Journal of Plant Physiology 169 (2012) 1571–1574 Contents lists available at SciVerse ScienceDirect Journal of Plant Physiology jou rn al h o mepage: www.elsevier.de/jplph Short communication A type-B response regulator drives the expression of the hydroxymethylbutenyl diphosphate synthase gene in periwinkle Olivia Ginis, Audrey Oudin ∗ , Grégory Guirimand, Mouadh Chebbi, Vincent Courdavault, Gaëlle Glévarec, Nicolas Papon, Joel Crèche, Martine Courtois Université Franc ¸ ois Rabelais de Tours, EA 2106 “Biomolécules et Biotechnologies Végétales” – 31 avenue Monge, 37200 Tours, France a r t i c l e i n f o Article history: Received 7 March 2012 Received in revised form 9 July 2012 Accepted 20 July 2012 Keywords: Catharanthus roseus Cytokinin Hydroxymethylbutenyl diphosphate synthase gene promoter Methyl erythritol phosphate pathway Type-B response regulator a b s t r a c t In plant cytokinin (CK) signaling, type-B response regulators (RRs) act as major players in orchestrat- ing the transcriptome changes in response to CK. However, their direct targets are poorly known. The identification of putative type-ARR1 motifs located within the promoter of the CK-responsive hydroxyl methyl butenyl diphosphate synthase (HDS) gene from the methyl erythritol phosphate (MEP) pathway prompted us to investigate the ability of a previously isolated periwinkle type-B RR (CrRR5) that presents high homologies with ARR1 to interact with the promoter. Electrophoretic mobility shift assays (EMSAs) demonstrated that the CrRR5 DNA-binding domain binds specifically type-ARR1 motifs within the HDS promoter. We also established through yellow fluorescent protein (YFP) imaging the targeting of CrRR5 into cell nucleus in accordance with its putative function of transcription factor. In transient assays per- formed on periwinkle cells cultivated with CK, overexpression of the full-length CrRR5 or a truncated CrRR5 engineering a constitutive active form (35S:DDK) did not affect the HDS promoter activity that reached a threshold. By contrast, in absence of CK, overexpression of CrRR5DDK enhanced promoter activity up to the threshold level observed in cells grown with CK. Our results strongly suggest that CrRR5 directly transactivates the HDS promoter. CrRR5 is the first identified transcription factor mediating the CK signaling that targets a gene from the MEP pathway involved in isoprenoid metabolism. Moreover, CrRR5 could play a role in a regulatory mechanism controlling CK homeostasis in periwinkle cells. © 2012 Elsevier GmbH. All rights reserved. Introduction Cytokinins (CK) play crucial roles in diverse aspects of plant growth and development as well as in the control of secondary metabolite biosynthetic pathways (Décendit et al., 1992; Deikman and Hammer, 1995; Mok and Mok, 2001; Argueso et al., 2009). CK signaling involves phosphoryl transfers from histidine kinase receptors up to receiver domain of the type-B response regula- tors (RRs). These type-B RRs possess a C-terminal output domain containing a DNA-binding motif (GARP) followed by a transac- tivation domain (Imamura et al., 1998; Sakai et al., 2001) and act as transcription factors regulating CK-responsive gene expres- sion. In plants, emerging evidences indicate that the mediation of CK responses relies on complex transcriptional cascades in which Abbreviations: CFP, cyan fluorescent protein; CK, cytokinin; EMSA, elec- trophoretic mobility shift assay; GST, gluthatione-S-transferase; HDS, hydroxyl methyl butenyl diphosphate synthase; HMBPP, hydroxyl methyl butenyl diphos- phate; IPP, isopentenyl diphosphate; MEP, methyl erythritol phosphate; RR, response regulator; YFP, yellow fluorescent protein. ∗ Corresponding author. Tel.: +33 247 36 72 15; fax: +33 247 27 66 60. E-mail address: audrey.oudin@univ-tours.fr (A. Oudin). type-B RRs are considered as key elements conducting transcrip- tome changes triggered by this hormone (Argueso et al., 2010). However, despite the large number of gene subsets regulated by CK (Rashotte et al., 2003; Brenner et al., 2005; Kiba et al., 2005; Taniguchi et al., 2007), the identity of genes that are direct targets of type-B RRs remains poorly known. The Madagascar periwinkle (Catharanthus roseus) is a highly investigated medicinal plant producing monoterpenoid indole alkaloids (MIAs). MIAs derive from the iridoid glucoside secolo- ganin whose terpenoid moiety is provided by the plastidial methyl erythritol phosphate (MEP) pathway that synthesizes isopentenyl diphosphate (IPP) and dimethylallyl diphosphate for the produc- tion of various isoprenoids including monoterpenes and some CK. Although the metabolic flux through the MEP pathway is highly regulated by transcriptional mechanisms in response to different signals including CK, no transcription factors regulating the expres- sion of MEP pathway genes have been identified to date (Oudin et al., 2007; Cordoba et al., 2009). Interestingly, in C. roseus, the biosynthesis of MIAs is enhanced by CK that act positively on the expression of several MEP pathway genes and especially of hydroxyl methyl butenyl diphosphate (HMBPP) synthase (HDS) gene that encodes the fifth enzyme of this pathway (Décendit et al., 1992; Oudin et al., 2007). Besides being an intermediate of the 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.07.008