|Scooped by PMG|
11 type-B response regulators (type-B ARRs), and some of them were shown to bind in vitro to the core cytokinin response motif (CRM) 5′-(A/G)GAT(T/C)-3′ or, in case of ARR1, to an extended motif (ECRM), 5′-AAGAT(T/C)TT-3′. Here we obtained in planta proof for the functionality of the latter motif. Promoter deletion analysis of the primary cytokinin response gene ARR6 showed that a combination of two extended motifs within the promoter is required for mediating the full transcriptional activation by ARR1 and other type-B ARRs. CRMs were found to be overrepresented in the vicinity of ECRMs in the promoters of cytokinin-regulated genes suggesting their functional relevance. Moreover, an evolutionary conserved 27 bp-long region between -220 and -193 bp was identified and shown to be required for the full activation by type-B ARRs and the response to cytokinin. This novel enhancer is not bound by the DNA binding domain of ARR1, indicating that additional proteins might be involved in mediating the transcriptional cytokinin response. Furthermore, genome-wide expression profiling identified genes, among them ARR16, whose induction by cytokinin depends on both ARR1 and specific other type-B ARRs. This together with the ECRM/CRM sequence clustering indicates cooperative action of different type-B ARRs for the activation of particular target genes.