This paper reviews and discusses strategies for the use of thermal imaging for studies of stomatal conductance in the field and compares techniques for image collection and analysis. Measurements were taken under a range of environmental conditions and on sunlit and shaded canopies to illustrate the variability of temperatures and derived stress indices. A simple procedure is presented for correcting for calibration drift within the images from the low-cost thermal imager used (SnapShot 225, Infrared Solutions, Inc.). The use of wet and dry reference surfaces as thresholds to eliminate the inclusion of non-leaf material in the analysis of canopy temperature is discussed. An index that is proportional to stomatal conductance was compared with stomatal measurements with a porometer. The advantages and disadvantages of a possible new approach to the use of thermal imagery for the detection of stomatal closure in grapevine canopies, based on an analysis of the temperature of shaded leaves, rather than sunlit leaves, are discussed. Evidence is presented that the temperature of reference surfaces exposed within the canopy can be affected by the canopy water status.

Stomatal regulation is a key determinant of plant photosynthesis and water relations, influencing plant survival, adaptation, and growth. Stomata sense the surrounding environment and respond rapidly to abiotic and biotic stresses. Stomatal conductance to water vapour (g s) and/or transpiration (E) are therefore valuable physiological parameters to be monitored in plant and agricultural sciences. However, leaf gas exchange measurements involve contact with leaves and often interfere with leaf functioning. Besides, they are time consuming and are limited by the sampling characteristics (e.g. sample size and/or the high number of samples required). Remote and rapid means to assess g s or E are thus particularly valuable for physiologists, agronomists, and ecologists. Transpiration influences the leaf energy balance and, consequently, leaf temperature (T leaf). As a result, thermal imaging makes it possible to estimate or quantify g s and E. Thermal imaging has been successfully used in a wide range of conditions and with diverse plant species. The technique can be applied at different scales (e.g. from single seedlings/leaves through whole trees or field crops to regions), providing great potential to study plant-environment interactions and specific phenomena such as abnormal stomatal closure, genotypic variation in stress tolerance, and the impact of different management strategies on crop water status. Nevertheless, environmental variability (e.g. in light intensity, temperature, relative humidity, wind speed) affects the accuracy of thermal imaging measurements. This review presents and discusses the advantages of thermal imaging applications to plant science, agriculture, and ecology, as well as its limitations and possible approaches to minimize them, by highlighting examples from previous and ongoing research.

Temperatures of leaves or canopies can be used as indicators of stomatal closure in response to soil water deficit. In 2 years of field experiments with grapevines (Vitis vinifera L., cvs Castelão and Aragonês), it was found that thermal imaging can distinguish between irrigated and non-irrigated canopies, and even between deficit irrigation treatments. Average canopy temperature was inversely correlated with stomatal conductance measured with a porometer. Variation of the distribution of temperatures within canopies was not found to be a reliable indicator of stress. A large degree of variation between images was found in reference 'wet' and 'dry' leaves used in the first year for the calculation of an index proportional to stomatal conductance. In the second year, fully irrigated (FI) (100% Et(c)) and non-irrigated (NI) canopies were used as alternatives to wet and dry leaves. A crop water stress index utilizing these FI and NI 'references', where stressed canopies have the highest values and non-stressed canopies have the lowest values, was found to be a suitable measure for detecting stress. It is suggested that the average temperatures of areas of canopies containing several leaves may be more useful for distinguishing between irrigation treatments than the temperatures of individual leaves. Average temperatures over several leaves per canopy may be expected to reduce the impact of variation in leaf angles. The results are discussed in relation to the application of thermal imaging to irrigation scheduling and monitoring crop performance.

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 ( g s ) 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 g s 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 g s 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 g s 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 noncontact nature and its ability to sample large numbers of leaves, are most apparent. Although the results suggest that estimates of the absolute magnitude of g s 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.

Temperature of leaves or canopies can be used as an indicator of stomatal aperture, which is considered a sensitive response to soil water deficit. In this paper we analyse the robustness and sensitivity of thermal imaging for detecting changes in stomatal conductance and leaf water status in a range of plant species. Thermal imaging successfully distinguished between irrigated and non-irrigated plants of a variety of species under greenhouse or controlled chamber conditions, with strong correlations between thermal indices and stomatal conductance measured by porometry. Our results also highlighted issues that need to be addressed in order to be confident of always detecting drought stress using this technique. These include variability in leaf angles and the limited reliability of 'wet' and 'dry' leaves to represent leaves with stomata fully open or stomata fully closed. These results should assist the design of protocols for application in crop production or ecosystem monitoring.Abbreviations -ANOVA, analysis of variance; CWSI, crop water stress index; g s , stomatal conductance to water; T, temperature.