Abscisic acid and epibrassinolide reprogram barley drought responses across multiple regulatory layers

Abstract Abscisic acid (ABA) is a key stress hormone, and brassinosteroids (BRs) modulate hormonal networks and stress physiology. However, how they shape drought responses in cereals remains unclear. Therefore, we aimed to investigate more precisely the mechanisms underlying ABA- and BR-mediated drought responses and their cross-regulation. Barley plants were preconditioned with ABA or epibrassinolide (epiBL) before drought and analyzed at the hormonal, gene expression, microRNA, and physiological level. Hormonal preconditioning shifted the balance between developmental and stress-related hormonal programs during drought. Within growth-associated hormones gibberellin GA 1 and auxin remained suppressed, whereas cytokinin responses were isopentenyladenine-dominated. Stress-related hormones showed treatment-specific induction patterns; jasmonic acid was the most ABA- and epiBL-responsive. ABA preferentially influenced HvD1 expression, linked to G-protein-mediated non-canonical BR signaling, rather than the canonical HvBZR1 pathway. Contrastingly, epiBL constrained ABA biosynthetic capacity through HvNCED1 . Selected microRNAs further support reciprocal ABA–BR coupling at the post-transcriptional level under drought. Physiologically, ABA treatments separated flexible acclimation from stress-prioritized photoprotection, whereas epiBL shifted the response from canonical PSII excitation at low levels to rapid stress signaling activation at higher levels. These findings elucidated multilayered ABA- and epiBL-driven responses of barley to drought, establishing a regulatory architecture spanning hormonal dynamics, transcriptional, post-transcriptional, and physiological regulation.