Quantitative Assessment of Heat Shock-Induced Ferroptosis-Like Cell Death via Electrolyte Leakage in Arabidopsis thaliana Seedlings.

We present a protocol to allow continuous assessment of cell death in Arabidopsis thaliana (L.) seedlings by measuring the release of electrolytes from dying cells upon heat shock. The electrolyte leakage assay is a well-established method to quantify the extent of cell death of plant tissues exposed to pathogen infection, since the activation of the immune response leads to compromised membrane integrity and to the release of ions from the dying cell. This prolonged release of electrolytes is considered a hallmark of regulated cell death in plants. Heat shock in plants induces ferroptosis-like cell death, which can be suppressed either pharmacologically, using inhibitors such as ferrostatin, or genetically through knockout of ferroptosis-related genes. Here, we have adapted the electrolyte leakage assay to quantify cell death in young Arabidopsis seedlings exposed to a heat shock previously shown to induce ferroptosis-like cell death. We also illustrate how this method can be used to assess activation of ferroptosis-like cell death in whole Arabidopsis seedlings using ferrostatin or knockout mutants of potential gene candidates involved in ferroptosis-like cell death. Key features • This protocol does not require any technical experience apart from gentle handling of young seedlings and is less labor-intensive than microscopy-based cell death evaluation. • Builds upon existing methods to quantify the extent of cell death upon immune response in whole seedlings subjected to heat stress. • Only requires a conductivity meter and allows the assessment of continuous cell death using multiple parallel replicates. • The protocol demonstrates how heat shock-induced ferroptosis-like cell death can be inhibited pharmacologically or genetically in whole seedlings, supported with quantitative data.