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

Zhenhua Liu,Na Liu,Xuehuan Dai,Fengning Xiang,Xiangzheng Liu,Shuo Li,Juan Li

doi:10.1105/tpc.19.00022

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 (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.

Highlights

In contrast to most animal cells, plant cells typically maintain totipotency, allowing regeneration of a wide variety of explant types into whole plants through judicious in vitro culture conditions (Birnbaum and Sanchez Alvarado, 2008)
A transient expression assay in Arabidopsis protoplasts showed that the luciferase signal was stronger in both the CLV3pro:LUC + 35Spro:ARR1 and ProCLV3:LUC + 35Spro:ARR12 samples compared to the CLV3pro:LUC + empty vector (Figures 5F and 5G)
Pathways involved in hormone signal transduction and indole alkaloid synthesis were well represented among the differentially transcribed genes (DTGs) in arr1 versus the wild type (Figure 7B)

Summary

Introduction

In contrast to most animal cells, plant cells typically maintain totipotency, allowing regeneration of a wide variety of explant types into whole plants through judicious in vitro culture conditions (Birnbaum and Sanchez Alvarado, 2008). During the shoot induction phase, a number of shoot meristem-associated genes become transcriptionally active, including WUSCHEL (WUS), CLAVAT3 (CLV3), and SHOOT MERISTEMLESS (STM) (Gordon et al, 2007; Atta et al, 2009; Chatfield et al, 2013). Type-B ARRs participate in a variety of developmental processes in a largely functionally redundant manner (Mason et al, 2005; Moubayidin et al, 2010; Zhao et al, 2010). Evidence from the in vitro shoot regeneration system supports the functional redundancy of type-B ARRs in cytokinin responses and cytokinin-induced shoot regeneration. The roles of individual type-B ARRs in shoot regeneration are not fully understood, and the repression of shoot regeneration by type-B ARRs has not been previously reported

Methods

Results

Conclusion