Expression of candidate Arabidopsis ferroptosis marker genes. (a) Arabidopsis roots from 6-d-old seedlings were preincubated with 1 µM Fer-1, 10 µM CPX, or DMSO (control). Cell death was induced by treating roots at 55°C for 10 min, and roots were stained with Sytox green 6 h after treatment. The images show that cells in the main root undergo cell death that is prevented by ferroptosis inhibitors. Bars, 50 µm. (b) Changes in expression of candidate genes that might be associated with plant ferroptosis in A. thaliana roots 2 h after HS at 55°C for 10 min. mRNA levels are expressed as a fold-change ratio between the different conditions and were analyzed by one-way analysis of variance (ANOVA). D, DMSO; nd, not determined. (c) mRNA expression levels of KOD determined by real-time quantitative PCR in A. thaliana roots in response to 55°C for 2 h. Data are from three independent biological replicates and presented as mean ± SD. Data were analyzed by one-way analysis of variance. *, P < 0.05. Significance is indicated relative to the treatment at 55°C plus the ferroptosis inhibitor Fer-1.