BackgroundThermography is a popular tool to assess plant water-use behavior, as plant temperature is influenced by transpiration rate, and is commonly used in field experiments to detect plant water deficit. Its application in indoor automated phenotyping platforms is still limited and mainly focuses on differences in plant temperature between genotypes or treatments, instead of estimating stomatal conductance or transpiration rate. In this study, the transferability of commonly used thermography analysis protocols from the field to greenhouse phenotyping platforms was evaluated. In addition, the added value of combining thermal infrared (TIR) with hyperspectral imaging to monitor drought effects on plant transpiration rate (E) was evaluated.ResultsThe sensitivity of commonly used TIR indices to detect drought-induced and genotypic differences in water status was investigated in eight maize inbred lines in the automated phenotyping platform PHENOVISION. Indices that normalized plant temperature for vapor pressure deficit and/or air temperature at the time of imaging were most sensitive to drought and could detect genotypic differences in the plants’ water-use behavior. However, these indices were not strongly correlated to stomatal conductance and E. The canopy temperature depression index, the crop water stress index and the simplified stomatal conductance index were more suitable to monitor these traits, and were consequently used to develop empirical E prediction models by combining them with hyperspectral indices and/or environmental variables. Different modeling strategies were evaluated, including single index-based, machine learning and mechanistic models. Model comparison showed that combining multiple TIR indices in a random forest model can improve E prediction accuracy, and that the contribution of the hyperspectral data is limited when multiple indices are used. However, the empirical models trained on one genotype were not transferable to all eight inbred lines.ConclusionOverall, this study demonstrates that existing TIR indices can be used to monitor drought stress and develop E prediction models in an indoor setup, as long as the indices normalize plant temperature for ambient air temperature or relative humidity.