Journal of Experimental Botany, Vol. 53, No. 376, pp. 1989-1990,
September 1, 2002
© 2002 Oxford University Press
Expression analysis in plant and cell suspensions of CrCKR1, a cDNA encoding a histidine kinase receptor homologue in Catharanthus roseus (L.) G. Don.
Received 24 April 2002; Accepted 4 June 2002
EA 2106, Biomolécules et Biotechnologies Végétales, Université de Tours, 31 Avenue Monge, F-37200 Tours, France
2 Present address and to whom correspondence should be sent: Laboratoire de Biologie Moléculaire et Biochimie Végétale, UFR des Sciences Pharmaceutiques, 31 Avenue Monge, F-37200 Tours, France. Fax: +33 2 47 27 66 60. E-mail: creche{at}univ-tours.fr
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A full length cDNA (CrCKR1) encoding a hybrid histidine kinase was isolated from a Catharanthus roseus cDNA library. The kinase belongs to the subfamily of cytokinin receptors represented by CRE1/AHK4/WOL in Arabidopsis thaliana. In cell suspensions, the expression of CrCKR1 is not affected by various stress and hormonal treatments but is stimulated in cells continuously exposed to cytokinin. In plants, CrCKR1 is strongly expressed only in the petals of mature flowers. These data suggest that CrCKR1 could take part in the mechanisms leading to the production of secondary metabolites in C. roseus.
Key words: Key words: Catharanthus roseus, cytokinin receptors, histidine kinase.
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In bacteria, fungi and plants, some adaptive responses are mediated through signalling mechanisms, referred to as histidine–aspartate kinases. The basic two-component systems involve the autophosphorylation of a conserved histidine residue in a sensor, histidine kinase, then the transfer of the phosphate to a conserved aspartate in a receiver protein. The more complex multistep His–Asp–His–Asp phosphorelays usually involve four sequential phosphorylation events through three proteins: a hybrid histidine kinase in which the histidine kinase is fused to the receiver domain, a histidine phosphotransfer module and an aspartate-containing response regulator (see Stock et al., 2000, for a review). The recent completion of the Arabidopsis genome revealed 17 histidine kinases, and the first plant hybrid histidine kinases to be characterized (namely ETR1 and CKI1 in Arabidopsis thaliana) were proposed to be ethylene and cytokinin receptors (Chang et al., 1993; Kakimoto, 1996).
Among other functions, cytokinins can regulate some secondary metabolites pathways, including anthocyanin synthesis in maize (Piazza et al., 2002) and terpenoid indole alkaloid (TIA) production in C. roseus suspension cells (Décendit et al., 1992; Yahia et al., 1998). In order to investigate the underlying mechanisms that control the effect of cytokinins on TIA accumulation in periwinkle, a search was made for elements involved in the transduction of cytokinin signal. A full length 4040 bp cDNA, CrCKR1, was isolated from a C. roseus cDNA library using a PCR strategy based on protein conserved domains. The predicted protein (1041 amino acids; calculated molecular mass: 116 kDa) is 50–60% identical with the A. thaliana cytokinin receptors represented by CRE1/AHK4/WOL (Fig. 1) which is involved in the control of vascular development (Mahonen et al., 2000). Its putative structure is shown in Fig. 2. The N-terminal sensing region contains two 
-helices that span the plasma membrane and delimit an extracellular loop containing the CHASE domain required for low molecular mass hormone binding (Anantharaman and Aravind, 2001). The transmitter domain exhibits all the boxes highly conserved within the histidine kinase superfamily, i.e. the H box essential for histidine phosphorylation and the N/G1/F/G2 boxes representing the catalytic ATP-binding domain. The C-terminal region contains two receiver domains organized in tandem as typically found in A. thaliana CRE1/AHK4/WOL. Southern analysis revealed only one CrCKR1 copy in the C. roseus genome (data not shown).
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Since it has been reported that some genes encoding hybrid histidine kinases were rapidly induced by stress and phytohormones in plants (Urao et al., 1999; Hua et al., 1998), it was tested whether treatments with 2,4-dichlorophenoxyacetic acid, jasmonic acid, ethylene, zeatin or NaCl could regulate the expression of CrCKR1 in periwinkle cell suspensions. The concentrations of the different compounds used were those that have been shown previously to affect TIA biosynthesis in C. roseus cells (Décendit et al., 1992; Yahia et al., 1998; Gantet et al., 1998). Figure 3A indicates that these treatments did not significantly modify CrCKR1 transcript accumulation after 1 h. By contrast, exogenous cytokinin treatments during three subculture cycles induced CrCKR1 transcript accumulation (Fig. 3B). This was correlated with a 2-fold increase in alkaloid production (data not shown). A strong accumulation of transcripts was also observed in transgenic cell lines overexpressing the Agrobacterium tumefaciens ipt gene and producing high levels of endogenous cytokinins (Fig. 3C) (Garnier et al., 1996). In plant, a high CrCKR1 transcript steady-state level was found in the anthocyanin accumulating petals (Fig. 3D). These data suggest that CrCKR1 gene expression is positively regulated by a long-term exposure to cytokinin, which was shown to enhance alkaloid production in the cells cultivated in vitro (Décendit et al., 1992; Yahia et al., 1998). Interestingly, CrCKR1 expression also correlated with anthocyanin biosynthesis in plants, which is known in other models to be positively regulated by cytokinins (Piazza et al., 2002). This may suggest that, in C. roseus, the CRE1/AHK4/WOL cytokinin receptor homologue, CrCKR1, could take part in the mechanisms leading to secondary metabolite production in response to cytokinin. This hypothesis has now to be investigated further.
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Acknowledgement |
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We thank the ‘Ministère de l’Education Nationale, de la Recherche et de la Technologie’ for financial funds and a scholarship for NP.
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