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.     

Thermotolerance of Leaf Discs from Four Isoprene-Emitting Species Is Not Enhanced by Exposure to Exogenous Isoprene

The effects of exogenously supplied isoprene on chlorophyll fluorescence characteristics were examined in leaf discs of four isoprene-emitting plant species, kudzu (Pueraria lobata [Willd.] Ohwi.), velvet bean (Mucuna sp.), quaking aspen (Populus tremuloides Michx.), and pussy willow (Salix discolor Muhl). Isoprene, supplied to the leaves at either 18 μL L⁻¹ in compressed air or 21 μL L⁻¹ in N₂, had no effect on the temperature at which minimal fluorescence exhibited an upward inflection during controlled increases in leaf-disc temperature. During exposure to 1008 μmol photons m⁻² s⁻¹ in an N₂ atmosphere, 21 μL L⁻¹ isoprene had no effect on the thermally induced inflection of steady-state fluorescence. The maximum quantum efficiency of photosystem II photochemistry decreased sharply as leaf-disc temperature was increased; however, this decrease was unaffected by exposure of leaf discs to 21 μL L⁻¹ isoprene. Therefore, there were no discernible effects of isoprene on the occurrence of symptoms of high-temperature damage to thylakoid membranes. Our data do not support the hypothesis that isoprene enhances leaf thermotolerance.     

A leaf disc method for measuring cuticular conductance

This paper describes a new method for the measurement of cuticularconductance (g_o;) using a leaf disc sealed in a specially-designedenvelope. Conductances for astomatous (adaxial) and stomatous(abaxial) surfaces of beech {Fagus sylvatica L.) were determinedfrom measurements of water flux. Leaf discs were punched outfrom attached leaves and placed inside individual envelopesthat provided a water supply. Water flux from an exposed epidermalsurface of the leaf discs was measured gravimetrically. Allmeasurements were made under darkness. Conductance of the adaxialsurface was referred to as g_c, whereas conductance of the abaxialsurface was considered as a minimum leaf surface conductance()- The main advantage of this method is that it enables measurement of g_c and from leaf samples with intact cuticles and a highrelative water content [RWC) for periods of up to 12 d. Conductancesof leaf discs in envelopes were compared with those of wholeleaves and leaf discs without envelopes. Data demonstratinga strong positive relationship between conductance and RWC ispresented. Key words: Cuticular conductance, leaf disc, relative water content     

Continuous monitoring of plant water potential

Plant water potential was monitored continuously with a Wescor HR-33T dewpoint hygrometer in conjunction with a L51 chamber. This commercial instrument was modified by replacing the AC-DC mains power converter with one stabilized by zener diode controlled transistors. The thermocouple sensor and electrical lead needed to be thermally insulated to prevent spurious signals. For rapid response and faithful tracking a low resistance for water vapor movement between leaf and sensor had to be provided. This could be effected by removing the epidermis either by peeling or abrasion with fine carborundum cloth. A variety of rapid plant water potential responses to external stimuli could be followed in a range of crop plants (sunflower (Helianthus annuus L., var. Hysun 30); safflower (Carthamus tinctorious L., var. Gila); soybean (Glycine max L., var. Clark); wheat (Triticum aestivum L., var. Egret). These included light dark changes, leaf excision, applied pressure to or anaerobiosis of the root system. Water uptake by the plant (safflower, soybean) mirrored that for water potential changes including times when plant water status (soybean) was undergoing cyclical changes.     

Ratio of cut surface area to leaf sample volume for water potential measurements by thermocouple psychrometers

Evaporative losses from the cut edge of leaf samples are of considerable importance in measurements of leaf water potential using thermocouple psychrometers. The ratio of cut surface area to leaf sample volume (area to volume ratio) has been used to give an estimate of possible effects of evaporative loss in relation to sample size. A wide range of sample sizes with different area to volume ratios has been used. Our results using Glycine max L. Merr. cv Bragg indicate that leaf samples with area to volume values less than 0.2 square millimeter per cubic millimeter give psychrometric leaf water potential measurements that compare favorably with pressure chamber measurements.     

Effect of altered sink: source ratio on photosynthetic metabolism of source leaves

When seven crop species were grown under identical environmental conditions, decreased sink:source ratio led to a decreased photosynthetic rate within 1 to 3 days in Cucumis sativus L., Gossypium hirsutum L., and Raphanus sativus L., but not in Capsicum annuum L., Solanum melongena L., Phaseolus vulgaris L., or Ricinus communis L. The decrease was not associated with stomatal closure. In cotton and cucumber, sink removal led to an increase in starch and sugar content, in glucose 6-phosphate and fructose 6-phosphate pools, and in the proportion of ¹⁴C detected in sugar phosphates and UDPglucose following ¹⁴CO₂ supply. When mannose was supplied to leaf discs to sequester cytoplasmic inorganic phosphate, promotion of starch synthesis, and inhibition of CO₂ fixation, were observed in control discs, but not in discs from treated plants. Phosphate buffer reduced starch synthesis in the latter, but not the former discs. The findings suggest that sink removal led to a decreased ratio inorganic phosphate:phosphorylated compounds. In beans ¹⁴C in sugar phosphates increased following sink removal, but without sucrose accumulation, suggesting tighter feedback control of sugar level. Starch accumulated to higher levels than in the other plants, but CO₂ fixation rate was constant for several days.     

Efficiency and regulation of water transport in some woody and herbaceous species

The efficiency with which plants transport water is related to the water potential differences required to drive water fluxes from the soil to the leaf. A comparative study of two woody and three herbaceous species (Citrus sinensis L. cv. Koethen, Pyrus kawakami L., Helianthus annuus L. cv. Mammoth Russian, Capsicum frutescens L. cv. Yolo Wonder, and Sesamum indicum L. cv. Glauca) indicated contrasts in water transport efficiency. Depression of leaf water potential in response to transpiration increases was found in the woody species; the herbaceous species, however, had more efficient water transport systems and presented no measurable response of leaf water potential to transpiration changes. Different maximum transpiration rates under the same climatic conditions were observed with different species and may be accounted for by stomatal response to humidity gradients between leaf and air. Leaf diffusion resistance in sesame increased markedly as the humidity gradient was increased, while leaf resistance of sunflower responded less to humidity. Stomata appeared to respond directly to the humidity gradient because changes in leaf water potential were not detected when leaf resistance increased or decreased.     

Potassium Deficiency-induced Changes in Stomatal Behavior, Leaf Water Potentials, and Root System Permeability in Beta vulgaris L

Studies of the water relations of potassium deficient sugarbeet plants (Beta vulgaris L.) revealed two factors for stomatal closure. One component of stomatal closure was reversible by floating leaf discs on distilled water to relieve the water deficit in the leaves; the other component was reversible in the light by floating the leaf discs on KCl solution for 1 hour or more. Potassium-activated stomatal opening in the light was observed when the guard cells were surrounded by their normal environment of epidermal and mesophyll cells, just as observed by previous workers for epidermal strips. Leaf water potentials, like stomatal apertures, appear to be strongly related to leaf potassium concentration. Potassium-deficient plants have a greatly decreased root permeability to water, and the implications of this effect on stomatal aperture and leaf water potential are discussed. In contrast, petiole permeability to water is unaffected by potassium treatment.     

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid ofAgrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2–5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation,L. chilense LA1969 andL. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.The Hebrew University of Jerusalem, Faculty of Agriculture, Department of Field and Vegetable Crops     

Measurement and effects of gel matric potential and expressibility on production of morphogenic callus by cultured sugarbeet leaf discs

During studies to optimize production of morphogenic callus from cultured leaf discs of sugarbeet (Beta vulgaris L.) large differences were observed associated with the gelling agent employed. Water availability, as determined mainly by gel matric potential, was found to be the dominant factor. A simple method was devised to measure the relative matric potential of different gels. A precisely moistened filter-paper disc was placed on the gel surface, allowed to equilibrate, removed and weighed. The relative gain or loss of water from the paper disc was a measure of the matric potential of the gel and varied with both gel type and concentration. Leaf disc expansion and production of callus-derived embryos and shoots were shown to be directly proportional to gel matric potential. Water availability may also be affected by the ease with which liquid is expressed from gels in response to localized pressure caused by explant expansion and contortion. This property, called gel expressibility, was easily measured with a weight and capillary pipette and shown also to vary with gel type and concentration. Validity of the technique for measuring relative matric potential was verified physiologically by culturing leaf discs on filter-paper overlays to eliminate expressibility differences among gels. Additionally, comparison of leaf disc growth on uncovered gel surfaces versus filter-paper overlays demonstrated the contribution of liquid expression to overall water availability. Expression of liquid by explants on uncovered gel surfaces greatly enhanced the production of morphogenic callus.     

Water relations of cotton plants under nitrogen deficiency

Nitrogen deficiency in cotton plants (Gossypium hirsutum L.) increased the threshold water potentials for both stomatal closure and leaf senescence (defined as loss of chlorophyll and protein) during drought. These studies attempted to answer two questions: (1) What is the basis for the N/water interaction on senescence? (2) Is there a direct relationship between stomatal closure and senescence? Young and old leaves from N-deficient and N-sufficient plants maintained their relative sensitivities to water stress when excised leaf discs were floated on solutions of polyethylene glycol in dim light. In this leaf disc system, both leaf aging and N deficiency increased the threshold water potential for senescence. Leaf aging and N deficiency also decreased the concentration of exogenous abscisic acid necessary to initiate senescence in discs. A role for cytokinins in controlling senescence could not be clearly shown. In young leaves of both N-deficient and N-sufficient plants, stomata closed at water potentials much higher than those causing senescence. During leaf aging, the water potentials causing senescence increased more than those causing stomatal closure. The two processes thus occurred at about the same potentials in the oldest leaves. These data argue against a general cause-and-effect relationship between stomatal closure and senescence. Rather, each process apparently responded independently to absicsic acid accumulated during drought.     

Proline Oxidase and Water Stress-induced Proline Accumulation in Spinach Leaves

Spinach (Spinacia oleracea L.) leaf discs accumulated free proline when exposed to polyethylene glycol solutions of water potential less than −10 bars. At −20 bars, the accumulation was 11 micromoles per gram original fresh weight in a 24-hour period.     

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.     

Apple leaf senescence: leaf disc compared to attached leaf

Attached apple leaves (Pyrus malus L., Golden Delicious) began to lose protein in early August as the first sign of senescence. Apple leaf discs prepared from samples before early August gained protein for up to 7 days after detachment. After early August, the loss of protein from leaf discs was no greater than the loss from attached leaves in 7 days. The loss of chlorophyll from leaf discs began over 2 months before attached leaves began to lose chlorophyll naturally and before leaf discs lost protein. Leaf discs from presenescent leaves did not senesce significantly faster when maintained in darkness instead of 12 hours of light. In general, the loss of protein and chlorophyll from apple leaf discs after 7 days was much less than for most other leaf types studied.     

Incorporation of C-Leucine into Apple Leaf Protein and Its Inhibition by Protein Synthesis Inhibitors during Growth and Senescence

Of the total ¹⁴C-leucine taken up by intact apple (Pyrus malus L., Golden Delicious) leaf discs, 44 to 62% is incorporated into protein from June to early October. Of this amount, an average of 35% is released by mild, room temperature acid hydrolysis. Prior to mid-August when leaf protein begins to decline, 15 to 20% of the ¹⁴C-leucine incorporated into protein occurs in water-(buffer) soluble protein, of which only 3% is released by mild acid hydrolysis. After mid-August, 40% of the label in protein occurs in soluble protein. The specific radio-activity of the soluble protein increases by 4- to 5-fold after mid-August, while that of total protein increases by less than 2-fold. In presenescent leaves (before the decline of protein in August) 20 micrograms per milliliter cycloheximide inhibits the incorporation of ¹⁴C-leucine into protein by 71%, and 20 micrograms per milliliter chloramphenicol inhibits it by 30%. In senescing leaves, cycloheximide inhibits ¹⁴C-leucine by 85% or more, while chloramphenicol inhibits it by less than 15%. Coincident to the initial decline of leaf protein, chloramphenicol greatly loses its ability to inhibit the incorporation of ¹⁴C-leucine into apple leaf protein. At all leaf ages, chloramphenicol increases the loss of chlorophyll from apple leaf discs. The effect of cycloheximide on leaf disc senescence changes with leaf age: in early season samples, it increases the loss of chlorophyll; in mid-season samples, it has no effect; and in late season samples, it retards the loss of chlorophyll.     

Water Stress in Sunflower (Helianthus annuus L.): I. Effect on Plant Development

Development of sunflower plants grown in pots under controlledconditions was monitored at frequent intervals. Two separatewater stresses, which reached a minimum of –1.7 MPa, wereimposed at specified developmental stages on two separate batchesof plants. Leaf initiation and leaf unfolding were significantlyreduced by water stress. Inflorescence primordium initiation,involucre bract formation, disc and disc floret developmentwere not affected. The mitotic index was reduced by water stressin unfolding leaves from 5 to 0.5 per cent. In other leaves,the mitotic index was reduced to a lesser extent. This reducedmitotic index helps explain reduction in leaf development. Thetime between seed germination and inflorescence primordium initiationremained relatively constant despite the reduced leaf numberassociated with water stress. The discussion evaluates the complicationsthat this may cause in the comparison of leaves in the upperhalf of the leaf profile. Helianthus annuus L., sunflower, water stress, mitotic index, leaf development     

Effects of drought stress on the evergreen Quercus ilex L., the deciduous Q. robur L. and their hybrid Q. × turneri Willd.

Five-year-old trees of deciduous Quercus robur L., evergreen Q. ilex L., and their semideciduous hybrid, Q. × turneri Willd. (var. pseudoturneri), growing in pots, were subjected to drought stress by withholding water for 18–22 days, until leaf water potentials decreased below ?2 MPa. Gas-exchange rates, oxygen evolution, and modulated chlorophyll (Chl) fluorescence measurements revealed that by strong stomata closure and declining photosynthetic capacity down to approximately 50%, all three taxa responded with strongly reduced photosynthesis rates. In Q. robur, photochemical quenching of the drought-stressed plants was much lower than in nonstressed controls. Dissection of the occurring events in the photosynthetic electron transport chain by fast Chl fluorescence induction analysis with the JIP-test were discussed.     

The Measurement of Stomatal Responses to Stimuli in Leaves and Leaf Discs

A comparison has been made of stomatal responses in intact leaves,leaf discs supplied with water via their cut edges and leafdiscs floating on water. Xanthium pennsylvanicum leaf discswatered via their cut edges appeared to be more turgid thanintact leaves; this considerably slowed down the rate of stomatalopening but it slightly increased the final steady-state stomatalopening. When the water potential of such leaf discs was loweredby pre-treatment with mannitol solutions rates of stomatal openingincreased whereas maximum steady-state openings decreased. In tobacco leaf discs floating on water the stomata in contactwith water were wider open than those in contact with normalair and they did not respond to treatment with carbon dioxide-freeair. The rate of photosynthesis was severely reduced in tobaccoleaf discs floating with the lower epidermis on water, mostprobably owing to the slow rate of diffusion of carbon dioxidein water. By floating such discs on osmotica the degree of stomatalopening was increased, however, a response to treatment withcarbon dioxide-free air was still not measurable. It is postulatedthat, on account of the relative unavailability of carbon dioxidefrom the water, the carbon dioxide concentration in the substomatalcavities of the lower surface is abnormally low, irrespectiveof whether ordinary air or carbon dioxide-free air is availableto the upper surface. A comparison between porometer readings and measurements ofsiliconerubber impressions of stomatal pores taken from insidethe porometer cup confirmed that the silicone-rubber impressionmethod of assessing stomatal responses to stimuli has severelimitations, especially at small stomatal apertures.     

Predictions of vegetation change following fire in a great salt lake marsh

A Gleasonian model of succession (van der Valk, 1981) was used to predict the species composition of a freshwater marsh following fire. Seed bank samples from 5 vegetation types (Distichlis spicata (L.) Greene, Scirpus lacustris L., S. maritimus L., Typha spp., open water) were used in conjuction with plant life history characteristics to make predictions of post-fire species composition for each vegetation type and the entire study area. In general, predictions for specific vegetation types were not good, but were more accurate for the entire area. Stronge correlations for specific vegetation types may have been prevented by considering marsh vegetation types as discrete units, statistical sampling problems, competitive interactions, or more specific germination requirements of some species.     

Measurement of CO(2) and H(2)O Vapor Exchange in Spinach Leaf Discs : Effects of Orthophosphate

A leaf chamber has been designed which allows the measurement of both CO₂ and water vapor exchange in Spinacia oleracea leaf discs. The center of the disc lies within a cylindrical gas chamber and its margins are enclosed within a cavity through which water or various metabolites can be pumped. In saturating light and normal atmospheres, the leaf discs have a relatively low resistance to H₂O vapor transfer (r_w = 1.87 seconds per centimeter) and can support high rates of photosynthesis for several hours. The abaxial surface of a disc had a higher resistance to water vapor transfer (r_w = 3.22 seconds per centimeter) than the adaxial (r_w = 2.45 seconds per centimeter) despite having a higher stomatal frequency (abaxial, 105/square millimeter; adaxial, 58/square millimeter). In 2% O₂, the discs required an internal concentration of CO₂ of 115 microliters per liter to support one-half of the maximal velocity of apparent photosynthesis (average value, 66 milligrams CO₂ per square decimeter per hour). In 20% O₂, the comparable values are 156 microliters per liter and 56 milligrams CO₂ per square decimeter per hour. In air, apparent photosynthesis saturated at intensities (750 microeinsteins per square meter per second) well below that of daylight but, when the internal CO₂ was raised to 700 to 900 microliters per liter, photosynthesis was not saturated even at daylight intensities (2025 microeinsteins per square meter per second). The distribution of Prussian blue crystals, formed after ferrocyanide feeding, showed that water entered the disc via the vasculature. When 25-minute pulses of orthophosphate were provided in the feeding solution, there were concentration-dependent increases in both r_w and r_m leading to inhibition of photosynthesis. The orthophosphate-dependent inhibitions were reversible.