Vacuolar H^(+)-ATPase and BZR1 form a feedback loop to regulate the homeostasis of BR signaling inArabidopsis

Brassinosteroid(BR)is a vital plant hormone that regulates plant growth anddevelopment.BRASSINAZOLE RESISTANT1(BZR1)is a key transcription factor in BR signaling,and its nucleocytoplasmic localization is crucial for BR signaling.However,the mechanisms that regulate BzR1 nucleocytoplasmic distribution and thus the homeostasis of BR signaling remain largely unclear.The vacuole is the largest organelle in mature plantcells and plays a key role in maintenance of cell ular pH,storage of intracellular substances,and transport ofions.In this study,weuncovered anovel mechanismof BR signaling homeostasis regulatedbythe vacuolar H+-ATPase(V-ATPase)and BZR1 feedback loop.Our results revealed that the vha-a2 vha-a3 mutant(vha2,lacking V-ATPase activity)exhibits enhanced BR signaling with increased total amount of BZR1,nuclearlocalized BZR1,and the ratio of BZR1/phosphorylated BZR1 in the nucleus.Further biochemical assays revealed that VHA-a2 and VHA-a3 of V-ATPase interact with the BZR1 protein through a domain that is conserved across multiple species.VHA-a2 and VHA-a3 negatively regulate BR signaling by interacting with BzR1 and promoting its retention in the tonoplast.Interestingly,a series of molecular analyses demonstrated that nuclear-localized BZR1 could bind directlyto specific motifs in the promoters of VHA-a2 andVHAa3topromote their expression.Taken together,these results suggest that V-ATPase and BzR1 mayforma feedback regulatory loop to maintain thehomeostasis of BR signaling in Arabidopsis,providing new insights into vacuole-mediated regulation of hormone signaling.