OsMYBR17 enhances thermotolerance in rice by activating OsLEA4 and reinforcing antioxidant defense

Abstract Heat stress (HS) severely threatens rice (Oryza sativa) growth, development and yield formation; elucidating regulatory mechanisms underlying rice thermotolerance is essential. Here, we dissected the role of OsMYBR17, a previously uncharacterized R2R3-MYB transcription factor, in the heat-stress responses of rice using gain- and loss-of-function approaches combined with DNA affinity purification sequencing (DAP-seq) and RNA sequencing (RNA-seq). The results indicated that OsMYBR17 was induced by HS. Overexpression of OsMYBR17 markedly enhanced seedling thermotolerance, whereas CRISPR/Cas9-mediated knockout lines exhibited pronounced heat hypersensitivity. OsMYBR17-overexpression plants accumulated less reactive oxygen species (ROS) and exhibited reduced lipid peroxidation, as reflected by lower malondialdehyde (MDA) levels, accompanied by elevated ascorbate peroxidase (APX) activity. Consistently, chloroplast structural integrity and chlorophyll-associated signals were better maintained in overexpression lines under prolonged heat exposure, while opposite trends were observed in knockout mutants. Integrative analysis of DAP-seq and RNA-seq identified multiple heat- and oxidative stress-associated candidate targets regulated by OsMYBR17, among which OsLEA4 emerged as a prominent target. Further analyses demonstrated that OsMYBR17 recognized specific cis-elements in the OsLEA4 promoter and directly activated its transcription. Functional analysis of OsLEA4 gain- and loss-of-function materials, together with genetic analysis in the OsMYBR17-overexpression background, further supported its positive contribution to thermotolerance. Collectively, our findings establish an OsMYBR17–OsLEA4 regulatory module that enhances rice heat tolerance and is associated with improved redox homeostasis and chloroplast stability under HS. This study advances our understanding of the molecular basis of heat-stress tolerance in rice and highlights OsMYBR17 and OsLEA4 as potential targets for developing heat-resilient rice cultivars.