ROS-mediated interplay between brassinosteroids and gibberellic acids antagonistically modulates asymmetric periclinal cell division leading to middle cortex formation in <i>Arabidopsis</i> roots

Asymmetric cell divisions (ACDs) in the root ground tissue of <i>Arabidopsis thaliana</i> are essential for middle cortex (MC) formation, which contributes to root architecture and environmental adaptability. Here, we demonstrate that brassinosteroids (BRs) and gibberellins (GAs) antagonistically regulate MC formation via reactive oxygen species (ROS). Brassinolide (BL, a BR) or paclobutrazol (PAC, a GA biosynthesis inhibitor) promoted MC formation and sporadic periclinal cell divisions in root endodermal cell files, whereas brassinazole (BRZ, a BR biosynthesis inhibitor) or GA₃ suppressed them. Consistently, the BR-signaling gain-of-function mutant <i>bzr1</i>-<i>1D</i>, the GA-biosynthesis-deficient mutant <i>ga1</i>-<i>3</i>, and the GA-insensitive mutant <i>gai</i>-<i>1</i> exhibited elevated H₂O₂ levels and increased MC formation. Conversely, the BR-biosynthesis-deficient mutant <i>det2</i> and the GA-signaling-enhanced <i>rga</i>/<i>gai</i> double mutant showed reduced ROS accumulation and MC formation. BL or PAC further enhanced MC-forming effects, while BRZ or GA₃ diminished them. This antagonistic regulation of BRs and GAs on MC formation was further validated in double mutants: <i>ga1-3/bzr1-1D</i> displayed an additive promotion, while <i>ga1-3/det2</i> showed a diminished effect on MC formation. The ROS-deficient <i>rbohD/F</i> mutant exhibited reduced MC formation and attenuated responses to BL or PAC, and ROS scavenging by potassium iodide suppressed the MC-promoting effects of <i>bzr1-1D</i>, <i>ga1-3</i>, and <i>ga1-3/bzr1-1D</i>. These results identify ROS as a central integrator of BR-GA antagonism, linking hormonal regulation to SHR/SCR-mediated ACDs during MC development in <i>Arabidopsis</i> roots.