The Arabidopsis B‐type response regulator 18 homomerizes and positively regulates cytokinin responses

Abstract

Summary In higher plants, the two‐component system (TCS) is a signaling mechanism based on a His‐to‐Asp phosphorelay. The Arabidopsis TCS involves three different types of proteins, namely the histidine kinases (AHKs), the histidine phosphotransfer proteins (AHPs) and the response regulators (ARRs). The ARRs comprise three different families, namely A, B and C types, according to their protein structure. While some members of the B‐type family of ARRs have been studied extensively and reported to act as DNA‐binding transcriptional regulators, very limited information is available for other B‐type ARRs such as ARR18. In this study, we characterize in detail the molecular and functional properties of ARR18. ARR18 acts as a transcriptional regulator in plant cells and forms homodimers in planta as shown by FRET–FLIM studies. As demonstrated by mutational analysis, the aspartate at position 70 (D70) in the receiver domain of ARR18 acts as crucial phosphorylation site. The modification of D70 affects the response regulator’s ability to homodimerize and to activate its target genes. Furthermore, physiological investigations of Arabidopsis lines ectopically expressing ARR18 introduce ARR18 as a new member within the cytokinin‐regulated response pathway regulating root elongation.

Homodimerization of Ehd1 Is Required to Induce Flowering in Rice

“…Cytokinin strongly induces type-A RR genes and stabilizes their proteins (Brandstatter and Kieber, 1998;D'Agostino et al, 2000;To et al, 2007;Tsai et al, 2012). By contrast, type-B RR genes are not induced by cytokinin, but their proteins have transcriptional activity that is enhanced by cytokinin-stimulated phosphorylation of the R domain (Hass et al, 2004;Kim et al, 2006;Veerabagu et al, 2012). In addition, the subcellular localization of RR proteins is not affected by phosphorylation (Sakai et al, 2000;Tsai et al, 2012).…”

“…Both RR types are activated by phosphorylation of the middle Asp (D) in the conserved AspAsp-Lys (D-D-K) motif within the R domain (West and Stock, 2001;Hwang et al, 2002). This phosphorylation either increases or decreases the stability of RR proteins Kim et al, 2012;Liang et al, 2012) and also induces the homomerization of RRs through conformational changes (Fiedler and Weiss, 1995;Birck et al, 1999;Da Re et al, 1999;Anand et al, 2000;Veerabagu et al, 2012). Cytokinin strongly induces type-A RR genes and stabilizes their proteins (Brandstatter and Kieber, 1998;D'Agostino et al, 2000;To et al, 2007;Tsai et al, 2012).…”

Phytosulfokine Regulates Growth in Arabidopsis through a Response Module at the Plasma Membrane That Includes CYCLIC NUCLEOTIDE-GATED CHANNEL17, H+-ATPase, and BAK1

“…These binary vectors and p19 as gene silencing suppressor were transformed into Agrobacterium tumefaciens strain GV3101 and infiltrated into Nicotiana benthamiana leaves. The measurements were performed 3 d after infiltration using the SP8 laser scanning microscope (Leica Microsystems) with LAS AF and SymPhoTime software as described (Veerabagu et al, 2012). Before the FRET-FLIM measurement, the presence of the fluorophores was detected using 488-or 561-nm lasers for GFP or RFP excitation, respectively.…”

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

“…In a separate protoplast assay, ARR18 induced an ARR5:LUC construct by about 50% in the presence of cytokinin (Veerabagu et al, 2012). When ectopically overexpressed in transgenic plants, ARR18 increased the cytokinin sensitivity, and activated versions of ARR11, ARR18, and ARR19 induced a cytokinin-like response (Liang et al, 2012;Veerabagu et al, 2012).…”

“…Second, these previous studies were performed in a wild-type background, as opposed to the arr1 arr12 background, raising the possibility that their function is dependent in part on genes regulated through action of ARR1 and/ or ARR12. Alternatively, because ARR18 multimerizes (Veerabagu et al, 2012), a physical association with ARR1 and/or ARR12 may allow for indirect transcriptional regulation. Characterization of two-component signaling in plants is likely to be particularly susceptible to artifacts from overexpression based on the known potential for promiscuous interactions in two-component systems (Skerker et al, 2008;Bell et al, 2010;Schaller et al, 2011).…”