Exogenous cytokinin is critical for in vitro shoot regeneration. Proteins involved in the cytokinin signal transduction pathway, including type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), participate in shoot regeneration in Arabidopsis (Arabidopsis thaliana). Some type-B ARRs (e.g., ARR1 and ARR12) promote shoot regeneration by directly activating WUSCHEL (WUS) expression; however, it is unclear how type-B ARRs inhibit shoot regeneration. Here, we show that ARR12 is a central enhancer of callus formation and shoot regeneration, whereas ARR1 is a strong inhibitor of this process that counteracts the positive effect of ARR12. ARR1 indirectly represses CLAVATA3 (CLV3) expression in an ARR12-dependent manner via competing with ARR12 for binding to the CLV3 promoter, which contributes to its ARR12-dependent inhibitory effect on callus formation and shoot regeneration. In parallel, ARR1 inhibits shoot regeneration through transcriptional activation of INDOLE-3-ACETIC ACID INDUCIBLE17, an auxin response repressor gene, and the consequent indirect repression of WUS expression. Thus, type-B ARRs have diverse effects on callus formation and shoot regeneration. Our study reveals novel molecular pathways linking cytokinin signaling, the CLV3 regulator, and auxin signaling, and sheds light on the mechanism underlying cytokinin-regulated shoot regeneration.
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