S additional domains to interact using the substrate protein. The target proteins of many of the 700 F-box proteins of Arabidopsis usually are not known. The plant hormone cytokinin exerts its functions mainly via transcriptional activation of its principal target genes, that are activated by type-B response regulators (Sakai et al., 2000; Hwang and Sheen, 2001; Sakai et al., 2001). These are activated by phosphorylation after the cytokinin signal has been transduced from sensor histidine kinase receptors to the nucleus by a multi-step His-Asp phosphorelay signaling technique (Cyclofenil Epigenetic Reader Domain Werner and Schm ling, 2009; Kieber and Schaller, 2014). This pathway has been extensively studied and is now effectively characterized. In contrast, signaling downstream of this initial pathway is only partially known. Transcriptomic approaches have shed light on cytokininregulated genes (Rashotte et al., 2003; Brenner et al., 2005, 2012; Bhargava et al., 2013; Brenner and Schm ling, 2015). Besides some quick early cytokinin response genes supplying feedback towards the upstream cytokinin metabolic and signaling program (type-A response regulator genes), most of them may perhaps contribute to physiological and developmental downstream CP-465022 In Vivo responses of cytokinin (Argueso et al., 2009; Werner and Schm ling, 2009; Ha et al., 2012; Hwang et al., 2012; Vanstraelen and Benkov 2012; El-Showk et al., 2013; Kieber and Schaller, 2014). These cytokinin-regulated genes probably play a specific role in the execution on the many functions of cytokinin and are hence major candidates for additional investigation. Among these cytokinin responsive genes is CFB (Cytokinin-induced F-box encoding), which was found in a meta-analysis of cytokinin-related transcriptome data (Brenner and Schm ling, 2015) and encodes a putative F-box protein. In many hormonal pathways, polyubiquitination of target proteins by SCF-type E3 ligases mediated by certain F-box proteins plays a vital function, for instance, TIR1 (Gray et al., 2001; Dharmasiri et al., 2005; Kepinski and Leyser, 2005) and COI1 (Dai et al., 2002; Xu et al., 2002), regulating the auxin and jasmonic acid pathways, respectively. Handful of reports regarding the involvement of targeted protein degradation by the ubiquitin roteasome pathway and its functional relevance for cytokinin signaling happen to be published, and these that exist have partially contradictory results (Smalle et al., 1997; Yamada et al., 2004; Kim et al., 2013). Here, we present the characterization from the above-mentioned cytokinin-regulated gene, CFB. Overexpression of CFB triggered a pleiotropic phenotype with the improvement of albinotic tissue in the apical end in the inflorescence stem. The morphological, cytological, and chemical phenotypes of plants with enhanced CFB expression resembled these of the cycloartenol synthase mutant cas1-1 (Babiychuk et al., 2008a, 2008b). The phenotype and cytokinin-dependent hyperaccumulation on the CAS1 substrate 2,3-oxidosqualene in cas1-1 mutants suggests a hyperlink involving cytokinin signaling and sterol biosynthesis.Supplies and methodsPhylogenetic evaluation and analysis of protein structure Molecular phylogenetic analyses by the Maximum Likelihood method had been carried out using MEGA version 5.05 (http:www. megasoftware.net) (Tamura et al., 2011). The evolutionary history was inferred utilizing the Maximum Likelihood approach depending on the JTT matrix-based model (Jones et al., 1992). The bootstrap consensus tree inferred from 500 replicates (Felsenstein,.
