Potatos

1975) GANDAR AND TANNER: MEASURING WATER POTENTIALS 387

COMPARISON OF METHODS FOR MEASURING LEAF AND TUBER WATER POTENTIALS IN POTATOES 1

P. W. Gandar and C. B. Tanner 2

Abstract

Comparative methods for measuring water potential in leaves and tubers of potatoes (Solanum tuberosum L., var. Russet Burbank) are described.

For leaves drier than -3 bars, the pressure chamber gave estimates of water potential which were zero to three bars drier than potentials measured using thermocouple psychrometers. Pressure chamber readings ranged _+ 2.5 bars from psychrometer value for leaves wetter than -3 bars; the psychrometer measurement usually was drier than the pressure chamber when leaves were sampled in the evening.

With tubers , water potent ia l measu remen t s using in situ psychrometers and the pressure chamber agreed to within one bar, except in tubers drier than -7 bars, where there were discrepancies o f _ + 2.5 bars. However, if the interval between psychrometer insertion and water potential measurement were longer than 24 hours, serious errors arose in the psychrometer measurements, apparently from suberization of tissues surrounding the psychrometers which prevented vapor equilibrium.

Pressure chamber measurements of tubers also were compared with tuber water potentials determined from the weight changes of tuber samples immersed in graded sucrose osmotica. For tubers wetter than -5 bars, the osmotica method gave drier estimates than the pressure chamber; for tubers drier than -5 bars the reverse occurred. Causes for these discrepancies are discussed.

Resumen

Se describen m6todos comparativos para medir potenciales de agua en hojas y tub6rculos de papa (Solanum tuberosum L., var. Russet Burbank).

Para hojas contenidos de humedad menores que -3 bars, la cfimara de presi6n di6 estimados de potenciales de agua que estuvieron entre cero y tres bars por debajo de los potenciales medidos usando psic6metros de termocupla. Las lecturas de la cfimara de presi6n se desviaron+ 2.5 bars de los valores del psic6metro para hojas mils h6medas que -3 bars; las medidas del psic6metro di6 contenidos de humedad mils bajos que la cfimara de presi6n cuando las muestras de hojas se tomaron al anochecer.

1Contribution from the Department of Soil Science, University of Wisconsin. Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madi- son, Wisconsin, 53706, and by USDA Hatch funds. 2Research Assistant (presently Scientist, D.S.I.R., Palmerston North, New Zealand), and Professor of Soil Science, University of Wisconsin, Madison, Wisconsin, 53706. Received for publication April 14, 1975.

388 AMERICAN POTATO JOURNAL (Vol. 52

En tubrrculos, las demidas del potencial de agua usando psicdmetros en situ y la c/tmara de presirn concordaron dentro del limite de 1 bar, excepto en tubrrculos con humedad menor que -7 bars, donde hubieron discrepancias de ___ 2.5 bars.

Sin embargo, si el intervalo entre la inserci6n del psic6metro y la medida del potencial de agua fue mayor de 24 horas, serios errores sur-

g ieron en las medidas del psic6metro, aparentemente debido a la suberizaci6n de tejidos alrededor del psic6metro, impidiendo asi al equilib- rio del vapor.

Las medidas de la cfimara de presi6n para tubrrculos tambirn se compararon con potenciales de agua determinados por el cambio de peso de las muestras de tub4rculos sumergidos en soluciones osmdticas de sucrosa. Para tubrrculos con contenidos de humedad mayores que -5 bars, el mrtico dio estimados de humedad mils bajos que la cfimara de presirn; 1o opuesto ocurri6 en tubrrculos menos ht~medos que -5 bars. Se discuten las causas de estas discrepancias.

Introduction

Studies of crop response and growth during periods of water deficits require measurement of'plant water status. Few measurements of water status in leaves or tubers of potato have been reported. In a few experiments on potato, leaf relative water contents have been used as an index of response to changes in soil water supply or atmospheric evaporative de- mand (16, 27, 29, 32). In other cases, pressure chambers (19, 26), thermocouple psychrometers (11,27), and in situ hygrometers (3, 10) have been used to measure leaf water potentials (qJl). The only comparisons among any of these methods for potato leaves were made by Baughn and Tanner (3, 4). In their experiments, made indoors under low transpiration conditions, the pressure chamber gave estimates of qJ~ which were within +_ 1.5 bars of values measured using an in situ hygrometer, and psychrometer measurements which were 0 to -2 bars drier than the hygrometer. Similar comparisons, under field conditions have not been reported for potatoes.

Tuber water status has been measured in only a few experiments. Epstein and Grant (16), following the method of Meyer and Wallace (23), determined tuber water potential (Wt) from the weight changes of plugs of tuber tissue immersed in sucrose solutions with different osmotic potentials. Tuber water potentials have also been measured with soil psychrometers sealed in holes drilled in tubers (11), and with a pressure chamber (19). No comparisons have been made between these methods.

The purpose of this paper is to provide comparisons of different methods for measuring qJ~ and Wt in potatoes. Comparative measurements of water potential in leaves were made using a pressure chamber and


thermocouple psychrometer; in the case of tubers, we compare the pressure chamber method with graded osmotica determinations, and with in situ soil psychrometers.

Materials and Methods

Plant material--Leaves and tubers from Russet Burbank potatoes grown in a glasshouse and in the field at Hancock, Wisconsin (18) were used in these experiments.

Leaf measurements--Pressure chamber and psychrometer measurements were made on leaves from field-grown plants. Young (third to fifth from top) and old (tenth to fifteenth) leaves were washed and dried on

• -plants, prior to excision, to remove contaminants which might affect psy= chrometer readings (15). Measurements of leaf xylem water potential, qJ~x, were made with a pressure chamber as described by Gandar and Tanner (19). Once qJ~x had been measured, leaves were removed from the chamber and sampled for thermocouple psychrometer determinations of qJ~, as described by Baughn and Tanner (4).

Tuber m e a s u r e m e n t s - - T h e comparison between the pressure chamber, graded osmotica, and in situ psychrometer methods were made using glasshouse-grown plants. The tubers used in these experiments were 50 to 90 days old (from initiation). Their lengths ranged from five to ten centimeters.

Soil psychrometers (Wescor, Inc., Logan, Utah, 84321) were inserted into slightly undersized holes drilled in the apices or sides of tubers without detaching them from plants. Holes usually were rinsed with distilled water and swabbed dry before psychrometers were inserted. Psychrometers were sealed into tubers with a low melting-point wax. To minimize errors due to heat conduction, leads were coiled against the tubers. Tubers were then buried under sphagnum moss. Tuber water potentials were measured 18 hours to 10 days after psychrometers had been inserted, usually in the early afternoon. Once q6 had been determined with the psychrometers, tubers were excavated and their xylem water potentials, qJ~x, were measured in the pressure chamber (19). Tubers were then sampled for graded osmotica determination of tuber water potential.

We followed the method of Meyer and Wallace (23) for the graded osmotic measurements. Plugs 20 mm in length and 5 mm in diameter were cut from a tuber, rinsed in distilled water, and dried by gentle blotting between filter papers. After weighing, plugs were equilibrated in 0. I to 0.6 M sucrose solutions for four hours at 5 C. Plugs were then rinsed in distilled water, dried, and reweighed. Tuber water potential was found by inter- polating to the sucrose solution osmotic potential at which the weight of immersed tissue samples remained constant.


Results and Discussion

Pressure chamber vs. psychrometers for leaves~The comparison of ~×, measured with the pressure chamber, and qJl, obtained using psychrometers, is shown in Fig. 1. Data are presented as the difference A~, between tlJix and W~. Positive A ~ indicate that the pressure chamber estimate, W~x, is drier than the psychrometer value, W~. Data are plotted against W~x on the abscissa; since both qJ~x and qJ~ are subject to error, tlh might equally well have been used. Data have been separated according to the time of day in which samples were collected. We shall discuss the daytime results first, and then the morning and evening data.

The pressure chamber gave drier readings than the psychrometer for leaves collected during the day. This discrepancy would appear even larger if chamber values were corrected for the 1-bar osmotic potential of the xylem sap (3). Pressure chamber measurements which are drier than those of psychrometers have been observed in a number ofother species (2, 7). However, comparisons of psychrometers and hygrometers, and of hygrometers and the pressure chamber by Baughn and Tanner (3, 4) suggest that psychrometer readings should be drier than pressure chamber readings for dry potato leaves. The difference between their data and ours may reflect differences between glasshouse-grown and field-grown plants. However, it is equally likely that psychrometer-pressure chamber differences resulted from our measurement procedure. Psychrometer and pressure chamber measurements were made on the same leaves. From excision until insertion of samples in psychrometers, leaves were covered with a damp cloth to prevent evaporation. This procedure did not affect the pressure chamber readings (19). However, if moisture from the cloth permeated the cuticle or remained on the surfaces of leaves, psychrometer readings would have been wetter than Baughn and Tanner's results. Ap- parently this occurred, for the psychrometers approached equilibrium from the wet side rather than the dry, as is usually the case. This systematic wetting appeared in all our psychrometer measurements, including those for morning and evening leaves. However, had we eliminated this error by discarding the damp cloth, the pressure chamber-psychrometer comparison might not have been improved, for vapor sorption by psychrometer walls and cuticle causes measurements to be too dry (4, 24). All other errors, such as evaporation and sap loss, which would cause dry pressure chamber readings, should produce similar shifts in the psychrometer measurement.

In contrast to the daytime data, the high water potential data during morning and evening shows both positive and negative AtlJ (Fig. 1). Some of this scatter arises because pressure chamber endpoints are less clear, and psychrometer measurements less reliable, at high water potentials. Part of the scatter may also arise from growth effects in the psychrometers.

1975) G A N D A R A N D T A N N E R : M E A S U R I N G W A T E R P O T E N T I A L S 391

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o • Morning, old leaves tx Morning, young leaves

o • Daytime leaves • Evening, old leaves

o Evening, young leaves

I I I I I I | I

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PRESSURE-CHAMBER POTENTIAL (bars)

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FIG. 1. C o m p a r i s o n of p r e s su re c h a m b e r and t h e r m o c o u p l e p s y c h r o m e t e r m e a s u r e m e n t s of leaf wa te r potent ial . A q j = qJ~-W~x = W(psych rome te r ) _tp (p ressure chamber) . Posit ive AlP indicate a drier p r e s su re c h a m b e r es t imate .

In separate experiments (18), we have established that nearly all the expansion growth of well-watered, field-grown potato leaves occurs in the late afternoon and evening. We believe that the tendency for negative AW (psychrometer drier than pressure chamber), evident in the young-leaf evening data of Fig. I is attributable to the expansion of leaf samples within the psychrometers, as described by Bayer (8) and Tinklin (31). Such expansion would occur at the expense of sample turgor, with the result that W~ is lowered. Negative Aq J were not obtained for the young morning, or the old evening and daytime leaves, because these leaves were not expand- ing.

Pressure chamber vs. in situ psychrometer for tubers---The relationship between pressure chamber measurements of Wtx, and in situ psychrometer measurements of Wt, is shown in Fig. 2. Data have been separated according to the length of time between insertion ofpsychromet- ers and measurement ofq/t. The 18- to 24-hour, and the 24- to 72-hour data provide comparisons of measurements made on the same tuber. The 5- to


10-day data were obtained by comparing psychrometer Wt values with measurements of W~x rather than Wtx. The implicit assumption that Wtx WJx was tested in a separate experiment in which we measured qs]x and qJtx of drying plants. We found (18) that under the low transpiration conditions in our glasshouse, that ~ x were 0.7 __+ 0.1 bar drier than qJtx. This difference was small enough to justify treating the W~x-Wt comparison as if it were tlJtx-~t. We have not corrected the pressure chamber readings in Fig. 2 for the osmotic potential of the tuber xylem sap. For a comparable set of tubers, we found that the average xylem sap osmotic potential was 1.5___ 0.1 bar (19). If this amount were added to the pressure chamber readings in Fig. 2, all except one of the AW values would be positive (chamber drier than psychrometer).

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FIG, 2. Comparison of pressure chamber and in sitt~ psychrometer measurements of tuber water potential. AKu = q/t-I.IJtx = i t / (psychrometer) - ~ (pressure chamber). Positive Akv indicate a drier pressure chamber estimate. Data are separated according to the length of time between insertion of psychrometers and measurement of tlJt.


In the 18- to 24-hour data of Fig. 2, there is good agreement between the chamber and psychrometer, except for tubers drier than -7 bars. The agreement between chamber and psychrometer for the 24- to 72-hour data, and particularly, the 5- to 10-day data, is poor. Positive AtI t values show that psychrometers gave much wetter readings than the pressure chamber. We believe that this is caused by gradual suberization of the tissues surrounding the psychrometer cup which slows, and eventually prevents, the attainment of water equilibrium between tuber and psychrometer. The efficacy of suberized tissue in reducing water vapor loss from tuber disks has been demonstrated (20). Wet psychrometer readings in drying tubers arise because the imposition of a suberized barrier reduces water transfer from the psychrometer cup to the surrounding tissue, leaving the vapor pressure in the cup erroneously high.

We examined the development of suberin in tissue surrounding psychrometers by staining hand-sections with 0.05% toluidine blue, a dye which colors suberized cell walls blue (6). We found that about 50% of the surface of the hole was suberized 24 hours after insertion of psychrometers; by 48 hours, suberization of the surface was almost complete and transverse walls in the top cell layer were partly suberized; after eight days, suberization of most of the cells in the first two layers of tissue surrounding the psychrometers had occurred. Comparable rates of suberization have been reported (5, 20). This rate of suberization is in keeping with the decline in psychrometer performance with time.

In addition to suberization, there are other sources of error in our pressure chamber-psychrometer comparison. The main error in the pressure chamber method is overestimation of the tuber endpoint caused by high rates of pressurization or by lack of equilibrium within the tuber (19). In both cases, ~tx would appear too dry. We minimized the first error by using rates of pressurization of less than 0.05 bar sec _1 as endpoints were approached. The second error should not be important under the low transpiration conditions of our glasshouse experiments.

There are a number of difficulties in using soil thermocouple psychrometers to measure tuber water potentialin situ. Spurious readings will be obtained if the temperature of the thermocouple differs from that of the tissue whose water potential is being measured. Temperature errors could arise from heat conduction down the lead wires, from thermal gradients along the axis of the psychrometer (22), and from the increased respiratory heat production in the wound healing process (6, 21), which follows insertion of psychrometers. We attempted to reduce the first two errors by wrapping the psychrometer leads against the tuber and by the use of insulation. Nothing could be done about respiratory heat production within the tuber. When psychrometers were in the dry-bulb condition we observed offsets of from 5 to 15~tV. These suggest that temperature increases, due to wound-induced respiration, were of the order of 0.2 C (25). We


compensated for this heat production by nulling the psychrometer output before each reading.

Errors caused by air gaps between the ceramic cup and surrounding tissue could also affect in situ psychrometer measurements. We ensured a tight fit between the psychrometer and the tuber by using an undersized hole initially, but with time, gaps could have developed. In a limited trial, we inserted psychrometers, from which the ceramic cups had been removed, into tubers. No significant improvement in the performance of these psychrometers was apparent.

Pressure chamber vs. graded osmotica method---The comparison between tuber water potentials obtained using the pressure chamber, and from weight changes in graded osmotica, is shown in Fig. 3. There is a clear trend: in tubers in which Wtx is wetter than about -5 bars, the graded osmotica estimate is drier than the pressure chamber measurement; for tubers drier than -5 bars, the converse holds.

Discrepancies shown could arise from errors in either, or both methods. However, in view of the agreement between the pressure

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FIG. 3. Comparison of pressure chamber and graded osmotica determinations of tuber water potential. AtlJ =tlJ (graded osmotica) - t14 (pressure chamber). Positive AW indicate a drier pressure chamber estimate.


chamber and psychrometer in the 18- to 24-hour data of Fig. 2, we have some confidence that there are no major systematic errors in the pressure chamber method. Therefore, the graded osmotica method appears to give unreliable measurements of tuber water potential.

The basic assumption in the graded osmotica method is that changes in the weights of tissue samples are caused by the uptake or loss of water and not by the exchange of solutes. Uptake of solutes would cause overestimation of the final weights of tissue samples so that the tuber water potential would appear too dry. Experiments have shown that about 25% of the volume of disks of tuber tissue is occupied by external solution following equilibration in osmotica (17, 30). This volume includes intercellular spaces, in which external solution replaces air, and cell wails, in which external solution replaces water (1, 23, 28). In our experiments, penetration of sucrose solution into intercellular spaces and cell walls could, at the outside, cause a one bar underestimate (more negative) of tuber water potential. Thus, solute uptake can explain only part of the negative Attt shown in Fig. 3.

Overestimates of final weights (and underestimates of water potentials) in the graded osmotica method could also be caused by uptake of external solution associated with the expansion of tissue samples. It has been established that disks of tuber tissue can show weight gains associated with cell enlargement when immersed in osmotica (W >-5 bars) and water (12, 30). These weight gains depend upon auxin-induced changes in the extensibility of cell walls, and upon the oxidative metabolism of the disks. Cell enlargement also could occur for physical reasons. In an intact tuber, cells are constrained by surrounding cells. In a tissue sample, these constraints are partially removed and expansion is possible (9). Because of the short time and low temperatures used for tissue equilibration in our experiments, removal of physical constraints seems a more likely cause for cell enlargement than metabolically-linked uptake. In our experiments, a 5% increase in tissue volume would cause a 2.5-bar underestimate of tuber water potential.

Although solute uptake and cell enlargement could explain discrepancies between the pressure chamber and the graded osmotica method for tubers wetter than -5 bars, these mechanisms cannot account for the positive AW shown for drier tubers. Here, other mechanisms must have an overriding effect. One possibility is loss of solutes from tissue plugs to the external solution (13), which could cause overestimates of tuber water potential. However, although the loss of electrolytes from tuber disks drier than -6 bars increases (14), this effect is probably more than offset by sucrose uptake. Another possibility is that dry (but not plasmolysed) tubers lose part of their ability to rehydrate on immersion in water in the same manner as disks plasmolysed in osmotica (17). If this happened to tubers drier than -5 bars, smaller weight gains in hypotonic solutions would be


expected. This would cause overestimates of graded osmotica water potentials, and hence, the positive AW of Fig. 3. However, we doubt that this is a complete explanation for the discrepancies between the pressure chamber and graded osmotica determinations of tuber water potential for dry tubers.

Both the graded osmotica and pressure chamber methods require destructive sampling. In view of the ambiguities in the graded osmotica method we recommend the faster, simpler pressure chamber method for routine measurements of tuber water potentials.

Acknowledgment

Psychrometer measurements of leaf water potential were made by J. W. Baughn.

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