results and discussion
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Preliminary results showed that when was measured either by the pressure chamber or by the leaf-cutter psychrometer there was no difference in among the three or four adjacent covered leaves. Subsequently all psychrometer comparisons were based on leaves sampled above and below the leaf assigned to the pressure chamber. There was considerable sample variation in the measured by psychrometry, resulting in a poor 1:1 relationship between measured by psychrometry and that by the pressure chamber (Fig. 1A) . Even when psychrometer samples were taken from the same leaf, there was considerable variation between samples (data not shown). Because the psychrometric samples were taken in a humidified chamber and within 60 to 120 s of one another, it was unlikely that substantial variation was caused by differences in evaporative losses from the samples (Brown and Oosterhuis, 1992) or arose from real variations in within the nontranspiring leaf. For some samples, darkening of the tissue around the disc perimeter was observed when the tissue was removed from the psychrometer chamber after the measurements of were completed, indicating that the psychrometer cutting edge may have damaged the leaf disc during sampling.
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Fig. 1 Relationship between leaf measured with the pressure chamber (P) and that measured with the leaf-cutter psychrometer (L) when the sample was obtained with (A) the cutter of the psychrometer (L = 0.45P3 + 0.75P2 + 0.67P - 0.34; r2 = 0.68); (B) a new pair of razor blades (L = 1.02P + 0.11; r2 = 0.84); (C) a disposable biopsy punch (L = 1.19P + 0.23; r2 = 0.92); and (D) a biopsy punch, where the leaf was subsequently damaged with a nail punch (L = 0.23P3 - 0.50P2 - 0.89P - 0.54; r2 = 0.90). Values in (C) were the undamaged controls for (D); additional sets of data for undamaged discs obtained with the biopsy punch gave similar results to (C). Values are means ± 1 SE of 2 (C and D) or 3 (A and B) separate samples. The dotted line is the 1:1 line
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When leaflet segments of a similar size were sampled with a pair of new razor blades and carefully placed by hand into the psychrometer chamber, the variation between samples taken from nearby leaves was markedly reduced, and the relationship between the measured with the pressure chamber and that with the psychrometer was not significantly different from the 1:1 line (Fig. 1B). Similarly, when a new biopsy punch was used, the standard errors were small and the relationship between the measured by the two methods was not significantly different from the 1:1 line (Fig. 1C).
When four leaf discs were taken from the same leaf with a biopsy punch and two were deliberately damaged with a nail punch, the undamaged discs gave the same results as those shown in Fig. 1C; the relationship for the damaged discs is shown in Fig. 1D. Even though the calculated damaged area was only 15% of the area of the disc, the damaged discs had a substantially higher variation in , particularly at intermediate values of as measured by the pressure chamber (Fig. 1D). In a separate experiment it was shown that transferring the disc from the biopsy punch to the psychrometer chamber with forceps was sufficient to damage the disc and caused a 0.2 MPa increase in compared with carefully lifting the discs into the psychrometer chamber by hand or with a flat spatula (data not shown).
The cause of the variability in the psychrometer measurements appears to be cell sap released during leaf excision and damage. Barrs and Kramer (1969) reported that increases in determined psychrometrically were associated with tissue damage (slicing) and both increases and decreases in were reported by Oosterhuis and Wullschleger (1987), depending upon the plant species used and the method of damage. Since for any turgid cell the s must be lower than the total , and because cell damage results in the release of cell contents, it is reasonable to expect a decrease in the of a tissue when cells are damaged. Barrs and Kramer (1969), however, presented evidence that the solutes released from the damaged cells were actively reabsorbed, presumably by the undamaged cells, and resulted in an increase in the total of the tissue. This is expected, even with no change in the total water content of the tissue, because of the high volumetric elastic modulus () of plant cells. For instance, the measured of 10 MPa in chickpea (Duda, 1998) means that there will be a 1.0 MPa increase in turgor (p) for a relative cell volume (V/V) increase of 0.1. The measured s of the leaf tissue used in this study was -0.76 ± 0.08 MPa. Thus, if 15 % of the cells were damaged (as was the case when damaged by the leaf punch), and assuming all of the solutes but none of the water from these damaged cells were taken up by the remaining undamaged cells, then the s of the remaining cells should decrease by about 15 %, or by about 0.1 MPa. However, the water contained by the damaged cells would also be available for uptake, and represents a potential V/V of 0.15 for the remaining cells. Assuming an of 10 MPa, it would only require a V/V of 0.011 to increase p by the same amount as the initial decrease in s (0.11 MPa), so additional water would be available to further hydrate the remaining cells, causing an increase in cell and tissue .
The magnitude of the error in will depend on a number of factors, such as the relative magnitudes of and s, the relationship between and p and the degree to which cell solutes behave ideally. It is clear from our study, however, that even moderate amounts of tissue damage should be avoided when comparing psychrometric to other methods for measuring . It is interesting to note that when damage was minimized, good agreement was obtained between values of measured by psychrometry and those by the pressure chamber method (Fig. 1B and 1C), even though the area of the tissue samples used in our study (2 x 0.20 cm2) was small and had a low ratio of surface area to cut edge (about 0.13 cm2/cm). For comparison, Barrs and Kramer (1969) used standard and small samples of 15.8 and 2.43 cm2 area, respectively, the small samples having a ratio of surface area to cut edge of about 0.34 cm2/cm. Hence, sample excision itself may have a minimal effect on , provided a suitable excision method is used.
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Conclusions
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In some cases, leaf-cutter psychrometers appear to cause enough damage to substantially modify the of leaf tissue. We recommend sample excision with new biopsy punches or razor blades. Insertion into the psychrometer chamber also requires care to ensure that no damage occurs when transferring the tissue to the chamber and that loss of water is minimized by making the transfer in a humidified chamber. With these precautions, we conclude that leaf-cutter psychrometers can measure the of nontranspiring leaves as reliably as the pressure chamber.
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ACKNOWLEDGMENTS
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K.A. Shackel thanks the Cooperative Research Centre for Legumes in Mediterranean Agriculture (CLIMA) for financial support and the University of California, Davis, for sabbatical leave to undertake this research. Drs. T.P. Bolger, J.A. Palta, R. Chapman, A.G. Condon, and M.H. Behboudian are thanked for their comments on the manuscript.
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NOTES
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Research conducted at the Cooperative Res. Cent. for Legumes in Mediterranean Agric., Univ. of Western Australia.
Received for publication May 13, 1999.
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REFERENCES
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- Barrs H.D., Kramer P.J. Water potential increase in sliced leaf tissue as a cause of error in vapor phase determinations of water potential. Plant Physiol. 1969;44:959-964.[Abstract/Free Full Text]
- Begg J.E., Turner N.C. Water potential gradients in field tobacco. Plant Physiol. 1970;46:343-346.[Abstract/Free Full Text]
- Boyer J.S. Measuring the water status of plants and soils. San Diego, CA: Academic Press, 1995.
- Brown R.W. New technique for measuring the water potential of detached leaf samples. Agron. J. 1976;68:432-434.[Abstract/Free Full Text]
- Brown R.W., Oosterhuis D.M. Measuring plant and soil water potentials with thermocouple psychrometers: Some concerns. Agron. J. 1992;84:78-86.[Abstract/Free Full Text]
- Duda, R. 1998. Characterization of drought resistance in chickpea (Cicer arietinum). M.S. thesis, Univ. of Hannover, Germany.
- McCutchan H., Shackel K.A. Stem-water potential as a sensitive indicator of water stress in prune trees (Prunus domestica L. cv. French). J. Am. Soc. Hortic. Sci. 1992;117:607-611.[Abstract/Free Full Text]
- Oosterhuis, D.M., and S.D. Wullschleger. 1987. The use of leaf discs in thermocouple psychrometers for measurement of water potential. p. 77–81. In R.J. Hanks and R.W. Brown (ed.) Proc. Intl. Conf. on Measurement of Soil and Plant Water Status, Vol. 2. Plants. Utah State Univ., Logan, UT. 6–10 July 1987. Utah State Univ. Agric. Exp. Stn., Logan.
- Shackel K.A. Direct measurement of turgor and osmotic potential in individual epidermal cells. Plant Physiol. 1987;83:719-722.[Abstract/Free Full Text]
- Turner N.C. Measurement of plant water status by the pressure chamber technique. Irrig. Sci. 1988;9:289-308.
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ABSTRACT
INTRODUCTION
