Respiratory burst oxidase homologue-dependent H2O2 and chloroplast H2O2 are essential for the maintenance of acquired thermotolerance during recovery after acclimation

Thermotolerance is improved by heat stress (HS) acclimation, and the thermotolerance level is "remembered" by plants. However, the underlying signalling mechanisms remain largely unknown. Here, we showed NADPH oxidase-mediated H2O2 (NADPH-H2O2), and chloroplast-H2O2 promoted the sustained expression of HS-responsive genes and programmed cell death (PCD) genes, respectively, during recovery after HS acclimation. When spraying the NADPH oxidase inhibitor, diphenylene iodonium, after HS acclimation, the NADPH-H2O2 level significantly decreased, resulting in a decrease in the expression of HS-responsive genes and the loss of maintenance of acquired thermotolerance (MAT). In contrast, compared with HS acclimation, NADPH-H2O2 declined but chloroplast-H2O2 further enhanced during recovery after HS over-acclimation, resulting in the reduced expression of HS-responsive genes and substantial production of PCD. Notably, the further inhibition of NADPH-H2O2 after HS over-acclimation also inhibited chloroplast-H2O2, alleviating the severe PCD and surpassing the MAT of HS over-acclimation treatment. Due to the change in subcellular H2O2 after HS acclimation, the tomato seedlings maintained a constant H2O2 level during recovery, resulting in stable and lower total H2O2 levels during a tester HS challenge conducted after recovery. We conclude that tomato seedlings increase their MAT by enhancing NADPH-H2O2 content and controlling chloroplast-H2O2 production during recovery, which enhances the expression of HS-responsive genes and balances PCD levels, respectively.