Final Report Summary - IMPRESS (Imaging of plant responses to environmental stress)
Work performed to achieve the projects objectives Objective 1 Thermal and chlorophyll fluorescence imaging (or point, non-imaging chlorophyll fluorescence measurements) were combined to investigate the impacts of abiotic stress on photosynthesis and transpiration at the leaf level. The imaging techniques were applied to detect spatial and temporal responses to a range of environmental stresses in photosynthetic reactions and transpiration and stomatal conductance of leaves of tobacco plants with altered capacity to scavenge ROS, as a result of transformation to express manganese superoxide dismutase (MnSOD), glutathione reductase (GR), dehydroascorbate reductase (DHAR) or glutathione-S-transferase (GST) in the chloroplasts. Transplastomic and control plants were grown in replicated experiments in controlled environment units. The plants were exposed to a range of environmental stresses (high temperature, low temperature, and limited water availability). Severe and mild stress were compared with control conditions; in addition recovery of stressed plants was monitored in the case of cold stress. Thermal and fluorescence and standard reflectance images of selected leaves were taken with appropriate cameras and light pulses. Reference artificial 'leaves' were included in each thermal image to allow calculation of thermal indices. All the leaves being imaged were kept flat to ensure that variation in leaf temperature related entirely to transpiration and not to the angle of the leaves. Non-imaging techniques were included to ensure the validity of the more novel imaging techniques and to develop a broad understanding of the response of different transformants to stress. Objective 2 Thermal imaging was used to investigate the impacts of environmental stress on photosynthesis and transpiration at the plant level. Imaging was applied to detect spatial and temporal responses to environmental stress in transpiration and stomatal conductance of different genotypes of rye grass (as a model monocot). The different genotypes were grown together in a glasshouse. A similar system for imaging as described for Objective 1 was used, but whole plants rather than individual leaves were imaged. In the field, thermal imaging was also applied to screen for variation between rye-grass genotypes and clover genotypes (ryegrass and clover being important forage crops). Objective 3 Thermal imaging and non-imaging chlorophyll fluorescence measurement were combined to investigate the impacts of environmental stress on photosynthesis and transpiration at the crop level. In addition to measurement of individual plants, aerial thermal and hyperspectral images were obtained over a vineyard that included three experiments in which different levels of irrigation were imposed. This work was carried out in a commercial vineyard, in collaboration with scientists at the Universidad de la Rioja. In addition, thermal imaging was used to determine the impact of different potassium inputs on transpiration of Miscanthus crops, and different nitrogen inputs on transpiration of winter wheat. Main results Objective 1 Chlorophyll fluorescence imaging showed that transplastomic tobacco lines over-expressing dehydroascorbate reductase, glutathione reductase, glutathione-S-transferase, or glutathione reductase and glutathione-S-transferase together, had superior tolerance of cold temperatures (4ºC) under relatively high photosynthetically active radiation (PAR) (250 µmol m?2 s?1). This effect was not seen when PAR was reduced to 80 µmol m?2 s?1, and a series of experiments at different air temperatures indicated a complex relationship between ROS-scavenging, air temperature, PAR, and age and acclimation of leaves. Enhanced ROS-scavenging capacity was not seen to enhance tolerance of high temperature, very high PAR, or drought. Objective 2 No difference between genotypes has been detected in field plots of ryegrass or clover, when assessed using thermal imaging, suggesting that where water and nutrients are not limiting, the stomatal conductance of all genotypes is similar. This contrasts with results from other species. In a greenhouse with additional lighting, however, differences have been found between rye-grass genotypes/cultivars, suggesting that where light is not limiting, some genotypes transpire at higher rates than others. This may indicate potential for more rapid growth. Moreover, differences in response to a drought treatment are emerging, and are being further explored in a PhD studentship. Objective 3 The temperature of grapevine canopies obtained from an aerial thermal image was found to correlate inversely with stem water potential, a standard measure of vine water status, when data from three plots at different locations in the vineyard are assessed together. This is encouraging for the application of thermal imaging to detecting stress over relatively large areas of crops. In one of the plots, however, this relationship was not maintained. This may be a result of the difficulty of separating soil and leaf temperatures in the relatively low resolution image obtained. If soil temperatures are not removed, variation in soil temperature would lead to variation in the derived canopy temperatures. In fact, substantial variation was found in soil temperature depending on shading from neighbouring vines. With respect to thermal images taken facing the vine row or looking down on the vine row from a grape-harvester, canopy temperature was inversely correlated with stomatal conductance when images were taken over a relatively short period of time, in one plot. This has been shown before for images taken facing the vine row, but the suitability of images of the top of the canopy had not previously been determined. This validation of thermal images of the top of the vine rows is important in the context of scaling up to aerial imaging. This relationship would not be expected to be maintained over different times of day, several dates, and different plots. When reference surfaces were included in each image, however, environmental variation was largely removed by deriving an index of stomatal conductance, thus allowing detection of water stress even over several plots, dates, and different times of day. A new type of reference surfaces was used in this work, making use of a sensor designed for other purposes which has two artificial leaves, one of which is kept wet by means of a wick in a small reservoir of water. Conclusions and Scientific impact Interaction of various stresses with PAR and age and acclimation of leaves has been found to impact on whether or not, or to what extent, enhanced ROS-scavenging improves stress tolerance. This should be taken into account in future research on ROS Variation between rye-grass genotypes/cultivars in drought tolerance is of interest for the dairy and beef industries both in Ireland and further a field, and is being further explored in a PhD studentship Aerial thermal imaging holds potential for assessing crop water status over large areas; with non-continuous crops the impact of mixing of leaf and soil temperatures within pixels requires further investigation, or alternatively high resolution imaging must be used Standardisation of reference surfaces for calculation of thermal indices would allow comparison of different experiments and could be a key step in moving thermal imaging of crops towards routine commercial application. The type of reference surfaces explored in this project were found to be reliable, are easily portable, but should be further improved for this purpose e.g. by increasing the size of the artificial leaves to more closely mimic crop leaves Socio-economic impact The MC fellowship led directly to a new PhD fellowship being funded, on thermal imaging of ryegrass The MC fellow was invited main lecturer at the international workshop The use of thermal imaging for drought stress detection, Chiang Mai University, Thailand, 1-5 Nov 2010, at which several scientists and representatives from the public sector and agricultural and water companies were trained in the applications of thermal imaging, particularly in irrigation scheduling Contact details Professor Philip Dix: Philip.dix@nuim.ie Dr Olga Grant: olga.grant@ucd.ie