BackgroundGlobal warming is a major challenge for plant survival and growth. Understanding the molecular mechanisms by which higher plants sense and adapt to upsurges in the ambient temperature is essential for developing strategies to enhance plant tolerance to heat stress. Here, we designed a heat-responsiveArabidopsis thalianareporter line that allows an in-depth investigation of the mechanisms underlying the accumulation of protective heat-shock proteins (HSPs) in response to high temperature.MethodsA transgenicArabidopsis thalianareporter line named “Heat-Inducible Bioluminescence And Toxicity” (HIBAT) was designed to express from a conditional heat-inducible promoter, a fusion gene encoding for nanoluciferase andd-amino acid oxidase, whose expression is toxic in the presence ofd-valine. HIBAT seedlings were exposed to different heat treatments in presence or absence ofd-valine and analyzed for survival rate, bioluminescence and HSP gene expression.ResultsWhereas at 22 °C, HIBAT seedlings grew unaffected byd-valine, and all survived iterative heat treatments withoutd-valine, 98% died following heat treatments ond-valine. The HSP17.3B promoter was highly specific to heat as it remained unresponsive to various plant hormones, Flagellin, H2O2, osmotic stress and high salt. RNAseq analysis of heat-treated HIBAT seedlings showed a strong correlation with expression profiles of two wild type lines, confirming that HIBAT does not significantly differ from its Col-0 parent. Using HIBAT, a forward genetic screen revealed candidate loss-of-function mutants, apparently defective either at accumulating HSPs at high temperature or at repressing HSP accumulation at non-heat-shock temperatures.ConclusionHIBAT is a valuable candidate tool to identifyArabidopsismutants defective in the response to high temperature stress. It opens new avenues for future research on the regulation of HSP expression and for understanding the mechanisms of plant acquired thermotolerance.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13007-023-01033-x.