Journal of Experimental Botany, Vol. 54, No. 388, pp. 1793-1795,
July 1, 2003
© 2003 Oxford University Press
Differential expression of two type-A response regulators in plants and cell cultures of Catharanthus roseus (L.) G. Don*
Received 21 February 2003; Accepted 7 April 2003
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EA 2106, Biomolécules et Biotechnologies Végétales, Université de Tours, 31 Avenue Monge, F-37200 Tours, France
* The sequences of CrRR2 and CrRR3 were deposited in the GenBank database under the accession numbers AF534889 and AF534890, respectively. 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 02 47 27 66 60. E-mail: creche{at}univ-tours.fr
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Abstract |
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Two full-length cDNAs named CrRR2 and CrRR3 have been isolated by PCR from a C. roseus cDNA library. The first one encodes a 154 amino acid putative protein with a high percentage of identity with the Arabidopsis thaliana response regulators ARR16 and ARR17, and is not expressed in C. roseus organs and cell cultures. The second one encodes a 188 amino acid ORF sharing the highest homologies with the A. thaliana ARR8 and ARR9 response regulators. Its expression is root-specific and the transcripts are transiently up-regulated after trans-zeatin treatment in C. roseus suspension cells. CrRR3 protein might be involved in the cytokinin-enhanced alkaloid production in C. roseus cell cultures.
Key words: Catharanthus roseus, cytokinin, response regulators.
Cytokinins are plant growth regulators with pleiotropic functions, including the regulation of biosynthesis for some secondary metabolites such as anthocyanins in Arabidopsis thaliana plants (Deikman and Hammer, 1995) and terpenoid indole alkaloids (TIAs) in C. roseus cell cultures (Yahia et al., 1998). Recently, significant progress toward understanding cytokinin signalling has been made with the discovery of multistep His–Asp phosphorelays in A. thaliana (Hwang and Sheen, 2001). The isolation of CrCKR1 (GenBank accession number AY092025 [GenBank] ) and CrHpt1 (GenBank accession number AF346308 [GenBank] ) that encodes a histidine kinase cytokinin receptor and a histidine-containing phosphotransfer domain, respectively, in C. roseus has previously been reported, and it is suggested that these proteins could take part in the mechanisms leading to the cytokinin-enhanced TIA production in cell suspension (Papon et al., 2002). To characterize the downstream components of the C. roseus cytokinin transduction pathway further, the isolation of genes encoding cytokinin-inducible response regulators (RR) was undertaken.
Two full length cDNAs named CrRR2 and CrRR3 were isolated by a PCR strategy based on the screening of conserved protein domains. CrRR2 encodes a 154 amino acid putative protein (calculated MM: 17.2 kDa) whereas CrRR3 encodes a 188 amino acid predicted protein (MM: 21.3 kDa). For both proteins, the search for sequence homologies in databases confirmed the presence of the DDK signature found in all the Arabidopsis receiver domains (D’Agostino et al., 2000) (Fig. 1). The highest percentage of identity (57%) between CrRR2 and other RRs was found with two proteins from A. thaliana, namely ARR16 (GenBank accession number AF305721 [GenBank] ) and ARR17 (GenBank accession number AF305722 [GenBank] ). As compared with ARR16 and ARR17, CrRR2 also exhibits a very short C-terminal extension, but it lacks the potential N-myristoylation site found near the N-terminus. The CrRR3 protein highly resembles two A. thaliana RRs (68.7% identity with amino acids 9–142 of ARR8/ATRR3, GenBank accession number AB010917 [GenBank] ; 64.4% identity with amino acids 9–163 of ARR9/ATRR4, GenBank accession number AB010918 [GenBank] ; D’Agostino et al., 2000), but is shorter than ARR9 due to several amino acid deletions in the C-terminal end (for example between amino acids 144–167). CrRR3 also has significant homologies with the putative response regulator ZmRR6 from Zea mays. CrRR2 and CrRR3 do not share identities with the receiver domains found in DNA-binding type-B RRs (Immamura et al., 1999) and thus belong to the type-A RR-subfamily. Southern analysis indicated that the corresponding genes are present in multiple copies in the C. roseus genome (data not shown).
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To investigate the regulation of expression of CrRR2 and CrRR3 genes, the accumulation of their transcripts in whole plants and in suspension cells of C. roseus were studied. CrRR2 and CrRR3 gene expression differed considerably. As it was impossible to detect any CrRR2 transcripts by northern blot analysis even after several weeks of exposure (not shown), it may be concluded that the CrRR2 gene is expressed at a very low level, if any, in the plant and that treatment by cytokinins does not trigger its transcription in suspension cells. Interestingly, ARR17 transcripts could not be detected in A. thaliana seedlings treated by cytokinins (D’Agostino et al., 2000). By contrast, the CrRR3 gene was clearly expressed in C. roseus. In suspension cells, transcript amounts remained unchanged after treatment with NaCl, abscisic acid or jasmonic acid, but was increased with trans-zeatin (Fig. 2A). Dose-dependent studies showed a maximum of CrRR3 transcripts at a concentration of 10–6 M zeatin, with an increase already observed at 10–9 M (Fig. 2B). Kinetic studies showed that the transcript level was maximum after 1 h of zeatin treatment, then progressively returned to the steady-state control level within 6 h (Fig. 2C). In the plant, CrRR3 transcripts were rather undetectable in petals, ovaries, stem apexes, mature leaves, and stems, but were present in the roots (Fig. 2D).
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Among the 11 type-A response regulators reported in A. thaliana, ARR8, ARR9, ARR16, and ARR17 have been examined less than others. However, ARR8 was previously reported to be specifically expressed in roots (Urao et al., 1998) and recent findings suggest that this tissue-specific expression may reflect an important role in cytokinin signalling (Osakabe et al., 2002). In C. roseus, CrRR3 is also strictly expressed in the roots, i.e. organs that biosynthesize a large pallet of TIAs (De Luca and Laflamme, 2001). In cell cultures, cytokinins enhance TIA production (Yahia et al., 1998) and, at least with the cell line CR6, a transient treatment with zeatin at the beginning of the culture is sufficient to trigger TIA accumulation in the cells 2 weeks later (Kodja et al., 1989). Thus, while the CrRR2 protein seems not to be involved in the enhancing effect of cytokinin on TIA accumulation, CrRR3 and possibly other type-A response regulators might be primary effectors of the cytokinin effect in C. roseus cell cultures.
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Acknowledgements |
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This research was supported by the ‘Ministère de l’Education Nationale, de la Recherche et de la Technologie’ (MENRT, France). A doctoral grant was awarded by the MENRT to NP. We are grateful to Dr J Memelink (University of Leiden, the Netherlands) who kindly provided the oriented cDNA library.
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