Abstract
Plants have evolved a complex regulatory network to cope with heat stress (HS), which includes microRNAs (miRNAs). However, the roles of the entire miRNA biogenesis machinery in HS responses remain unclear. Here, we show that HS induces the majority of miRNAs primarily through the enhanced nuclear localization of HYPONASTIC LEAVES 1 (HYL1), rather than by upregulating MIR gene transcription in Arabidopsis (Arabidopsis thaliana). Disruption of miRNA biogenesis increases plant susceptibility to HS. We also demonstrate that HYL1 phosphorylation modulates its nuclear localization during HS, which is critical for miRNA induction and thermotolerance. MAP KINASE3 (MPK3) phosphorylates and stabilizes the phosphatase C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1), while CPL1 inhibits MPK3 activity, creating a feedback loop that regulates HYL1 phosphorylation. Disruption of MPK3 function results in increased nuclear HYL1 levels and miRNA production, conferring enhanced HS tolerance to mpk3 mutants. These findings reveal a mechanism by which plants enhance miRNA biogenesis during HS, offering insights into the regulatory networks that govern plant thermotolerance.