Drought stress limits plant development and reproduction. Multiple mechanisms in plants are activated to respond to stress. The MYC2 transcription factor is a core regulator of the jasmonate (JA) pathway and plays a vital role in the crosstalk between abscisic acid (ABA) and JA. In this study, we found that SlMYC2 responded to drought stress and regulated stomatal aperture in tomato (Solanum lycopersicum). Overexpression of SlMYC2 repressed SlCHS1 expression and decreased the flavonol content, increased the reactive oxygen species (ROS) content in guard cells and promoted the accumulation of JA and ABA in leaves. Additionally, silencing the SlCHS1 gene produced a phenotype that was similar to that of the MYC2-overexpressing (MYC2-OE) strain, especially in terms of stomatal dynamics and ROS levels. Finally, we confirmed that SlMYC2 directly repressed the expression of SlCHS1. Our study revealed that SlMYC2 drove stomatal closure by modulating the accumulation of flavonol and the JA and ABA contents, helping us decipher the mechanism of stomatal movement under drought stress.

SUMMARY The jasmonic acid (JA) signaling pathway is involved in the plant response to drought stress. JA and other hormones synergistically regulate the drought response in plants. However, the molecular mechanism underlying this synergism remains poorly defined. In the present study, transcriptome analyses of guard cells and quantitative PCR experiments revealed that MYC2 negatively regulated the negative regulator of ABA signaling, SlPP2C1, and the type‐B response regulator in the cytokinin pathway, SlRR26, and this negative regulation was direct. SlRR26 overexpression reduced drought tolerance in transgenic tomatoes, whereas slrr26cr lines were more tolerant to drought. SlRR26 negatively modulated reactive oxygen species levels in stomata and stomatal closure through RobhB. Moreover, SlRR26 overexpression counteracted JA‐mediated stomatal closure, suggesting that SlRR26 played a negative role in the JA‐mediated drought response. These findings suggest that MYC2 plays a key role in JA‐regulated stomatal closure under drought stress by inhibiting SlPP2C1 and SlRR26.