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Published online 1 September 1999
Published in Agron J 91:859-862 (1999)
© 1999 American Society of Agronomy
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Agronomy Journal 91:859-862 (1999)
© 1999 American Society of Agronomy

AGROCLIMATOLOGY

Response of In Situ Leaf Psychrometer to Cuticle Removal by Abrasion

Colin S. Campbella and Kevin J. McInnesa

a Dep. of Soil and Crop Science, Texas A&M University, College Station, TX 77843-2474 USA

k-mcinnes{at}tamu.edu


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results and discussion
 Conclusion
 REFERENCES
 
In situ measurements of leaf water potential by thermocouple psychrometers are useful in monitoring the energy state of water in plants. Excessive equilibration times caused by low vapor conductance across the leaf cuticle and vapor sorption in the psychrometer chamber often make in situ sampling difficult. Several methods of removing the waxy leaf cuticle have been shown to increase water vapor conductance across the epidermis of the leaf, but their relative effectiveness has not been compared and equilibration times are unknown. Six wax removal treatments were compared with an untreated control to determine the most effective technique for increasing water vapor conductance across the leaf surface. The mean time for the leaf psychrometer to reach an equilibrated water potential was estimated for the treatment that produced the greatest conductance. Abrasion treatment of leaves with 600 grit sandpaper produced the greatest increase in cuticular conductance, resulting in a mean equilibration time for psychrometer measurements of 7.5 min.

Abbreviations: aw, water activity • M, mol kg-1 • PPFD, photosynthetic photon flux density • rpm, revolutions per minute • teq, equilibration time • {psi}, water potential


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results and discussion
 Conclusion
 REFERENCES
 
MEASUREMENT OF WATER VAPOR in equilibrium with leaf water using wet-bulb thermocouple psychrometry remains a useful technique for determining plant water potential ({psi}) (Savage and Wiebe, 1989). Widespread use of in situ psychrometers has been limited by excessive measurement time. Estimates of equilibration times for psychrometric measurements range from 0.25 to 2.5 h (Campbell, 1985; Neumann and Thurtell, 1972; Savage and Wiebe, 1989; Wullschleger and Oosterhuis, 1987). The leaf's natural barrier to water vapor movement across the epidermis or waxy cuticle causes extremely slow equilibration of vapor pressure in the psychrometer chamber (Bennett and Cortes, 1985; Savage et al., 1984). To overcome this constraint, equilibration times have been reduced by removing the waxy cuticle from the epidermis of the leaf (Wullschleger and Oosterhuis, 1987), but no experiments have been conducted to determine the best technique.

Procedures for removing the waxy cuticle and allowing more rapid water diffusion through the epidermis, even with stomatal closure, are most promising. Organic solvents such as chloroform have been used to dissolve and remove cuticular waxes (Bewick et al., 1993; Martin, 1960). Neumann and Thurtell (1972) tested different organic solvents on leaf surfaces and found that only xylene increased vapor conductance while leaving the leaf viable following treatment. Mechanical removal of the leaf cuticle by abrasion has also been used to increase leaf conductance. Savage et al. (1984) abraded the cuticle with a cotton swab, but were unsatisfied with the results. Savage and Wiebe (1989) cited other abrasion techniques, including using a slurry of grit (Oosterhuis et al., 1983; Savage et al., 1983), scratching with a razor blade (Richter, 1978), and scraping with emery cloth (Schaefer et al., 1986).

Attempting to improve psychrometric measurements by increasing conductance through abrasive removal of cuticular wax is not without problems. Destruction of the leaf's epidermal vapor barrier makes leaf cells susceptible to rapid changes in water potential from desiccation. Wullschleger and Oosterhuis (1987) suggested that abrasion may also increase variability in water potential measurements among leaf samples if epidermal cell walls were ruptured. In addition, the action of abrasion may fill stomata with wax, reducing their conductance (Oosterhuis et al., 1988).

These concerns about abrasion appear to be surmountable, for the following reasons. First, rapid attachment of the thermocouple psychrometer to the leaf would stop desiccation if the treated area were small. Second, application of distilled water during abrasion followed by careful drying with tissue paper would dilute and remove any cell sap from epidermal cells ruptured by abrasion, while having little effect on water potential (Nelsen et al., 1978). Wullschleger and Oosterhuis (1987) estimated the decrease in water potential from cell rupture to be about -0.07 MPa, within the inherent ±0.1 MPa error for measurements using modern in situ thermocouple psychrometers (Campbell and Campbell, 1974). Finally, clogging of stomata with wax is of little concern, because the stomatal contribution to vapor diffusion is minimal compared with the rest of the abraded surface.

While vapor equilibrium in a thermocouple psychrometer chamber is affected in large part by leaf conductance, it also depends on the composition and condition of the chamber walls. Lambert and van Schilfgaarde (1965) identified metallic surfaces as the most effective in reducing equilibration times. Contamination and oxidation of metallic chamber surfaces increase equilibration times, but may be minimized by frequent cleaning of the chamber (Brown and Oosterhuis, 1992).

Our objectives were to compare several techniques for removing cuticular wax, to identify which produces the greatest increase in cuticular conductance, and then to estimate the equilibration time for thermocouple psychrometers used on leaves treated with the most effective technique.


   Materials and methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results and discussion
 Conclusion
 REFERENCES
 
The experiments were conducted in two parts. First, cuticle removal techniques were compared using four plant species. Six removal treatments and a control treatment were applied to three mature leaves on three plants of each species. To estimate cuticular conductance, surface conductance for each treatment was measured using a diffusion porometer in a laboratory under low-light conditions. In the second part of the experiment, vapor equilibration in psychrometers were compared and analyzed, under laboratory conditions, for three types of samples, each with a different surface conductance. The samples supplying the different conductances were: salt solutions, untreated leaves, and leaves treated with the most effective wax removal technique. The thermocouple psychrometer output was modeled to estimate equilibration time.

Cuticle Removal
The four plant species used were corn (Zea mays L.), cotton (Gossypium hirsutum L.), sunflower (Helianthus annuus L.), and wax leaf ligustrum (Ligustrum japonicum Thunb. cv. Texanum). Plants were grown separately in 3.8-L pots with a 2:1 mixture of organic potting soil and fritted clay (Absorb-n-Dry, Balcones Mineral Corp., Flatonia, TX) (see van Bavel et al., 1978). When not in use for experimentation, plants were kept in a growth chamber, where they were exposed to 12 h of light (photosynthetic photon flux density, PPFD, of 250 µmol m-2 s-1) per day and watered with deionized water to excess every other day.

Treatments were selected to compare wax removal methods reported in the literature. Four organic solvents were chosen: xylene (Neumann and Thurtell, 1972), chloroform (Bewick et al., 1993; Martin, 1960), n-butanol, and dichloromethane (R.C. Ronald, personal communication, 1996). The organic solvents were applied using a cotton swab (5 mm diam.) (Cotton Tail Absorbent Tipped Applicator, Citmed Corp., Citronelle, AL) tightened into the chuck of a hand drill (Dremel Moto-Tool, Model 370, Dremel Manufacturing Co., Racine, WI). The cotton swab was wetted with solvent and spun for 1 s at approximately 28000 rpm to remove the excess liquid. With the axis of the swab held horizontal to the leaf surface and the drill rotating at approximately 5000 rpm, the swab was moved in a circular motion around an area of 80 mm2 for 15 s, applying a force of approximately 1.5 N to the leaf. A finger was used to support the back of the treatment area to prevent puncture of the leaf. After treatment, the leaf was blotted with a lintless tissue (Kimwipe EX-L, Kimberly-Clark Corp., Roswell, GA) to remove any excess solvent, then allowed to dry for 15 s. No water was applied to the treatment area.

Abrasion was tested as another removal technique. A clean 40- by 20-mm piece of 600 grit sandpaper (<15 µm) was folded in half and used for abrasion (Turner et al., 1984). A drop of water was applied to the leaf to aid in abrading the surface, then the area was lightly sanded using ten 20-mm back-and-forth strokes. A slight pressure exerted by the finger on the opposite side of the leaf in the treatment area was used to improve contact between the epidermis and sandpaper. After abrasion, the water was immediately blotted with a lintless tissue and the area was left to dry for 15 s.

The effectiveness of the solvent and abrasion treatments at increasing cuticular conductance were compared with a dry cotton swab treatment and a control. The cotton swab treatment involved spinning a new, dry swab on the leaf surface in the same manner as the solvent treatments (Savage et al., 1984). The control treatment did nothing to the leaf surface.

The cuticular conductance of the selected plant species was measured on the adaxial side (top) of mature leaves using a diffusion porometer (LI-1600, LI-COR, Lincoln, NE) under low light, laboratory conditions (<5 µmol m-2 s-1). The low natural conductance of the adaxial side of the leaf helped differentiate between wax removal techniques by minimizing conductance due to stomata. Three leaves per plant and three plants per species were tested using each wax removal technique. Adjacent leaves were used for treatments where leaf size limited the surface available for testing. The three youngest mature leaves on each plant were chosen for testing with all species except wax leaf ligustrum, where we used the youngest mature leaves on randomly chosen branches. An analysis of variance was performed on the vapor conductance data for each plant species. Mean values for each treatment were compared within plant species using Tukey's pairwise comparison (HSD) to find the treatment that caused the greatest increase in conductance.

Psychrometer Calibration and Cleaning
Five thermocouple psychrometers (Model L-51; Wescor, Logan, UT) were calibrated in an isothermal block regulated at 25°C with a water bath. Salt solutions of 0.05, 0.2, and 0.5 m (mol kg-1) KCl, with water potentials of -0.232 MPa (0.999 water activity aw), -0.901 MPa (0.993 aw), and -2.242 MPa (0.984 aw) (Robinson and Stokes, 1955), respectively, were sealed in turn under each psychrometer chamber. A final microvolt reading was recorded after 10 to 15 min of equilibration. A calibration equation relating microvolt readings to known water potential values for each leaf psychrometer was determined by linear regression.

We cleaned each psychrometer under a 10x binocular microscope prior to testing to remove contaminants that might act as vapor sinks during equilibration (Brown and Oosterhuis, 1992). Filling the chamber with ethanol, we carefully wiped the inside walls using a toothpick with a small amount of cotton spun tightly around the tip until all visible residue was removed. Under the microscope, we also observed that well-used psychrometers had residue buildup on the copper thermocouple mounting posts. The tip of a syringe needle (23 gauge) was used to carefully scrape away the dark-colored residue until the bright metallic copper was visible. After cleaning, the chamber was flushed with purified water and air dried. We repeated the cleaning after 10 to 20 equilibration tests, to guard against abnormally long equilibration times caused by contamination.

Psychrometer Equilibration
An experimental chamber was constructed in the laboratory to control temperature and supply an average PFD of 500 µmol m-2 s-1 to the plant. Inside the chamber, thermocouple psychrometers were attached to treated leaves without the use of a sealing compound (Savage et al., 1983), and left to equilibrate. Dataloggers (Model CR7X; Campbell Scientific, Logan, UT) recorded leaf water potential ({psi}) readings once per minute. During analyses of psychrometer equilibration, plants were placed in the experimental chamber individually and not watered during the several days of repeated testing.

Response to salt solutions was evaluated to examine psychrometer equilibration when attached to a static vapor source with no cuticular resistance, essentially presenting conditions for the minimum possible equilibration time. Three of the calibrated psychrometers were used to test equilibration times of three different salt concentrations, 0.05, 0.2, and 0.5 M KCl, producing water potentials of -0.232 MPa (0.999 aw), -0.901 MPa (0.993 aw), and -2.242 MPa (0.984 aw), respectively.

We also compared the relative equilibration times of two psychrometers sealed to a corn leaf, one over an abraded area and the other over an untreated area. The psychrometer over the untreated leaf tissue was placed on the abaxial (bottom) side of the leaf, where most of the stomata occur, to allow tracking of plant {psi} with no effect of cuticle removal. The other psychrometer was placed on an abraded area on the adaxial side of the same leaf. The test started in a light period and continued into a dark period, to ensure complete equilibration and allow sufficient time to track changes in plant {psi}.

Leaf psychrometers were attached to abraded leaves and the output was modeled to estimate equilibration time. Five different psychrometers were repeatedly equilibrated, and output curves were analyzed to find a mean time constant ({tau}) value. We assumed that psychrometer equilibration curves follow a first-order decay running asymptotic to the actual {psi} value. The first-order decay model was

(1)
where {psi}m, {psi}i, and {psi}f are modeled, initial, and equilibrated water potential, respectively, t (min) is time after psychrometer attachment, and {tau} (min) is the time constant. Sum of the squared error between {psi}m and psychrometeric water potential ({psi}p) was minimized at 1-min intervals over the equilibration time by altering values of {psi}i, {psi}f, and {tau} with Levenburg–Marquardt nonlinear least squares analysis (NLLS). Using the assumption of first-order decay and {psi}i, {psi}f, and {tau} from the NLLS, we then solved for the time (teq) until {psi}m reached ±0.05 MPa of {psi}f. The value of ±0.05 MPa is half the ±0.1 MPa inherent limit of precision of psychrometer measurements reported by Campbell and Campbell (1974). The teq value was assumed to be the time necessary for the psychrometer to reach equilibration. An analysis of variance was perform on the teq values and individual psychrometer means were compared using Tukey's pairwise comparison (HSD).


   Results and discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
Conclusion
 REFERENCES
 
Abrasion with sandpaper was the most effective treatment for increasing cuticular conductance in all plants (Table 1) . The mean conductances for the sandpaper-treated leaves were consistently >10 mm s-1 in all species. Conductances for the control were often too small to measure with the diffusion porometer. Analysis with the naked eye at 1 wk after abrasion showed only minimal leaf damage from the sandpaper treatment, appearing as small whitish spots across the abraded area. The treatment did not affect the viability of the leaf.


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  		Table 1 Mean cuticular conductances with six methods of leaf wax removal for four plant species

 
Treatments using organic solvents were far less effective at increasing vapor conductances. Chloroform did not perform as well as expected from the tests reported by Bewick et al. (1993) and Martin (1960), causing brown, brittle areas to appear in the treated area of the leaf within 2 d. The apparent effectiveness of xylene at removing the waxy cuticle as observed by Neumann and Thurtell (1972) was not evident. Xylene failed to increase vapor conductance over the control for all but corn leaves in our tests.

Two of the four plant species were chosen to evaluate effects of the treatments on the thermocouple psychrometer equilibration time: Wax leaf ligustrum, because it separated the useful treatment (600 grit sandpaper) from all other treatments (Table 1), and corn, because of the ready availability of plants.

Arithmetic mean teq calculated for three psychrometers equilibrated over salt solutions varied considerably (Fig. 1) . Through 35 tests, individual arithmetic mean teq for three psychrometers was 1.5, 2.3, and 10.3 min. The last teq was statistically different from the first two at the 0.05 level, suggesting that teq over salt solution is unique to specific psychrometers.



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  		Fig. 1 Psychrometer equilibration over 0.05 M (mol kg-1) KCl salt standard solution. Each equilibration is modeled by a first-order equation (Eq. [1]), and fitted at 1-min intervals using nonlinear least squares analysis (solid lines). Equilibration times (teq) for measurements 1, 2, and 3 were 0.9, 2.7, and 10.0 min. The dashed lines represent ±0.05 MPa

 
The psychrometer over the abraded section of corn leaf reached apparent equilibrium in about 10 min, whereas the untreated psychrometer took more than 1 h (Fig. 2) . After reaching equilibration with the leaf {psi} (approximately -0.5 MPa), the output curves tracked each other through plant water potential changes. The test illustrates two important points. First, leaf psychrometers that have reached equilibrium give similar psychrometric {psi} measurements, regardless of whether placed on abraded or unabraded leaf surfaces. This provides some evidence against the criticism that abrasion can lead to erroneous {psi} readings (Turner et al., 1984). Second, it shows the much more rapid equilibration time for psychrometers over abraded leaf surfaces compared with those over unabraded surfaces.



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  		Fig. 2 Simultaneous equilibration of leaf psychrometers attached to corn. One psychrometer was attached to a surface without abrasion, the other was attached after abrasion with sandpaper. The experimental chamber light was turned on 8 h prior to the test and turned off approximately 4 h into the test. The approximate water potential ({psi}) of the corn leaf during the light period is between -0.5 and -0.6 MPa

 
Equilibration times (teq) for psychrometers attached to 259 different abraded leaves were tightly grouped (Fig. 3) . The combined arithmetic mean teq for all the tests was 7.5 min, which provides a conservative estimate of typical time to equilibration. Although significant differences were seen in individual mean teq during the salt solution equilibration, mean teq values for individual psychrometers over plant samples were not statistically different at the 0.01 level. In addition, mean teq values for psychrometers attached to sections of abraded leaf were not statistically different from any one of the mean salt solutions teq at the 0.01 level.



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  		Fig. 3 Frequency distribution of equilibration times (teq) for four psychrometers equilibrated over 259 different abraded corn and wax leaf ligustrum leaves. The arithmetic mean teq for all four psychrometers is 7.5 min

 

   Conclusion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 Conclusion
REFERENCES
 
Rapid in situ water potential measurements were possible by abrading leaf surfaces if psychrometers were carefully maintained. Similar equilibration times should also be obtainable in the field if temperature gradients are minimized (Savage et al., 1983; Turner et al., 1984; Savage and Cass, 1984). If the teq for psychrometers over salt solutions is assumed to be the minimum possible equilibration time for a sample, the similarity between mean salt solution and abraded leaf teq suggests that the sandpaper abrasion treatment produced the maximum possible cuticular conductance.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 Conclusion
 REFERENCES
 
Research conducted by the Texas Agric. Exp. Stn., The Texas A&M Univ. System.

Received for publication August 12, 1998.
   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 Conclusion
 REFERENCES
 




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