Systems analysis of long-term heat stress responses in the C₄ grass Setaria viridis

Many C₄ plants are used as food and fodder crops and often display improved resource use efficiency compared to C₃ plants. However, the response of C₄ plants to future extreme conditions such as heatwaves is less understood. Here, Setaria viridis, an emerging C₄ model grass, was grown under long-term high-temperature stress for 2 wk (42 °C, compared to 28 °C). This resulted in stunted growth, but surprisingly had little impact on leaf thickness, leaf area-based photosynthetic rates, and bundle sheath leakiness. Dark respiration rates increased, and there were major alterations in carbon and nitrogen metabolism in the heat-stressed plants. Abscisic acid and indole-3-acetic acid–amino acid conjugates accumulated in the heat-stressed plants, consistent with transcriptional changes. Leaf transcriptomics, proteomics, and metabolomics analyses were carried out and mapped onto the metabolic pathways of photosynthesis, respiration, carbon/nitrogen metabolism, and phytohormone biosynthesis and signaling. An in-depth analysis of correlations between transcripts and their corresponding proteins revealed strong differences between groups in the strengths and signs of correlations. Overall, many stress signaling pathways were upregulated, consistent with multiple signals leading to reduced plant growth. A systems-based model of the plant response to long-term heat stress is presented based on the oxidative stress, phytohormone, and sugar signaling pathways.