Design of anArabidopsis thalianareporter line to detect heat-sensing and signaling mutants

Abstract

Background: Global 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 special heat-responsive Arabidopsis thaliana reporter line that allowed an in-depth investigation of the mechanisms underlying the accumulation of protective heat-shock proteins (HSPs) in response to high temperature. Methods: A transgenic Arabidopsis thaliana reporter line named "Heat-Inducible Bioluminescence And Toxicity" (HIBAT) was designed to express from a conditional heat-inducible promoter, a fusion gene encoding for nanoluciferase and D-amino acid oxidase, whose expression was found to be toxic only in the presence of D-valine. HIBAT seedlings were exposed to different heat treatments in presence or absence of D-valine and analyzed for survival rate, bioluminescence and HSP gene expression. Results: Whereas at 22C, HIBAT seedlings grew unaffected by D-valine, and all survived following iterative heat treatments without D-valine, 98% died following heat treatments on D-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. Confirming that HIBAT does not significantly differ from its Col-0 parent, RNAseq analysis of heat-treated seedlings showed a strong correlation between the two lines. Using HIBAT, a forward genetic screen revealed candidate loss-of-function mutants defective either at accumulating HSPs at high temperature or at repressing HSP accumulation at low, non-heat-shock temperatures. Conclusion: This study adds insights into the molecular mechanisms by which higher plants sense and adapt to rapid elevations of ambient temperatures. HIBAT was valuable tool to identify Arabidopsis mutants defective in the response to high temperature stress. Our findings open new avenues for future research on the regulation of HSP expression and understanding their role in the onset of plant acquired thermotolerance