Journal of Experimental Botany, Vol. 53, No. 378, pp. 2283-2285,
November 1, 2002
© 2002 Oxford University Press
Characterization of a strawberry cDNA clone homologous to calcium-dependent protein kinases that is expressed during fruit ripening and affected by low temperature
Received 19 March 2002; Accepted 9 July 2002
Departmento de Fisiología y Biodiversidad Molecular, Instituto de Biología Molecular de Barcelona, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
Abbreviation: CDPK: calcium-dependent protein kinase.
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A full-length cDNA clone (FaCDPK1) encoding a calcium-dependent protein kinase (CDPK) has been isolated from a strawberry fruit cDNA library. FaCDPK1 contains the basic features of CDPKs: a catalytic kinase domain linked to a regulatory calmodulin-like domain by a junction sequence that has been shown to act as an autoinhibitory pseudosubstrate. Although the calmodulin-like domain of CDPKs typically contains four EF-hand calcium-binding motifs, FaCDPK1 was predicted to contain only three EF-hand motifs. FaCDPK1 gene expression was observed in roots, stolons, meristems, flowers, and leaves. FaCDPK1 mRNA was not detected in young fruits, but accumulated as fruit turned to white, suggesting a role for this gene in the developing strawberry fruit. In ripe fruit the levels of transcript increased in response to low temperature.
Key words: Key words: CDPK, cold treatment, strawberry, fruit.
In plants, it has been established that calcium acts as a second messenger in the transduction of several environmental and hormonal signals that affect many aspects of plant growth and development. Intracellular calcium fluxes are believed to be transduced to biological responses through the activation of several calcium-regulated proteins such as calcium-dependent protein kinases (CDPK) (for reviews see Poovaiah and Reddy, 1993; Roberts, 1993; Bush, 1995; Harmon et al., 2000). A cDNA clone designated FaCDPK1 (deposited in the GenBank under the Accession No. AF035944) has been isolated from a strawberry (Fragariax ananassa Duch cv. Pajaro) fruit cDNA library. The analysis of the FaCDPK1 sequence revealed that it is highly homologous to plant CDPKs. Several CDPK cDNAs have been isolated from different plant tissues and from protozoans (for a review see Harmon et al., 2000). However, although CDPK activity had been previously reported in apple and mango fruit (Battey and Venis, 1988; Frylinck and Dubery, 1998), this is the first report of a cDNA encoding a CDPK expressed in fruit.
CDPKs are characterized by a protein kinase domain highly homologous to mammalian calcium/calmodulin-dependent protein kinase type II (CaMKII) linked to a C-terminal calcium-binding regulatory domain that contains four EF-hand structures found in calmodulins. Both domains are linked by a highly conserved junction sequence that has been shown to act as an autoinhibitory pseudosubstrate regulating kinase catalytic activity. Preceding the kinase domain, CDPK proteins have an N-terminal domain with variable length and no conserved sequence, which has been suggested to determine the cellular localization of the protein (for reviews see Roberts and Harmon, 1992; Harmon et al., 2000).
The cDNA clone FaCDPK1 is 1967 bp long and has a 1587 bp open reading frame encoding a predicted protein of 529 amino acids with a deduced molecular mass of 59.6 kDa and an isoelectric point of 6.1. The FaCDPK1 deduced protein shows high homology to plant CDPK proteins described in the databank, especially to Arabidopsis AtCDPK19 to which it shows 78% of identity (Hong et al., 1996). Alignment of the deduced FaCDPK1 protein with Arabidopsis AtCDPK19 and other CDPK proteins is shown in Fig. 1. The most conserved region between the FaCDPK1 protein and other CDPKs corresponds to the catalytic domain, from residue 52 to residue 315. The calmodulin-like domain, from residue 347, typically contains four EF-hand calcium binding motifs. However, when searching for motifs in the PROSITE program, only three EF-hands, corresponding to sites II, III and IV, were found to fit the consensus pattern. The presence of atypical EF-hands has been reported for other CDPKs and it is likely to affect their calcium-binding properties (Zhao et al., 1994). For example, three CDPKs from soybean have been shown to respond to different ranges of calcium concentration, suggesting that they play distinct roles in mediating responses to calcium signals (Lee et al., 1998).
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To assess the expression of FaCDPK1, total RNA was isolated from various vegetative tissues and from fruits at different stages of development and ripening. Achenes and the central fibrous core were removed from fruits and the remaining tissue was used for RNA extraction. RNA gel blots were hybridized with a random primed DNA probe synthesized from full length FaCDPK1 cDNA. As shown in Fig. 2A, FaCDPK1 is predominantly expressed in roots, but its transcript is also detected in other vegetative tissues, including stolons, meristems, flowers, and young and adult leaves. No differences were observed in the level of expression of FaCDPK1 in young compared to adult leaves. By contrast, FaCDPK1 expression in fruits (Fig. 2B) was found to be affected by the stage of development of the fruit as transcripts were first detected in white fruits, but no expression could be observed in younger fruits. In addition, the abundance of the FaCDPK1 transcript is slightly altered during fruit ripening. These results indicate a developmental regulation of FaCDPK1 in fruit. The expression pattern of FaCDPK1 in fruit suggests that the encoded CDPK has a role in the signalling events that regulate the process of strawberry fruit development. Other calcium-regulated proteins have been shown to be expressed in fruit such as tomato, pepper and strawberry (Wilkinson et al., 1995; Proust et al., 1996; Kiyosue and Ryan, 1997).
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It is believed that CDPKs are implicated in environmental and hormonal signal transduction. The expression of CDPK genes from different plant species is induced in response to stimuli such as cold, phytohormones, touch, light, salt stress, wounding or fungal elicitors. In order to examine the effect of low temperature on the expression of FaCDPK1, detached ripe fruit were kept at 4 °C for 5 h or 10 h. As shown in Fig. 2B, an increase in FaCDPK1 mRNA level is observed when ripe fruits are kept for 5 h at 4 °C compared to control fruits maintained at 22 °C. A further increase is observed after 10 h of cold treatment, which clearly indicates an induction of FaCDPK1 transcription by low temperature. The stimulatory effect of cold on the expression of CDPK genes has been reported in other plant species (Martín and Busconi, 2001; Tähtiharju et al., 1997; Urao et al., 1994; Monroy and Dhindsa, 1995). Recently, a connection between a CDPK and low temperature signalling has been proven in rice, where the over-expression of a single CDPK confers cold as well as salt/drought tolerance (Saijo et al., 2000).
The results presented in this paper suggest a role for a strawberry CDPK in fruit development as well as in the response of the fruit to low temperature. The generation of transgenic plants with altered CDPK levels may help to elucidate the role of calcium and CDPKs in fruit physiology.
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Acknowledgements |
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We thank Dr D Grierson for revision of the manuscript and Dr S Prat for advice and help with construction of the cDNA library. This work was supported by the grant ALI94-1031-CO3-01 from the Comisión Interministerial de Ciencia y Tecnología and by Carburos Metálicos SL.
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