The worldwide rise in heatwave frequency poses a threat to plant survival and productivity. Determining the new marker phenotypes that show reproducible response to heat stress and contribute to heat stress tolerance is becoming a priority. In this study, we describe a protocol focusing on the daily changes in plant morphology and photosynthetic performance after exposure to heat stress using an automated non-invasive phenotyping system. Heat stress exposure resulted in an acute reduction of quantum yield of photosystem II and increased leaf angle. In the longer term, exposure to heat also affected plant growth and morphology. By tracking the recovery period of WT and mutants impaired in thermotolerance (hsp101), we observed that the difference in maximum quantum yield, quenching, rosette size, and morphology. By examining the correlation across the traits throughout time, we observed that early changes in photochemical quenching corresponded with the rosette size at later stages, which suggests the contribution of quenching to overall heat tolerance. We also determined that 6h of heat stress provides the most informative insight in plant responses to heat, as it shows a clear separation between treated and non-treated plants as well as WT and hsp101. Our work streamlines future discoveries by providing an experimental protocol, data analysis pipeline and new phenotypes that could be used as targets in thermotolerance screenings.
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