A cytokinin–auxin antagonistic module participates in nitrogen-triggered tiller outgrowth in rice

Tillering is a key trait that shapes rice (Oryza sativa L.) shoot architecture and directly influences yield. While tiller bud formation is largely genetically determined, bud outgrowth into functional tillers is highly responsive to environmental cues. However, integration of environmental signals with genetic regulators to determine tiller bud fate remains poorly understood. Here, we investigated the effects of nitrogen on early stages of tiller bud outgrowth. Comprehensive phenotyping and temporal transcriptomic analyses demonstrated that both nitrate and ammonium promote bud outgrowth and elicit overlapping transcriptional responses, with nitrate acting more slowly. Gene regulatory network analysis identified phytohormone signaling as a key interface for nitrogen- triggered tiller outgrowth. Pharmacological and molecular experiments demonstrated the involvement of cytokinin-auxin antagonism in nitrogen-mediated tillering. Cytokinin promoted bud activation by repressing the critical bud dormancy regulators rice TEOSINTE BRANCHED 1 (OsTB1) and a homolog of PIN-FORMED 1 (OsPIN1a) through the Cytokinin Response Factors OsERF53/54. In contrast, auxin maintained dormancy by inducing OsTB1 and OsPIN1a expression through Auxin Response Factors OsARF11/16. Consistently, OsTB1 overexpression lines showed reduced responsiveness to nitrogen and hormone treatments, placing OsTB1 downstream of these convergent inputs. Sequence and gene expression differences in OsERF53/54, along with phenotypic variations across contrasting rice accessions, further substantiated the crucial roles of OsERF53/54 in nitrogen-mediated tillering. Together, we identify a key regulatory role of the cytokinin-auxin antagonistic module for integrating nitrogen signals to determine tiller bud fate. Adequate nitrogen promotes cytokinin signaling while attenuating auxin signaling and transport in tiller buds, thereby releasing dormancy and initiating bud outgrowth.