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.

外源细胞分裂素对于体外芽再生至关重要。参与细胞分裂素信号转导途径的蛋白质，包括B型拟南芥响应调节剂（ARR），参与拟南芥（Arabidopsis thaliana）的芽再生。某些B型ARR（例如ARR1和ARR12）通过直接激活WUSCHEL（WUS）表达来促进芽再生。但是，目前尚不清楚B型ARR如何抑制芽再生。在这里，我们显示ARR12是愈伤组织形成和芽再生的主要增强剂，而ARR1是该过程的强大抑制剂，抵消了ARR12的积极作用。ARR1间接抑制CLAVATA3（CLV3经由与ARR12竞争结合到在ARR12依赖性方式）的表达CLV3启动子，这有助于对愈伤组织形成及不定芽再生其ARR12依赖性抑制作用。同时，ARR1通过生长素应答抑制基因INDOLE-3-ACETIC INDUCIBLE17的转录激活以及由此间接抑制WUS表达来抑制芽再生。因此，B型ARR对愈伤组织形成和枝条再生具有多种影响。我们的研究揭示了连接细胞分裂素信号传导，CLV3调节剂和生长素信号传导的新型分子途径，并阐明了细胞分裂素调控的芽再生的潜在机制。