Journal of Experimental Botany, Vol. 53, No. 371, pp. 1223-1225,
May 2002
© 2002 Oxford University Press
Gene Note |
Characterization of two CTR-like protein kinases in Rosa hybrida and their expression during flower senescence and in response to ethylene
1Department of Agricultural Science, Horticulture, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
2Department of Horticultural Genetics and Biotechnology, The Mediterranean Agronomic Institute of Chania, PO Box 85, Chania, Crete 73100, Greece
3Department of Crop Science; The Swedish University of Agricultural Sciences, PO Box 44, S-230 53 Alnarp, Sweden
4Department of Ecology, Genetics and Microbiology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
Received 29 November 2001; Accepted 18 December 2001
Abstract
The expression of two CTR-gene homologues was investigated during flower senescence in two Rosa hybrida cultivars. A fragment of a gene for a protein kinase, termed RhCTR1 (GenBank Acc. No. AF271206), was amplified by PCR and used to isolate the corresponding full-length cDNA (Acc. No. AY032953) from a rose petal cDNA library. The protein RhCTR1 has 66% amino acid identity to Arabidopsis CTR1. A fragment of a second CTR homologue, termed RhCTR2 (Acc. No. AY029067) is 69% identical to the corresponding region of RhCTR1. RhCTR1 expression increased during flower senescence, while RhCTR2 was constitutively expressed during flower development. The expression of both RhCTR1 and RhCTR2 was increased in response to exogenous ethylene.
Key words: CTR-homologues, ethylene, flower senescence, post-harvest, Rosa hybrida.
The plant hormone ethylene is involved in the regulation of several physiological and developmental processes. In Arabidopsis, tomato and miniature roses expression studies of several ethylene receptor genes have shown that some of these genes are differentially expressed (review by Chang and Stadler, 2001; Müller et al., 2000a, b)
Several genes involved in the transduction of ethylene responses down-stream of the receptors have been identified in Arabidopsis. Among these are CTR1, which encodes a protein kinase with homology to the mammalian Raf protein kinase (MAPKK kinase) family. Loss-of-function CTR1 mutants result in a constitutive ethylene-response phenotype in the absence of ethylene, indicating that CTR1 negatively regulates the ethylene-response pathway (Kieber et al., 1993). In tomato, two Arabidopsis CTR homologues, TCTR1 and TCTR2, have been characterized (Lin et al., 1998). In Arabidopsis the CTR homologue EDR1 is involved in defence responses (Frye et al., 2001). In the present study with rose flowers, the sequences of a gene fragment and a full-length cDNA of a CTR1 homologue (RhCTR1) and a cDNA fragment of a CTR2-like protein kinase (RhCTR2) are presented.
The primers used for the amplification of a RhCTR1 gene fragment were 5'-CCN GAR TGG ATG GCN CC-3' (corresponding to amino acid residues 715–720 of Arabidopsis CTR1) and 5'-GGN CKY TTC CAN GGY TC-3' (corresponding to amino acid residues 791–796 of Arabidopsis CTR1). The primers used for amplification of a RhCTR2 cDNA fragment were 5'-GAR GTN TAY CAY GCN GAY TG-3' (corresponding to amino acid 682–688 of Arabidopsis EDR1) and 5'-TTN GGR TCN KTY TGC CAR CA-3' (corresponding to amino acid 903–909 of Arabidopsis EDR1). A gene fragment of 561 bp (246 bp+two introns) in size was amplified from a rose gene termed RhCTR1 (GenBank Acc. No. AF271206) and a cDNA fragment of 684 bp in size from a rose gene termed RhCTR2 (Acc. No. AY029067). The partial gene sequence of RhCTR1 has 94% amino acid identity to the corresponding region of Arabidopsis CTR1 and contains two introns at similar positions. The partial cDNA sequence of RhCTR2 has 90% and 87% amino acid identity to tomato TCTR2 and Arabidopsis EDR1, respectively. The identity between the deduced partial protein sequences of RhCTR1 and RhCTR2 is 69% (Fig. 1
).
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The fragment RhCTR1 was used to isolate the corresponding full-length cDNA (Acc. No. AY032953) from a rose cDNA library constructed from total RNA from petals of freshly opened flowers. The isolated RhCTR1 cDNA (Acc. No. AY032953) contains an open reading frame for a protein of 847 amino acids and a predicted molecular weight of 93 kDa. The full-length protein RhCTR1 is 66%, 59% and 35% identical to Arabidopsis CTR1, tomato CTR1, and tomato CTR2, respectively (Fig. 2
).
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RT-PCR was used to evaluate differences in RhCTR1 and RhCTR2 expression in petals of two miniature rose cultivars that had earlier been shown to exhibit differences in post-harvest life and ethylene sensitivity (Müller et al., 1998, 2000a, b). Total RNA was extracted from petals of R. hybrida ‘Vanilla’ and ‘Bronze’ at three developmental stages; bud (A), open flowers (B) and incipient senescence (C) (Fig. 3
), or from ‘Bronze’ flowers at stage B, with or without previous ethylene treatment. First strand cDNA synthesis was carried out using a first strand cDNA synthesis kit for RT-PCR (AMV) (Boehringer Mannheim) using oligo(dT)15 for the CTR gene and a specific reverse primer (5'-CCA CCA CCC ATA GAA TCA AGA AAG AG-3') for the control ribosomal RNA. The primers used for CTR1 were 5'-GAT GGC GCC AGA AGT CC-3' (forward) and 5'-GCC CAG CAA GCC TCA AT-3' (reverse). The primers for CTR2 were 5'-GTC GCG CTT GAA ACA TAA CA-3' (forward) and 5'-AAC AGG GGG ATC AAC TTC TTT-3' (reverse). The primers for 18S ribosomal RNA (GenBank Acc. No. RH18SRRNP) were 5'-CGG GGA GGT AGT GAC AAT AAA TAA CA-3' (forward) and 5'-CCA CCA CCC ATA GAA TCA AGA AAG AG-3' (reverse). The exponential range was determined by carrying out the PCR for 30 cycles on serial dilutions of a fixed quantity of cDNA (Murphy et al., 1990). PCR products were separated on a 1.5% agarose gel, EtBr stained, and photographed. The bands were quantified using Quantity One Software (Biorad, CA, USA). The density of each EtBr-stained CTR band was corrected by division with the density of the EtBr-stained 18S rRNA band from the same sample.
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The expression of RhCTR1 and RhCTR2 exhibited no cultivar differences. The abundance of RhCTR1 transcript in the petals of both cultivars was clearly increased after the visible onset of flower senescence. The gene RhCTR2 was constitutively and stably expressed during flower development in both cultivars. Additionally, in the cultivar ‘Bronze’ the expression of both the RhCTR1 and RhCTR2 was higher after ethylene (0.25 µl l-1 for 3 d) treatment than in untreated control flowers.
These results indicate an involvement of RhCTR1 and RhCTR2 in ethylene-induced senescence in rose flowers. However, the negative regulator model of ethylene receptor function (Chang and Stadler, 2001) leads to the prediction that up-regulation of ethylene receptor expression in response to ethylene would down-regulate ethylene responses. Binding of ethylene is believed to inactivate the receptors, which in turn inactivate CTR, allowing the ethylene response pathway to be activated. In previous studies with three members of the ethylene receptor family it was shown that ethylene responses correlated to increased ethylene receptor expression (Müller et al., 2000b). In the present study, it was found that exogenous ethylene increased expression of two CTR homologues and resulted in accelerated flower senescence. In addition, RhCTR1 was up-regulated in senescing flowers, coincident with the increase in ethylene production. Assuming that flowers become more ethylene-sensitive during senescence, the negative regulator model predicts, as in the case of ethylene receptors, decreased CTR expression as a function of developmental stage and in response to exogenous ethylene. The observations on the expression of ethylene receptors and CTR homologues in miniature roses are therefore in apparent contrast to the negative regulator model under the assumption that ethylene sensitivity increases during flower senescence and in response to ethylene. Waki et al. recently found that the expression pattern of EIL1, another gene of the ethylene signal transduction pathway, likewise is in contrast to the negative regulator model (Waki et al., 2001). Further studies are needed to elucidate the exact relationship between ethylene sensitivity and the expression of genes involved in ethylene signal transduction.
Acknowledgements
This research was supported by a grant to R Müller from The Danish Agricultural and Veterinary Research Council.
Footnotes
5 To whom correspondence should be addressed. E-mail: renate.muller{at}kvl.dk 
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