The Type-B Cytokinin Response Regulator ARR1 Inhibits Shoot Regeneration in an ARR12-Dependent Manner in Arabidopsis

Abstract

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 (<i>Arabidopsis thaliana</i>). Some type-B ARRs (e.g., ARR1 and ARR12) promote shoot regeneration by directly activating <i>WUSCHEL</i> (<i>WUS</i>) 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 <i>CLAVATA3</i> (<i>CLV3</i>) expression in an ARR12-dependent manner via competing with ARR12 for binding to the <i>CLV3</i> 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 <i>INDOLE-3-ACETIC ACID INDUCIBLE17</i>, an auxin response repressor gene, and the consequent indirect repression of <i>WUS</i> expression. Thus, type-B ARRs have diverse effects on callus formation and shoot regeneration. Our study reveals novel molecular pathways linking cytokinin signaling, the <i>CLV3</i> regulator, and auxin signaling, and sheds light on the mechanism underlying cytokinin-regulated shoot regeneration.