Use of thermography for quantitative studies of spatial and temporal variation of stomatal conductance over leaf surfaces

This paper describes a new approach to the calibration of thermal infrared measurements of leaf temperature for the estimation of stomatal conductance and illustrates its application to thermal imaging of plant leaves. The approach is based on a simple reformulation of the leaf energy balance equation that makes use of temperature measurements on reference surfaces of known conductance to water vapour. The use of reference surfaces is an alternative to the accurate measurement of all components of the leaf energy balance and is of potentially wide application in studies of stomatal behaviour. The resolution of the technique when applied to thermal images is evaluated and some results of using the approach in the laboratory for the study of stomatal behaviour in leaves of Phaseolus vulgaris L. are presented. Conductances calculated from infrared measurements were well correlated with estimates obtained using a diffusion porometer.     

A condensation type porometer for field use

A double cup transpiration porometer is described which simultaneously measures the resistance to water vapor diffusion from the upper and lower epidermises of a leaf. In use the cups are dried, the porometer is clamped on the leaf, and the transit time required for water vapor to condense on a cold (0 C) reflective surface in each cup is recorded. Since the sensing element remains at constant temperature, only transit time and leaf temperature are required to calculate leaf resistance.     

A Krypton Diffusion Porometer for the Direct Field Measurement of Stomatal Resistance

A stomatal diffusion porometer is described which measures directlythe diffusion of radioactive krypton through amphistomatousleaves. The porometer is relatively small and portable and iseasily used under field conditions. It consists of a miniaturediffusion chamber above an acrylic plastic reservoir which contains1200 cm³ of air enriched with ⁸⁵Kr. Geiger tubes in the diffusionchamber and in the reservoir monitor the relative concentrationsof 85Kr. Krypton is allowed to diffuse from the larger reservoirthrough the leaf into the diffusion chamber and the time forits concentration in relation to that in the reservoir to changebetween two fixed values is recorded. When this time lapse wascalibrated against known resistances a linear relationship,independent of temperature was found. Sources of error are analysedand some experiments are described in which the porometer wasused to measure diurnal changes in stomatal resistance. Resistancesof potted sunflower plants (Helianthus annuus L.) grown in agreenhouse were measured with both the krypton diffusion porometerand a condensation-type porometer and the results were usedto calculate both cuticular and stomatal resistances. Demonstrationof field measurements with the porometer include data from eucalyptustrees {Eucalyptus camaldulensis Dehn) and from an unirrigatedcotton crop {Gossypium hirsutum var. SJ 2) growing under semi-aridconditions. Stomatal conductance of the cotton crop during theopening phase was linearly related to solar radiation.     

A New Porometer Based upon the Electrical Current Produced by Guard Cells

Stomatal guard cells extrude protons when the stomata open.This gives rise to an electrical current which is proportionalto the degree of stomatal opening. An instrument has been developedto measure this leaf surface current which is, in effect, anew type of porometer. The performance of the new porometerhas been compared with that of a commercially available diffusionporometer and a close relationship between leaf surface currentand stomatal conductance was observed for all the species investigated.It is concluded that the instrument has several advantages overthe diffusion porometer, in particular, its small size and simplicityof operation, making it especially suitable for use in the field. Key words: Leaves, stomata, electrical currents, porometry     

A Porometer for Laboratory and Field Operation

A porometer is described which is easily and rapidly attachedto plant leaves and appears to be a versatile and sensitiveinstrument for laboratory or field measurement of leaf resistanceto air flow. In the laboratory it can be connected to a normalresistance porometer circuit, and in the field to a sphygrnomanometer.A theoretical relationship has been developed and tested whichenables the conversion of leaf resistance obtained with a sphygmomanometerto that obtained with a normal porometer circuit.     

The Diffusion Resistance and Water Status of Leaves of Beta vulgaris

Pot-grown sugar-beet plants were deprived of water in two experimentsuntil they wilted completely. Leaf resistance to water-vapourtransfer was measured directly with a diffusion porometer andcompared with values calculated from measurements of transpiration.There was good agreement between the two estimates. Leaf turgor-pressuredecreased only gradually due, apparently, to an increase intissue-solute concentration with increasing water deficit. Relationshipsbetween leaf water potential, soil water potential, and leafresistance were established and the effective control of transpirationby small changes in leaf resistance was clearly demonstrated.     

Developments in Mass Flow Porometry

A ventilated leaf attachment clamp is described with which lateralair flow can be measured separately from through flow. Stomatalconductances are expressed with dimensions: mm s^–1. Basedon parallel measurements with a diffusion porometer the relationbetween mass flow and diffusive flow conductances has been experimentallydetermined for three species. Records of stomatal movementsin air of different compositions are presented. Key words: Stomata, porometry, diffusive and mass flow conductance     

Transit Time Diffusion Porometer Calibration: An Analysis taking into Account Temperature Differences and Calibration Non-Linearity

A method is described that determines the mean calibration ofa transit time diffusion porometer from a limited amount ofcalibration data. The calibration includes calculation of thetemperature coefficients over the range encountered. The commonassumption of a linear relation between transit time and leafresistance was found to give large errors, particularly at lowresistances, and a method of fitting the relation found is described.The improvement in fit is shown to improve significantly theaccuracy of estimates of changes in leaf conductance. Key words: Porometer, Stomata     

Estimating stomatal conductance with thermal imagery

Most thermal methods for the study of drought responses in plant leaves are based on the calculation of 'stress indices'. This paper proposes and compares three main extensions of these for the direct estimation of absolute values of stomatal conductance to water vapour (gs) using infrared thermography (IRT). All methods use the measured leaf temperature and two environmental variables (air temperature and boundary layer resistance) as input. Additional variables required, depending on the method, are the temperatures of wet and dry reference surfaces, net radiation and relative humidity. The methods were compared using measured gs data from a vineyard in Southern Portugal. The errors in thermal estimates of conductance were of the same order as the measurement errors using a porometer. Observed variability was also compared with theoretical estimates of errors in estimated gs determined on the basis of the errors in the input variables (leaf temperature, boundary layer resistance, net radiation) and the partial derivatives of the energy balance equations used for the gs calculations. The full energy balance approach requires accurate estimates of net radiation absorbed, which may not be readily available in field conditions, so alternatives using reference surfaces are shown to have advantages. A new approach using a dry reference leaf is particularly robust and recommended for those studies where the specific advantages of thermal imagery, including its non-contact nature and its ability to sample large numbers of leaves, are most apparent. Although the results suggest that estimates of the absolute magnitude of gs are somewhat subjective, depending on the skill of the experimenter at selecting evenly exposed leaves, relative treatment differences in conductance are sensitively detected by different experimenters.     

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.     

Environmental and genetic components of stomatal behavior in two genotypes of upland cotton

Two parental lines of Upland cotton (Gossypium hirsutum L.) plus their reciprocal F₁, F₂, and backcross populations were field-tested for environmental and genetic components of leaf diffusive resistance. Leaf resistance was measured with a diffusion porometer three times each day during 6 days in August. A large, consistent environmental component was present during the morning, afternoon, and evening, but the genetic component appeared mainly during the afternoon. Leaf water potential indicated that afternoon was the period of highest water stress. Genetic analyses revealed that leaf resistance was associated with both additive and dominance genetic variances, with an estimated narrow sense heritability of 25%. High leaf resistance was completely dominant to low resistance.     

Diffusion and Quasi-diffusion Resistances in Relation to the Carboxylation Kinetics of Maize Leaves

A “quasi-diffusion resistance”, rq, is defined to accommodate the role which the thermochemical and photochemical phenomena of photosynthesis play in the control of CO2 fixation to the terminology and approach of the diffusion resistance analogue for the CO2 exchange of leaves. The relationship of rq to Rabinowitch's classical rectangular hyperbolic model of photosynthesis rate as a function of CO2 concen-tration at the carboxylating surface is discussed. Examination of Kmapp for phos-phopyruvate carboxylase (the predominant carboxylase in maize) suggests, as a reasonable hypothesis for light-saturated maize leaves, that rq may be essentially independent of ambient CO2-concentration up to at least 300 μ1/l. A corollary of the hypothesis is that an increase of diffusion resistance, rather than of rq, may account for the observed curvature of the response curves of light-saturated maize leaf photosynthesis to ambient CO2-concentration. An experiment carried out on fieldgrown maize plants, using a well controlled leaf chamber as a nitrous oxide diffusion porometer, gave evidence which strongly supported the hypothesis.     

Responses of apple leaf stomata to environmental factors

Abstract. Stomatal conductances ( g _s) were measured on the leaves of 3–4 year old Golden Delicious trees and of seedlings of two other cultivars. Measurements were made on container grown trees in the field with a diffusion porometer in 1975 and 1976, and in controlled conditions in a leaf chamber in the laboratory in 1976. Stomatal densities in the Golden Delicious leaves were assessed from scanning electron micrographs. Stomatal density on extension shoot leaves was higher than on other leaf types after June.
The response to irradiance shown by both the porometer and the leaf chamber results could be described by a rectangular hyperbola: where g _max is maximum conductance and β indicates the sensitivity of g_s to photon influx density ( Q _p). The values of β were in the range 60–90 μmol m⁻² s⁻¹.
There was no evidence that apple stomata are sensitive to temperature per se, but g _s was reduced by increasing leaf to air vapour pressure deficits ( D ). There was a linear relationship between g _s and D which was not attributable to feed-back to leaf water potential (ψ_L) as the latter did not affect g _s until a threshold of about −2.0 to −2.5 MPa was reached. Conductance generally declined with increasing ambient CO₂ concentration.     

Dark opening of stomata in successional trees

Summary Dark opening of stomata was found in shade intolerant tree species in three different sets of experiments. In the field, leaves of eight successional species were darkened in light-proof bags and leaf resistance measured with a diffusion porometer. White ash, quaking aspen and American beech were sampled more intensively using darkened cuvettes and a dew point hygrometer. In the lab, white ash seedlings were kept in constant darkness and temperature, and their leaves also monitored with a cuvette and hygrometer. The stomata of all sampled shade intolerant species showed a dark opening response, often to leaf resistance levels typical of illuminated leaves. Shade tolerant species did not. Evidence suggests that this response is endogenous and could contribute to the morning opening of stomata of species under moderate water stress growing in well illuminated habitats.     

The effect of temperature differences between porometer head and leaf surface on stomatal conductance measurements

It is generally known that instantaneous values of leaf conductance as measured with a dynamic porometer need to be corrected for the temperature difference, ΔT, between the porometer cup and the sampled leaf. Leaf conductances, obtained with a Delta-T AP4 dynamic porometer, with and without correction for ΔT are compared for a bush species (Guiera senegalensis) and two forb species (Jacquemontia tamnifolia and Mitracarpus scaber). With temperature differences predominantly varying within the ±2.5 °C recommended by the manufacturer, it appears that the differences between uncorrected and corrected conductances are very large, up to 100% on average, especially for the two forbs. Furthermore, it is shown that, using the Mitracarpus data, a relatively small error of ±0.5 °C in ΔT can cause a difference of 25–50% in the final conductance value, in particular for the high conductance range. An error of ±0.5 °C may easily occur: the accuracy of ΔT as measured by the thermistors in the porometer is 0.2 °C and the temperature variation within a leaf can be much larger. This result will have implications for upscaling of leaf conductances to canopy values or may explain why upscaled values appear not to correspond with down-scaled values, obtained from eddy correlation measurements and an inverted canopy transpiration model.     

Influence of soil water stress on evaporation, root absorption, and internal water status of cotton

Diurnal variations in leaf water potential, diffusion resistance, relative water content, stem diameter, leaf temperature, and energy balance components were measured in cotton (Gossypium hirsutum L. var. Lankart 57) during drought stress under field conditions. A plot of leaf water potential against either relative water content or stem diameter during the 24-hour period yielded a closed hysteresis loop. The relation between cell hydration and evaporation is discussed.     

Photosynthesis, Transpiration, Leaf Stomatal and Mesophyll Resistance Measurements by the Use of a Ventilated Diffusion Porometer

A ventilated diffusion porometer was modified and adapted for simultaneous measurements of leaf resistance and photosynthesis (using ¹⁴C). The system enables measurements to be made under field and laboratory conditions with different concentrations of CO₂ and vapor pressure gradients between the evaporating surfaces inside the leaf and the external atmosphere. The leaf is subjected to the porometer's atmosphere only for short periods (up to 30 seconds) and it is assumed that stomata are not affected. Establishing the linear regression of the effect of CO₂ concentration on net photosynthesis makes it possible to extrapolate for CO₂ compensation point, to calculate the overall resistance to CO₂ and the mesophyll resistance to CO₂.     

A Handpiece for the Simultaneous Measurement of Photosynthetic Rate and Leaf Diffusive Conductance: I. DESIGN

The design and construction of a handpiece for the simultaneousmeasurement of photosynthetic rate and diffusive conductanceof cereal leaves in almost natural conditions in the field isdescribed. Photosynthetic rate is found from the assimilationof ¹⁴CO₂ by part of the leaf which is temporarily enclosed ina small hand-held chamber. This chamber also acts as a stirred,steady-state water vapour diffusion porometer, allowing thesimultaneous measurement of relative humidity from which theleaf diffusive conductance is estimated. The instrument alsomeasures the leaf and air temperatures and incident photon fluxdensity. The important criteria of the performance of such ahandpiece are discussed, and the sensors which measure the physicalvariables of humidity, temperature, and photon flux densityare described. An automatic sequencing system built from logiccircuits which displays the measured values of these variablesand times the operations is also described.     

Studies in Stomatal Behaviour: V. THE ROLE OF CARBON DIOXIDE IN THE LIGHT RESPONSE OF STOMATA

It was found that stomata on illuminated leaves, both of Pelargoniumand wheat, opened much wider where the leaf surface was enclosedin a small volume of air, as in a normal porometer cup, thanelsewhere. This was shown for both species by the infiltrationmethod, and for Pelargonium by Lloyd's method and direct microscopicalobservation also. The effect was shown not to be due to pressure of the porometercup or glass plate on the leaf, or to temperature differences,nor directly to the lack of movement or high humidity of theenclosed air. A considerable body of data was collected which appeared tosupport the hypothesis that the wide opening was due to accumulationof some volatile substance produced by the leaf, but all theresults were also consistent with the view that it was causedby reduction in the carbon dioxide content of the enclosed airbelow the normal 0·03 per cent. owing to photosynthesis.Further crucial experiments with both the porometer and infiltrationmethods left virtually no doubt that the latter hypothesis wascorrect. This extreme sensitivity of stomata to carbon dioxide concentrationwithin the range 0·03 per cent. to zero is discussedin relation to their operation in nature, and a possible biologicaladvantage is suggested. The bearing of the effect upon porometer investigations is alsodiscussed and it is concluded that for all quantitative or semi-quantitativeexperimentation it is essential to use a cup detached betweenreadings, or at least swept with air such as surrounds the restof the leaf, and to have the upper leaf surface above the cuparea freely exposed or similarly swept. For qualitative investigationof the light response of stomata the traditional form of cupmay be used. The importance is stressed of allowing porometer readings toreach equilibrium under one set of conditions before changingto another, when investigating the ‘closing’ or‘opening’ effects of external factors. Several subsidiary effects, observed in the course of the investigation,are discussed; in particular an effect of humidity upon therate of response to other factors.     

Two types of ventilated porometers compared on broadleaf and coniferous species

Two ventilated porometers (diffusion and steady-state) were compared on four broadleaf and five coniferous species. The diffusion porometer gave consistently lower conductance values for both types of species, reflecting a direct stomatal response to low chamber humidity. At high conductance values, the porometers produced a linear and nearly equal response, but the diffusion porometer was less sensitive at low conductance values. This was due to lower air flow (20% of the velocity in the steady-state porometer) and water vapor sorption (by its acrylic plastic chamber). The broadleaf species had less variation (R² = 0.81) than did the coniferous species (R² = 0.61), but, with the latter, there was better correspondence between the two porometers, possibly due to sampling technique. Conductance values were clustered by species.