Chloroplast response to low leaf water potentials: I. Role of turgor

The effect of decreases in turgor on chloroplast activity was studied by measuring the photochemical activity of intact sunflower (Helianthus annuus L. cv. Russian Mammoth) leaves having low water potentials. Leaf turgor, calculated from leaf water potential and osmotic potential, was found to be affected by the dilution of cell contents by water in the cell walls, when osmotic potentials were measured with a thermocouple psychrometer. After the correction of measurements of leaf osmotic potential, both the thermocouple psychrometer and a pressure chamber indicated that turgor became zero in sunflower leaves at leaf water potentials of −10 bars. Since most of the loss in photochemical activity occurred at water potentials below −10 bars, it was concluded that turgor had little effect on the photochemical activity of the leaves.     

Occurrence of Inverted Water Potential Gradients between Soil and Bean Roots

Measurements with a pressure chamber were made of the xylem water potential of leaves, shoots and roots from bean plants (Pkaseolus vulgaris L. cv. Processor) grown with a 12 hour dark period and natural or artificial light conditions during the day. The water potentials were measured at the end of a dark period and during the light period. Measurements taken at the end of the dark period indicated normal potential gradients within the soil/plant system (leaf < shoot < root < soil), when the matric potential of soil water was relatively high (above ?0.02 bar), and the gradients then also remained normal during the day (natural light). When the soil water potential was ?1 bar or lower in the morning, however, the root xylem water potential was higher than the soil water potential; at very low soil water potentials (< ?4 bar) it remained higher during most of the day. In this case also leaf and shoot xylem water potentials were higher than the soil water potential in the early morning, although decreasing rapidly in daylight. Under artificial light, both leaf and root water potentials were higher than the soil water potential throughout the whole diurnal cycle when the latter potential was below ?4 bar. From measurements of stomatal diffusion resistance, transpiration, relative water content of leaves and of changes in the matric potential of soil water, it was concluded that when the matric potential of soil water was low, water could be taken up by the plant against a water potential gradient. Because leaf xylem water potential was always lower than root xylem water potential, the mechanism involved in the inversion of water potential gradient must be localized in the roots, and probably related to ion uptake. Symbols and abbreviations used in the text: Ψ: Plant water potential (thermocouple psychrometer); Ψx: Xylem water potential (pressure chamber); Ψs: Osmotic potential of xylem sap; Ψm: Matric potential of soil water; RWC: Relative water content.     

Comparison of the dye method with the thermocouple psychrometer for measuring leaf water potentials

The dye method for measuring water potential was examined and compared with the thermocouple psychrometer method in order to evaluate its usefulness for measuring leaf water potentials of forest trees and common laboratory plants. Psychrometer measurements are assumed to represent the true leaf water potentials. Because of the contamination of test solutions by cell sap and leaf surface residues, dye method values of most species varied about 1 to 5 bars from psychrometer values over the leaf water potential range of 0 to −30 bars. The dye method is useful for measuring changes and relative values in leaf potential. Because of species differences in the relationships of dye method values to true leaf water potentials, dye method values should be interpreted with caution when comparing different species or the same species growing in widely different environments. Despite its limitations the dye method has a usefulness to many workers because it is simple, requires no elaborate equipment, and can be used in both the laboratory and field.     

Relationship between leaf and xylem water potentials in rice plants

Leaf and xylem water potentials were measured in rice plantswith and without transpiration using a thermocouple psychrometerand a pressure chamber. The leaf water potential practicallycoincided with the xylem water potential in leaves without transpiration,while the latter was 3–5 bars lower when intense transpirationwas occurring. The pressure chamber should not be used to measureleaf water potential during intense transpiration in the field.The water status in transpiring leaves is discussed. (Received March 6, 1978; )     

Errors in measuring water potentials of small samples resulting from water adsorption by thermocouple psychrometer chambers

The adsorption of water by thermocouple psychrometer assemblies is known to cause errors in the determination of water potential. Experiments were conducted to evaluate the effect of sample size and psychrometer chamber volume on measured water potentials of leaf discs, leaf segments, and sodium chloride solutions. Reasonable agreement was found between soybean (Glycine max L. Merr.) leaf water potentials measured on 5-millimeter radius leaf discs and large leaf segments. Results indicated that while errors due to adsorption may be significant when using small volumes of tissue, if sufficient tissue is used the errors are negligible. Because of the relationship between water potential and volume in plant tissue, the errors due to adsorption were larger with turgid tissue. Large psychrometers which were sealed into the sample chamber with latex tubing appeared to adsorb more water than those sealed with flexible plastic tubing. Estimates are provided of the amounts of water adsorbed by two different psychrometer assemblies and the amount of tissue sufficient for accurate measurements of leaf water potential with these assemblies. It is also demonstrated that water adsorption problems may have generated low water potential values which in prior studies have been attributed to large cut surface area to volume ratios.     

Matric potentials of leaves

A pressure chamber was used to measure matric potentials of frozen and thawed leaves. Significant matric potentials were demonstrated in sunflower (Helianthus annuus L.), yew (Taxus cuspidata Sieb. and Zucc.), and rhododendron (Rhododendron roseum Rehd.). Matric potentials were particularly negative in rhododendron and were correlated with the amount of cell wall present and with the volume of water outside the leaf protoplasts at comparable matric potentials. It was concluded that matric forces in leaves are associated mainly with cell walls, at least within the physiological range of water contents. Calculations indicated that the water potential of the solution in the cell wall could be estimated for living tissue from the sum of matric and osmotic potentials acting on water outside the protoplasts.     

XYLEM WATER HOLDING CAPACITY AS A SOURCE OF ERROR IN WATER POTENTIAL ESTIMATES MADE WITH THE PRESSURE CHAMBER AND THERMOCOUPLE PSYCHROMETER

The pressure chamber and the thermocouple psychrometer often provide different values when used to estimate plant water potential. One hypothesis to explain the discrepancy between instruments is that water movement between the xylem and symplast occurs during pressurization in the pressure chamber. Pressure chamber and thermocouple psychrometer measurements of Pinus ponderosa (Laws.) seedling shoots and mature Quercus agrifolia (Nee) shoots showed that the discrepancy is greater for Quercus. It was hypothesized that the xylem water content-water potential relationship of these species would explain the magnitude of the discrepancy between instruments. The xylem water holding capacity alone, however, does not explain the difference between species. The larger discrepancy in Quercus is likely due to a greater volume of water held in the xylem relative to the volume held in the symplast.     

Relationship of water potential to growth of leaves

A thermocouple psychrometer that measures water potentials of intact leaves was used to study the water potentials at which leaves grow. Water potentials and water uptake during recovery from water deficits were measured simultaneously with leaves of sunflower (Helianthus annuus L.), tomato (Lycopersicon esculentum Mill.), papaya (Carica papaya L.), and Abutilon striatum Dickson. Recovery occurred in 2 phases. The first was associated with elimination of water deficits; the second with cell enlargement. The second phase was characterized by a steady rate of water uptake and a relatively constant leaf water potential. Enlargement was 70% irreversible and could be inhibited by puromycin and actinomycin D. During this time, leaves growing with their petioles in contact with pure water remained at a water potential of —1.5 to —2.5 bars regardless of the length of the experiment. It was not possible to obtain growing leaf tissue with a water potential of zero. It was concluded that leaves are not in equilibrium with the potential of the water which is absorbed during growth. The nonequilibrium is brought about by a resistance to water flow which requires a potential difference of 1.5 to 2.5 bars in order to supply water at the rate necessary for maximum growth.

Leaf growth occurred in sunflower only when leaf water potentials were above —3.5 bars. Sunflower leaves therefore require a minimum turgor for enlargement, in this instance equivalent to a turgor of about 6.5 bars. The high water potentials required for growth favored rapid leaf growth at night and reduced growth during the day.

     

Direct Demonstration of a Growth-Induced Water Potential Gradient

When transpiration is negligible, water potentials in growing tissues are less than those in mature tissues and have been predicted to form gradients that move water into the enlarging cells. To determine directly whether the gradients exist, we measured water potentials along the radius of stems of intact soybean (Glycine max [L.] Merr.) seedlings growing in vermiculite in a water-saturated atmosphere. The measurements were made in individual cells by first determining the turgor with a miniature pressure probe, then determining the osmotic potential of solution from the same cell, and finally summing the two potentials. The osmotic potentials were corrected for sample mixing in the probe. The measurements were checked with a thermocouple psychrometer that gave average tissue water potentials. In the elongating region, the water potential was highest near the xylem and lowest near the epidermis and in the center of the pith. In the basal, more mature region of the same stems, water potentials were near zero next to the xylem and throughout the tissue. These basal potentials reflected mostly the potential of the xylem, which extended into the elongating tissues. Thus, the high basal potential confirmed the high potential near the xylem in the elongating tissues. The psychrometer measurements for each tissue gave average potentials that agreed with the average of the cell potentials from the pressure probe. We conclude that a radial gradient was present in the elongating region that formed a water potential field in three dimensions around the xylem and that confirmed the predictions of Molz and Boyer (F.J. Molz and J.S. Boyer [1978] Plant Physiol 62: 423-429).     

Direct and indirect measurements of Phloem turgor pressure in white ash

Direct determinations and indirect calculations of phloem turgor pressure were compared in white ash (Fraxinus americana L.). Direct measurements of trunk phloem turgor were made using a modified Hammel-type phloem needle connected to a pressure transducer. Turgor at the site of the direct measurements was calculated from the osmotic potential of the phloem sap and from the water potential of the xylem. It was assumed that the water potentials of the phloem and xylem were close to equilibrium at any one trunk location, at least under certain conditions. The water potential of the xylem was determined from the osmotic potential of xylem sap and from the xylem tension of previously bagged leaves, measured with a pressure chamber. The xylem tension of bagged leaves on a branch adjacent to the site of the direct measurements was considered equivalent to the xylem tension of the trunk at that point. While both the direct and indirect measurements of phloem turgor showed clear diurnal changes, the directly measured pressures were consistently lower than the calculated values. It is not clear at present whether the discrepancy between the two values lies primarily in the calculated or in the measured pressures, and thus, the results from both methods as described here must be regarded as estimates of true phloem turgor.     

Effects of salt secretion on psychrometric determinations of water potential of cotton leaves

Thermocouple psychrometers gave lower estimates of water potential of cotton leaves than did a pressure chamber. This difference was considerable for turgid leaves, but progressively decreased for leaves with lower water potentials and fell to zero at water potentials below about −10 bars. The conductivity of washings from cotton leaves removed from the psychrometric equilibration chambers was related to the magnitude of this discrepancy in water potential, indicating that the discrepancy is due to salts on the leaf surface which make the psychrometric estimates too low. This error, which may be as great as 400 to 500%, cannot be eliminated by washing the leaves because salts may be secreted during the equilibration period. Therefore, a thermocouple psychrometer is not suitable for measuring the water potential of cotton leaves when it is above about −10 bars.     

Psychrometric Field Measurement of Water Potential Changes following Leaf Excision

In situ measurement of sudden leaf water potential changes has not been performed under field conditions. A laboratory investigation involving the measurement of leaf water potential prior to and 2 to 200 minutes after excision of citrus leaves (Citrus jambhiri) showed good linear correlation (r = 0.99) between in situ leaf psychrometer and Scholander pressure chamber measurements. Following this, a field investigation was conducted which involved psychrometric measurement prior to petiole excision and 1 minute after excision. Simultaneous pressure chamber measurements were performed on neighboring leaves prior to the time of excision and then on the psychrometer leaf about 2 minutes after excision. These data indicate that within the first 2 minutes after excision, psychrometer and pressure chamber measurements were linearly correlated (r = 0.97). Under high evaporative demand conditions, the rate of water potential decrease was between 250 and 700 kilopascals in the first minute after excision. These results show that the thermocouple psychrometer can be used as a dynamic and nondestructive field technique for monitoring leaf water potential.     

Pressure probe and isopiestic psychrometer measure similar turgor

Turgor measured with a miniature pressure probe was compared to that measured with an isopiestic thermocouple psychrometer in mature regions of soybean (Glycine max [L.] Merr.) stems. The probe measured turgor directly in cells of intact stems whereas the psychrometer measured the water potential and osmotic potential of excised stem segments and turgor was calculated by difference. When care was taken to prevent dehydration when working with the pressure probe, and diffusive resistance and dilution errors with the psychrometer, both methods gave similar values of turgor whether the plants were dehydrating or rehydrating. This finding, together with the previously demonstrated similarity in turgor measured with the isopiestic psychrometer and a pressure chamber, indicates that the pressure probe provides accurate measurements of turgor despite the need to penetrate the cell. On the other hand, it suggests that as long as precautions are taken to obtain accurate values for the water potential and osmotic potential, turgor can be determined by isopiestic psychrometry in tissues not accessible to the pressure probe for physical reasons.     

A rapid leaf-disc sampler for psychrometric water potential measurements

An instrument was designed which facilitates faster and more accurate sampling of leaf discs for psychrometric water potential measurements. The instrument consists of an aluminum housing, a spring-loaded plunger, and a modified brass-plated cork borer. The leaf-disc sampler was compared with the conventional method of sampling discs for measurement of leaf water potential with thermocouple psychrometers on a range of plant material including Gossypium hirsutum L., Zea mays L., and Begonia rex-cultorum L. The new sampler permitted a leaf disc to be excised and inserted into the psychrometer sample chamber in less than 7 seconds, which was more than twice as fast as the conventional method. This resulted in more accurate determinations of leaf water potential due to reduced evaporative water losses. The leaf-disc sampler also significantly reduced sample variability between individual measurements. This instrument can be used for many other laboratory and field measurements that necessitate leaf disc sampling.     

Measurement of the water potential of stored potato tubers

A method of measuring the water potential of stored potato tubers (Solanum tuberosum L.) was needed to investigate the relationship of bacterial soft rot in tubers to water potential. Pressure chamber measurements, while useful for tubers with functional stolons, cannot be made on stored tubers. Measurements could be made on excised tissue pieces in a hygrometer chamber and with hygrometers implanted into tubers. We report here our evaluation of these hygrometric methods using a comparison with the pressure chamber on tubers harvested with stolons intact.

In tubers of high water potential, measurements on excised tissue were as much as 0.5 megapascals lower than the pressure chamber, probably due to turgor-driven expansion of the sample when freed from constraints imposed by surrounding tissue. Good agreement (±0.05 megapascals) was found between the implanted hygrometer and the pressure chamber at potentials higher than −0.5 megapascals. At lower water potentials, both hygrometer measurements were higher than the pressure chamber. Respirational heating of the tissue contributed to the increase in the excised tissue samples, but not with the implanted hygrometers because of the hygrometer design. The osmotic pressure balanced the pressure chamber measurement of potential at −0.7 megapascals, but was too small to do so at lower potentials. At most, 25% of this discrepancy can be accounted for by dilution by apoplastic water. We believe that the pressure chamber measurement is too low at low water potentials and that the error is associated with air bubbles in the xylem. At low potentials air emerged from xylem vessels along with sap, and fewer xylem emitted sap as potentials decreased.

     

Water potential gradient in a tall sequoiadendron

With an elevator installed in a 90-meter tall Sequoiadendron to collect the samples, xylem pressure potential measurements were made approximately every 15 meters along 60 meters of the tree's height. The measured gradient was about −0.8 bar per 10 meters of height, i.e., less than the hydrostatic gradient. Correction of the xylem pressure potential data by calibration against a thermocouple psychrometer confirmed this result. Similar gradients are described in the literature in tall conifers at times of low transpiration, although a different sampling technique was used. If the data in the present study and those supporting it are typical, they imply a re-evaluation of either the use of the pressure chamber to estimate water potential or the present theories describing water transport in tall trees.     

Evaluation of water stress control with polyethylene glycols by analysis of guttation

The water relations of pepper plants (Capsicum frutescens L.) under conditions conducive to guttation were studied to evaluate the control of plant water stress with polyethylene glycols. The addition of polyethylene glycol 6000 to the nutrient solution resulted in water relations similar to those expected in soil at the same water potentials. Specifically, xylem pressure potential in the root and leaf became more negative during a 24-hour treatment period, while osmotic potential of the root xylem sap remained constant. The decrease in pressure potential was closely correlated with the decrease in osmotic potential of the nutrient solution. In contrast, the addition of polyethylene glycol 400 to the nutrient medium resulted in a reduction of osmotic potential in the root xylem sap; this osmotic adjustment in the xylem was large enough to establish an osmotic gradient for entry of water and cause guttation at a nutrient solution osmotic potential of −4.8 bars. Pressure potential in the root and leaf xylem became negative only at nutrient solution osmotic potentials lower than −4.8 bars. About half of the xylem osmotic adjustment in the presence of polyethylene glycol 400 was caused by increased accumulation of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the root xylem. These studies indicate that larger polyethylene glycol molecules such as polyethylene glycol 6000 are more useful for simulating soil water stress than smaller molecules such as polyethylene glycol 400.     

In situ field measurement of leaf water potential using thermocouple psychrometers

Thermocouple psychrometers are the only instruments which can measure the in situ water potential of intact leaves, and which can possibly be used to monitor leaf water potential. Unfortunately, their usefulness is limited by a number of difficulties, among them fluctuating temperatures and temperature gradients within the psychrometer, sealing of the psychrometer chamber to the leaf, shading of the leaf by the psychrometer, and resistance to water vapor diffusion by the cuticle when the stomates are closed. Using Citrus jambhiri, we have tested several psychrometer design and operational modifications and showed that in situ psychrometric measurements compared favorably with simultaneous Scholander pressure chamber measurements on neighboring leaves when the latter were corrected for the osmotic potential.     

Which water potential? Differences between isopiestic thermocouple psychrometer measurements of intact and excised plant materials

Water potentials of leaves from well-watered plants were measured. There were species-specific differences in both the total and the osmotic potentials of pea (Pisum sativum), tradescantia (Tradescantia versicolor), rose (Rosa hybrida), bitter lemon (Citrus aurantium) and olive (Olea europaea). With tradescantia the potential measured after the destruction of turgor by freezing was less negative than before, a result which suggests that the value obtained is not identical with the real osmotic potential of the leaf. detached leaves of all species showed less negative water potential readings, and those of pea even a less negative osmotic potential, when cut into five pieces than when measured intact. Application of vaseline to the cut surface of the leaves reduced this effect with rose and olive, though not with tradescantia and pea. Measurements were also made of the water potentials of comparable leaves of tradescantia and bitter lemon, attached to and detached from their plants; when bitter lemon leaves were detached and watered through their petioles which protruded outside the thermocouple chamber, their potential became considerably less negative than when the same leaves had been attached to well watered plants. However, similar leaves whose cut petioles were introduced into the thermocouple chamber registered an even less negative potential. The results are consistent with the hypothesis that when a leaf is cut off a plant, and even more so when it is cut into sections, the water previously held by matrix forces becomes available to dilute the “spilled” cell sap and to be absorbed by adjacent cells and thereby to increase their turgor and render the net water potential of the leaf less negative. Similarly, the apparent negative turgor of the succulent, tradescantia leaves is likely to be due to dilution of the osmotic component by cell wall water. The discrepancies between the readings of attached and detached leaves indicate a considerable whole-plant matrix component, and the results as a whole suglest that thermocouple psychrometer readings carried out on detached and even more on cut-up leaves may be artifacts and that it is desirable to determine water potentials on leaves attached to their plants. The work was supported by a Government of Israel Fellowship and was conducted at the Department of Pomology and Viticulture, Faculty of Agriculture of the Hebrew University of Jerusalem, Rehovot, Israel.     

Applications of the compensating pressure theory of water transport

Some predictions of the recently proposed theory of long-distance water transport in plants (the Compensating Pressure Theory) have been verified experimentally in sunflower leaves. The xylem sap cavitates early in the day under quite small water stress, and the compensating pressure P (applied as the tissue pressure of turgid cells) pushes water into embolized vessels, refilling them during active transpiration. The water potential, as measured by the pressure chamber or psychrometer, is not a measure of the pressure in the xylem, but (as predicted by the theory) a measure of the compensating pressure P. As transpiration increases, P is increased to provide more rapid embolism repair. In many leaf petioles this increase in P is achieved by the hydrolysis of starch in the starch sheath to soluble sugars. At night P falls as starch is reformed. A hypothesis is proposed to explain these observations by pressure-driven reverse osmosis of water from the ground parenchyma of the petiole. Similar processes occur in roots and are manifested as root pressure. The theory requires a pump to transfer water from the soil into the root xylem. A mechanism is proposed by which this pump may function, in which the endodermis acts as a one-way valve and a pressure-confining barrier. Rays and xylem parenchyma of wood act like the xylem parenchyma of petioles and roots to repair embolisms in trees. The postulated root pump permits a re-appraisal of the work done by evaporation during transpiration, leading to the proposal that in tall trees there is no hydrostatic gradient to be overcome in lifting water. Some published observations are re-interpreted in terms of the theory: doubt is cast on the validity of measurements of hydraulic conductance of wood; vulnerability curves are found not to measure the cavitation threshold of water in the xylem, but the osmotic pressure of the xylem parenchyma; if measures of xylem pressure and of hydraulic conductance are both suspect, the accepted view of the hydraulic architecture of trees needs drastic revision; observations that xylem feeding insects feed faster as the water potential becomes more negative are in accord with the theory; tyloses, which have been shown to form in vessels especially vulnerable to cavitation, are seen as necessary for the maintenance of P, and to conserve the supplementary refilling water. Far from being a metastable system on the edge of disaster, the water transport system of the xylem is ultrastable: robust and self-sustaining in response to many kinds of stress.