Functional Characterization of Type-B Response Regulators in the Arabidopsis Cytokinin Response    

Abstract

Cytokinins play critical roles in plant growth and development, with the transcriptional response to cytokinin being mediated by the type-B response regulators. In Arabidopsis (Arabidopsis thaliana), type-B response regulators (ARABIDOPSIS RESPONSE REGULATORS [ARRs]) form three subfamilies based on phylogenic analysis, with subfamily 1 having seven members and subfamilies 2 and 3 each having two members. Cytokinin responses are predominantly mediated by subfamily 1 members, with cytokinin-mediated effects on root growth and root meristem size correlating with type-B ARR expression levels. To determine which type-B ARRs can functionally substitute for the subfamily 1 members ARR1 or ARR12, we expressed different type-B ARRs from the ARR1 promoter and assayed their ability to rescue arr1 arr12 double mutant phenotypes. ARR1, as well as a subset of other subfamily 1 type-B ARRs, restore the cytokinin sensitivity to arr1 arr12. Expression of ARR10 from the ARR1 promoter results in cytokinin hypersensitivity and enhances shoot regeneration from callus tissue, correlating with enhanced stability of the ARR10 protein compared with the ARR1 protein. Examination of transfer DNA insertion mutants in subfamilies 2 and 3 revealed little effect on several well-characterized cytokinin responses. However, a member of subfamily 2, ARR21, restores cytokinin sensitivity to arr1 arr12 roots when expressed from the ARR1 promoter, indicating functional conservation of this divergent family member. Our results indicate that the type-B ARRs have diverged in function, such that some, but not all, can complement the arr1 arr12 mutant. In addition, our results indicate that type-B ARR expression profiles in the plant, along with posttranscriptional regulation, play significant roles in modulating their contribution to cytokinin signaling.

Type-B ARABIDOPSIS RESPONSE REGULATORs Specify the Shoot Stem Cell Niche by Dual Regulation of WUSCHEL

“…ARR1-, ARR10-, and ARR12-Regulated WUS Expression Plays Critical Roles in Shoot Regeneration Double and triple mutants of ARR1, ARR10, and ARR12 could not generate shoots under in vitro culture owing to cytokinin insensitivity (Hill et al, 2013;Ishida et al, 2008). Here, we used ethanol-induced artificial microRNAs to silence the transcription of ARR1, ARR10, and ARR12 at different stages of shoot formation.…”

“…Previous studies showed that shoot regeneration capacity was impaired in the arr1 single mutant, as well as its double and triple mutants with arr10 and arr12, indicating that type-B ARRs, which are key regulators in cytokinin signaling, are required for shoot regeneration in Arabidopsis (Ishida et al, 2008;Hill et al, 2013). To determine the roles of ARR in the regeneration of shoots in callus on SIM, the transcription of ARR1, ARR10, and ARR12 needs to be temporally repressed.…”

“…The callus was then transferred onto a cytokinin-rich shoot induction medium (SIM) to allow shoot induction. It has been shown that ARR1, ARR10, and ARR12 play primary roles in transducing cytokinin signaling (Sakai et al, 2001;Ishida et al, 2008;Hill et al, 2013). Therefore, to investigate how cytokinin regulates WUS expression and shoot regeneration, we first traced the spatiotemporal expression patterns of the three ARR genes using translational reporter lines.…”

“…The expression of Arabidopsis His kinases4, which encodes a cytokinin receptor, precedes and subsequently overlaps with that of WUS (Gordon et al, 2009). Mutations in type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), key regulators of primary cytokinin response genes, result in reduced shoot regeneration (Ishida et al, 2008;Hwang et al, 2012;Hill et al, 2013;Zhang et al, 2015). By contrast, overexpressing type-A ARRs encoding negative regulators of cytokinin signaling suppress shoot formation (Buechel et al, 2010).…”

Ethylene Inhibits Cell Proliferation of the Arabidopsis Root Meristem

“…We tested this hypothesis by examining the ethylene control of RAM size in type B arr mutants (Table I), focusing on LOF mutations of ARR1, ARR2, ARR10, and ARR12, which are the principle type B ARRs expressed in the root (Mason et al, 2004;Hill et al, 2013). We initially examined the triple mutants arr1-3 arr10-2 arr12-1 and arr2-2 arr10-2 arr12-1 (Mason et al, 2005), examining their responsiveness to 1 and 10 mL L 21 ethylene (Fig.…”