The citrus leaf cuticle in relation to measurement of leaf water potential using thermocouple psychrometers*

Abstract. Cuticular resistance to water vapour diffusion is an important aspect of thermocouple psychrometry and may introduce significant error in the measurement of leaf water potential (Ψ). The effect of the citrus (Citrus mitis Blanco) leaf cuticle on water vapour movement was studied using the times required for vapour pressure equilibration during thermocouple psychrometric measurement of Ψ. Cuticular abrasion with various carborundum powders was used to reduce the diffusive resistance of both the adaxial and abaxial leaf surfaces, and the extent of the disruption to the leaf was investigated with light and electron microscopy. Cuticular abrasion resulted in reduced equilibration times due to decreased cuticular resistance and greater water vapour movement between the leaf and the psychrometer chamber. Equilibration times were reduced from over 5 h in the unabraded control leaves to 1 h with cuticle abrasion. This was associated with the decrease in diffusive resistance with cuticular abrasion from over 55 s cm^?1 to less than 8 s cm^?1 for both the adaxial and abaxial leaf surfaces. Scanning electron micrographs of the abraded leaf tissue revealed considerable disruption of the stomatal ledge and of the guard cells, surface smoothing and displacement of waxes into the stomatal aperture, and damage to veins. Observations with the transmission electron microscope revealed frequent disruption of epidermal cell walls, and damage to both the cytoplasmic and vacuolar membranes.     

Electron Microscope Study of Cuticular Abrasion on Cotton Leaves in Relation to Water Potential Measurements

Wullschleger, S. D. and Oosterhuis, D. M. 1987. Electron microscopestudy of cuticular abrasion on cotton leaves in relation towater potential measurements.—J. exp. Bot 38: 660–667. Accurate determination of plant water potential using thermocouplepsychrometers requires vapour equilibrium between the tissuesample and the sensing psychrometer junction. Failure to achievethis equilibrium due to cuticular resistances to vapour movementmay introduce significant errors into psychrometrically measured. The effect of cuticular abrasion on W equilibration timesfor cotton (Gossypium hirsutum L.) was studied with three typesof thermocouple psychrometers, and the extent of surface andcuticular damage was determined with electron microscopy. Water vapour equilibration between the leaf and psychrometerchamber was achieved in approximately 4 h for unabraded controls,whereas abrasion of the leaf with carborundum powder consistentlyreduced equilibration times to below 2?5 h for all three typesof psychrometers. Microscopic observation of abraded leaf tissueindicated that substantial damage to surface structures occurredduring the cuticular abrasion process. Scanning electron micrographsrevealed localized cellular damage to anastomosing leaf veinsand physical disruption of both the stomatal complex and glandulartrichomes. Transverse sections viewed with a transmission electronmicroscope indicated substantial direct damage to the cuticlewith large sections of cell wall devoid of a cuticular layer.Although the exposed cell walls were intact, the lateral cellwalls were physically compressed and distorted during abrasion.In addition, the cytoplasmic and vacuolar membranes of the epidermalcells were also frequently ruptured. Evaluation of the damagefollowing abrasion indicated that the release of turgor by theaffected cells may contribute to increased sample variabilityand possibly to errors in measurements. Key words: Leaf water potential, cuticular resistance, thermocouple psychrometer     

Leaf water potentials measured with a pressure chamber

Leaf water potentials were estimated from the sum of the balancing pressure measured with a pressure chamber and the osmotic potential of the xylem sap in leafy shoots or leaves. When leaf water potentials in yew, rhododendron, and sunflower were compared with those measured with a thermocouple psychrometer known to indicate accurate values of leaf water potential, determinations were within ± 2 bars of the psychrometer measurements with sunflower and yew. In rhododendron. water potentials measured with the pressure chamber plus xylem sap were 2.5 bars less negative to 4 bars more negative than psychrometer measurements.

The discrepancies in the rhododendron measurements could be attributed, at least in part, to the filling of tissues other than xylem with xylem sap during measurements with the pressure chamber. It was concluded that, although stem characteristics may affect the measurements, pressure chamber determinations were sufficiently close to psychrometer measurements that the pressure chamber may be used for relative measurements of leaf water potentials, especially in sunflower and yew. For accurate determinations of leaf water potential, however, pressure chamber measurements must be calibrated with a thermocouple psychrometer.

     

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.     

The Relationship Between Leaf Thickness and Plant Water Potential

Leaf thickness was continuously measured in a wide range ofenvironments using a new type of displacement transducer whichis easy to set-up and automatically compensates for the effectsof temperature. Simultaneous measurements were made of waterpotential using either a psychrometer attached to the leaf petioleor a leaf pressure chamber. Thickness of leaves was a sensitiveindicator of plant water status but calibrations against anindependent method were necessary in every plant for accurateestimates of water potential. The relationship between leafthickness changes and water potential, measured in detachedleaves, was usually curvilinear and was strongly influencedby leaf age, stress history and, in young leaves, by the effectsof leaf growth. Leaf thickness growth was absent in mature cabbageleaves. Key words: Leaf thickness, plant water potential, psychrometer     

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.     

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.     

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.     

Improved Thermocouple Psychrometer for the Measurement of Plant and Soil Water Potential: I. THERMOCOUPLE PSYCHROMETRY AND AN IMPROVED INSTRUMENT DESIGN

This paper is the first of a series which describes: (a) thedesign and operation of a thermocouple psychrometer which providesimproved range of measurement, accuracy, and equilibration rateof water potential determinations of plant and soil samples,and which avoids or minimizes six sources of large error, oneor more of which occur with previous psychrometers and (b) studiesthat provide a better understanding of the thermocouple psychrometricmethod. The present paper describes the main difficulties encounteredwith the method, examines those due to absorption phenomena,and describes an improved thermocouple psychrometer which overcomesor reduces some of these difficulties. Improvements include: (i) chamber walls of stainless steel type316 and samples arranged in shielding geometries to reduce delaysin equilibration due to adsorption, (ii) sample holders of geometriesthat avoid or reduce errors of leaf resistance, adsorption andtissue damage when appropriate cooling periods and tissue segmentsof adequate size are used, (iii) shielding arrangement of respiringtissue samples to permit thermal equilibration in the regionsensed by the ‘active’ thermojunction, a new thermocoupleassembly for increased range of measurement of water potential,(v) sample holders that permit plant or soil samples to be accommodatedin the one chamber.     

Theoretical and experimental errors for in situ measurements of plant water potential

Errors in psychrometrically determined values of leaf water potential caused by tissue resistance to water vapor exchange and by lack of thermal equilibrium were evaluated using commercial in situ psychrometers (Wescor Inc., Logan, UT) on leaves of Tradescantia virginiana (L.). Theoretical errors in the dewpoint method of operation for these sensors were demonstrated. After correction for these errors, in situ measurements of leaf water potential indicated substantial errors caused by tissue resistance to water vapor exchange (4 to 6% reduction in apparent water potential per second of cooling time used) resulting from humidity depletions in the psychrometer chamber during the Peltier condensation process. These errors were avoided by use of a modified procedure for dewpoint measurement. Large changes in apparent water potential were caused by leaf and psychrometer exposure to moderate levels of irradiance. These changes were correlated with relatively small shifts in psychrometer zero offsets (−0.6 to −1.0 megapascals per microvolt), indicating substantial errors caused by nonisothermal conditions between the leaf and the psychrometer. Explicit correction for these errors is not possible with the current psychrometer design.     

The Effect of Leaf Water Potential, Leaf Temperature and Light Intensity on Leaf Diffusion Resistance and the Transpiration of Leaves of Malus sylvestris

The relationship between leaf resistance to water vapour diffusion and each of the factors leaf water potential, light intensity and leaf temperature was determined for leaves on seedling apple trees (Malus sylvestris Mill. cv. Granny Smith) in the laboratory. Leaf cuticular resistance was also determined and transpiration was measured on attached leaves for a range of conditions. Leaf resistance was shown to be independent of water potential until potential fell below — 19 bars after which leaf resistance increased rapidly. Exposure of leaves to CO₂-free air extended the range for which resistance was independent of water potential to — 30 bars. The light requirement for minimum leaf resistance was 10 to 20 W m^?2 and at light intensities exceeding these, leaf resistance was unaffected by light intensity. Optimum leaf temperature for minimum diffusion resistance was 23 ± 2°C. The rate of change measured in leaf resistance in leaves given a sudden change in leaf temperature increased as the magnitude of the temperature change increased. For a sudden change of 1°C in leaf temperature, diffusion resistance changed at a rate of 0.01 s cm^?1 min^?1 whilst for a 9°C leaf temperature change, diffusion resistance changed at a rate of 0.1 s cm^?1 min^?1. Cuticular resistance of these leaves was 125 s cm^?1 which is very high compared with resistances for open stomata of 1.5 to 4 s cm^?1 and 30 to 35 s cm^?1 for stomata closed in the dark. Transpiration was measured in attached apple leaves enclosed in a leaf chamber and exposed to a range of conditions of leaf temperature and ambient water vapour density. Peak transpiration of approximately 5 × 10^?6 g cm^?2 s^?1 occurred at a vapour density gradient from the leaf to the air of 12 to 14 g m^?3 after which transpiration declined due presumably to increased stomatal resistance. Leaves in CO₂-free air attained a peak transpiration of 11 × 10^?6 g cm^?2 s^?1 due to lower values of leaf resistance in CO₂ free air. Transpiration then declined in these leaves due to development of an internal leaf resistance (of up to 2 s cm^?1). The internal resistance was masked in leaves at normal CO₂ concentrations by the increase in stomatal resistance.     

Leaf Discs or Press Saps? A Comparison of Techniques for the Determination of Osmotic Potentials in Freeze-Thawed Leaf Material

Two standard methods for the determination of osmotic potentialsin plant organs, thermocouple hygrometry of press saps and offreeze-thawed leaf discs, were compared on leaves of ten species.Strictly parallel samples invariably showed lower (more negative)osmotic potentials for the leaf discs. Evidence is presentedthat osmotically-active solutes are not completely extractedduring the preparation of press saps and that the discs contain,on average, a smaller amount of apoplastic water than the saps.Hydrolysis of macromolecules in the discs was not responsiblefor the differences. The presence of the cutinized epidermisresults in a comparatively long equilibration time for leafdiscs, which may be reduced by appropriate measures. Key words: Thermocouple psychrometry, vapour pressure equilibration, leaf discs, press saps, solute extraction     

Measured and Calculated Transpiration in Trifolium repens under Different Water Potentials

The relationship between transpiration measured gravimetrically,a generalized transpiration equation, and the ratio VPD/r_leafwas investigated in Trifolium repens plants subjected to varyingwater potentials. Dawn leaf water potential was measured witha pressure chamber, leaf diffusion resistance with a diffusionporometer, leaf temperatures with a thermistor, and relativehumidity with an aspirated psychrometer. During drought transpirationrates determined by both methods were quite similar particularlyat the lowest water potentials. After rewatering calculatedrates were somewhat higher than measured ones. It is concludedthat transpiration calculated by the indirect method is a usefuland reasonable estimate of transpiration for single plants undervarying water potentials.     

Diurnal Changes in Citrus Leaf Thickness, Leaf Water Potential and Leaf to Air Temperature Difference

Diurnal changes in leaf water potential and leaf thickness ofwell-watered citrus trees were found to be highly correlated.Midday decreases in leaf thickness of about 30–35 µm reflected midday decreases in leaf water potential of about1.1–1.3 MPa from predawn values. Leaf water potentialwas also correlated with changes in leaf-to-air temperaturedifference and ambient vapour pressure deficit. Leaf thicknessas well as leaf to air temperature difference could possiblybe used to monitor leaf water status continuously as an indicatorof citrus tree water stress.     

Influence of Leaf Age, Soil Moisture, VPD and Time of Day on Leaf Conductance of Various Musa Genotypes in a Humid Forest-Moist Savanna Transition Site

Leaf age effects on the leaf conductance to water vapour diffusionof the adaxial and abaxial leaf surfaces were measured in themorning and in the afternoon on 17 different plantain and banana(Musa spp.) genotypes. The irradiance levels increased three-foldwhile leaf to air vapour pressure deficit levels increased two-to four-fold from morning to afternoon during the sampling periodin a field site located in the humid forest-moist savanna transitionzone of Nigeria. Conductance values were reduced in older, andsenescing leaves relative to the young and mature leaves bothin the morning and in the afternoon. Conductances were higherfor the abaxial leaf surfaces than the adaxial surface and higherin the afternoon than in the morning, with some genotypic differences.Lower values of leaf conductance to water vapour in the afternoonunder a short dry spell was sufficiently variable (P 0·05)among the test genotypes to indicate potential adaptation totransient dry conditions. Differential and relative leaf conductanceadjustments were noted among genotypes experiencing a shortdry spell versus non-limiting soil moisture conditions. Significantgenotypic differences were observed for leaf conductance amongthe 17 genotypes during the afternoon on the lower leaf surfaceof younger leaves. ABB cooking banana cultivars 'Fougamou' and'Bluggoe' might be potentially promising cultivars for transientdry conditions while AAB plantain 'Bobby Tannap' and one ofits hybrids TMPx 582-4 could be very sensitive to short dryspells according to this evaluation.Copyright 1994, 1999 AcademicPress Musa spp., Musa hybrids, adaxial leaf surface, abaxial leaf surface, stomatal response     

Comparison of water potentials measured by in situ psychrometry and pressure chamber in morphologically different species

Leaf water potentials measured by in situ psychrometry were compared with leaf water potentials measured by the pressure chamber technique at various values of water potential in Helianthus annuus, Helianthus nuttallii, Vigna unguiculata, Nerium oleander, Pistacia vera, and Corylus avellana. In V. unguiculata, the leaf water potentials measured by the in situ psychrometer oscillated at the same periodicity as, and proportional to, the leaf conductance. In all species, potentials measured by in situ psychrometers operating in the psychrometric mode were linearly correlated with potentials measured with the pressure chamber. However, the in situ psychrometers underestimated the leaf water potential in the two Helianthus species at low water potentials and overestimated the water potential in P. vera, N. oleander, and C. avellana. The underestimation in the two Helianthus species at low water potentials resulted from differences in water potential across the leaf. The overestimation in P. vera, N. oleander, and C. avellana was considered to arise from low epidermal conductances in these species even after abrasion of the cuticle. Pressure-volume studies with Lycopersicon esculentum showed that less water was expressed from distal than proximal leaflets when the whole leaf was slowly pressurized. The implication of this for water relations characteristics obtained by pressure-volume techniques is discussed. We conclude that in situ psychrometers are suitable for following dynamic changes in leaf water potential, but should be used with caution on leaves with low epidermal conductances.     

Temperature and Water Relations for Sun and Shade Leaves of a Desert Broadleaf, Hyptis emoryi

The temperature and water relations of sun versus shade leavesof Hyptis emoryi Torr. were evaluated from field measurementsmade in late summer. Throughout most of the day sun leaves hadhigher temperatures and higher resistances to water vapour diffusion,but lower transpiration rates and lower stem water potentials,than did shade leaves. Leaf absorptivity to solar irradiationwas less for 1.5-cm-long sun leaves (0.44) than for 4.0-cm shadeleaves (0.56). For both leaf types the stomatal resistance increasedas the water vapour concentration drop from the leaf to theair increased. Energy balance equations were used together with the measuredtemperature dependence of photosynthesis to predict the effectof variations in leaf absorptivity, length, and resistance onnet photosynthesis. The influence of leaf dimorphism on wholeplants was determined by calculating daily photosynthesis andtranspiration for plants with various percentages of sun andshade leaves. A hypothetical plant with all sun leaves in thesun had about twice the photosynthesis and half the transpirationratio as did plants with sun leaves in the shade or shade leavesin the sun or shade. Plants with both sun and shade leaves hadthe highest predicted photosynthesis per unit ground area. Thepossible adaptive significance of the seasonal variation insun and shade leaf percentages observed for individual H. emoryibushes is discussed in terms of water economy and photosynthesi     

Nitrogenase Activity, Photosynthesis and Nodule Water Potential in Soyabean Plants Experiencing Water Deprivation

The effects of water deprivation on the activity of nodulesof soyabean (Glycine max) have been investigated in controlledenvironments. The water potential of detached nodules was measuredusing a Wescor psychrometer and a specially designed ten chamberpsychrometer; each chamber was capable of holding up to sixnodules which allowed for simultaneous measurements of betweenten and 60 nodules. Nitrogenase activity (acetylene reduction)and respiration of intact, undisturbed nodulated roots was measuredin a flow-through gas system. The equilibration pattern of singlenodules in a Wescor chamber showed a distinct biphasic pattern.Differences between the pseudo- and the true equilibrium valuesof water potential suggest that water potential gradients closeto 01 MPa exist within the nodule tissue. Such gradients probablyreflect histological discontinuities. When detached noduleswere allowed to dry, decreases in water potential and diameterwere accompanied by increases in the resistance to water vapourloss through diffusion. These changes provide evidence for anatomicalmodifications which might provide some regulation of water loss. During a 7 d period of water deprivation there was a close relationshipbetween decreases in leaf and nodule water potential. Nitrogenaseactivity showed a 70% decrease during the first 4 d, whilstphotosynthesis only declined by 5%. It is suggested that waterstress exerts an influence on nitrogenase activity which isindependent of the rate of photosynthesis; it acts directlyon nodule activity through increases in the resistance to oxygendiffusion to the bacteroids. The data suggest that the linearrelationship between oxygen diffusion resistance and water potentialis more important than any reductions in photosynthate supply. Key words: Nitrogen fixation, water potential, photosynthesis     

A system for measuring leaf gas exchange based on regulating vapour pressure difference

A system for measurement of leaf gas exchange while regulating leaf to air vapour pressure difference has been developed; it comprises an assimilation chamber, leaf temperature controller, mass flow controller, dew point controller and personal computer. A relative humidity sensor and air and leaf temperature sensors, which are all used for regulating the vapour pressure difference, are mounted into the chamber. During the experiments, the computer continuously monitored the photosynthetic parameters and measurement conditions, so that accurate and intenstive measurements could be made.When measuring the light-response curve of CO₂ assimilation for single leaves, in order to regulate the vapour pressure difference, the leaf temperature and relative humidity in the chamber were separately and simultaneously controlled by changing the air temperature around the leaf and varying the air flow rate through the chamber, respectively. When the vapour pressure difference was regulated, net CO₂ assimilation, transpiration and leaf conductance for leaves of rice plant increased at high quantum flux density as compared with those values obtained when it was not regulated.When measuring the temperature-response curve of CO₂ assimilation, the regulation of vapour pressure difference was manipulated by the feed-forward control of the dew point temperature in the inlet air stream. As the vapour pressure difference was regulated at 12 mbar, the maximum rate of and the optimum temperature for CO₂ assimilation in rice leaves increased 5 molCO₂ m^–2 s^–1 and 5°C, respectively, as compared with those values obtained when the vapour pressure difference took its own course. This was reasoned to be due to the increase in leaf conductance and the decrease in transpiration rate. In addition, these results confirmed that stomatal conductance essentially increases with increasing leaf temperature under constant vapour pressure difference conditions, in other words, when the influence of the vapour pressure difference is removed.This system may be used successfully to measure inter- and intra-specific differences and characteristics of leaf gas exchange in plants with a high degree of accuracy.Abbreviations A CO₂ assimilation rate - A_max Maximum rate of CO₂ assimilation - A_opt Optimum teperature for CO₂ assimilation - CTWB Controlled-temperature water bath - DPC Dew point controller - E Transpiration rate; gl, leaf conductance - HCC Humidity control circuit - IRGA Infrared gas analyzer - LT Leaf temperature - LTC Leaf temperature controller - MFC Mass flow controller - QFD Quantum flux density - RH Relative humidity - RHC Relative humidity controller - VPD Vapour pressure difference - CO₂ Difference of CO₂ concentration between inlet and outlet air     

Water potential in excised leaf tissue: comparison of a commercial dew point hygrometer and a thermocouple psychrometer on soybean, wheat, and barley

Leaf water potential (Ψ_leaf) determinations were made on excised leaf samples using a commercial dew point hygrometer (Wescor Inc., Logan, Utah) and a thermocouple psychrometer operated in the isopiestic mode. With soybean leaves (Glycine max L.), there was good agreement between instruments; equilibration times were 2 to 3 hours. With cereals (Triticum aestivum L. and Hordeum vulgare L.), agreement between instruments was poor for moderately wilted leaves when 7-mm-diameter punches were used in the hygrometer and 20-mm slices were used in the psychrometer, because the Ψ_leaf values from the dew point hygrometer were too high. Agreement was improved by replacing the 7-mm punch samples in the hygrometer by 13-mm slices, which had a lower cut edge to volume ratio. Equilibration times for cereals were normally 6 to 8 hours. Spuriously high Ψ_leaf values obtained with 7-mm leaf punches may be associated with the ion release and reabsorption that occur upon tissue excision; such errors evidently depend both on the species and on tissue water status.