Molecular cloning and expression of cDNAs encoding alcohol dehydrogenases from Vitis vinifera L. during berry development
Introduction
Alcohol dehydrogenase genes encode glycolytic enzyme (ADH, EC 1.1.1.1) that have been characterised at the molecular level in a wide range of flowering plants. Adh genes belong to small multigene families generally composed by two or three members [1], [2], [3], [4], with the exception of Arabidopsis that appears to have a single locus [5]. Much attention has been given to the induction of Adh gene expression and enzyme activity during anaerobiosis [6], [7]. There is also evidence that other stresses such as dehydration, low temperature, or chemical treatments induce Adh gene expression in a variety of plants [1], [8], [9]. The versatility of transcription from Adh promoter to respond to different stresses has been studied in details in Arabidopsis [10]. In addition, tissue specific and developmentally regulated Adh gene expression have been recently reported [11], [12], [13]. Altogether, these data indicate a central role for Adh in stress survival and organ development.
High ADH enzyme activity and Adh mRNA levels were observed in ripe Vitis vinifera L. berries [14]. ADH activity is induced at the onset of ripening, i.e. véraison and coordinated with berry development [15], [16]. The ADH induction in berries is apparently regulated at the transcriptional level as transcript abundance in other organs is rather limited [17]. Genomic analyses indicated that grapevine ADH is encoded by a small multigene family [17], [18]. In an effort to better understand the control of ripening in this non-climacteric fruit and to further elucidate the role of Adh in developing grape berries, the Adh isogenes involved in the berry ripening process were investigated. Adh cDNAs were cloned and expression of their corresponding transcripts was analysed throughout fruit development. Here we report the molecular characterisation of three divergent Adh-specific cDNAs from developing grape berries. Results showed the occurrence of three isogenes differentially expressed and exhibiting various biochemical properties. Thus, Adh gene expression during berry development is complex with three ADH gene products likely playing distinct metabolic roles.
Section snippets
RNA isolation and RT-PCR reactions
Total RNA was extracted from grape (Vitis vinifera L.) berries from the seedless cultivar Danuta (cross between Dattier de Beyrouth x Sultana Moscata) as described by Tesnière and Vayda [19]. Five g of ground, frozen berry tissue were added to the extraction buffer (200 mM Tris–HCl (pH 8.5) containing 300 mM LiCl, 10 mM Na2-EDTA, 1% (w/v) sodium deoxycholate, 1.5% (w/v) SDS, 1 mM ATA, 5 mM thiourea, 1% (v/v) NP-40 and 10 mM DTT) and homogenised. The extracts were centrifuged at 12 000×g for 15
Cloning and sequence analyses of three grapevine Adh cDNAs from grape berries
Internal segments (from exon 2 to exon 8) of grape berry Adh cDNAs were PCR amplified with primers corresponding to highly conserved regions of plant Adh sequences [2]. From these fragments (around 700 bp), 18 clones were examined. Sequence analyses indicated three similar but distinct partial Adh-like cDNAs. Consensus primers C (exon 7) and D (exon 4) were designed from these sequences (Table 1) and respectively used to obtain 3′- and 5′- ends of the three cDNAs using RACE techniques [20]. For
Discussion
ADH gene families have been well characterised in annual species, Adh from maize being the first gene cloned [26]. However, little is known on Adh in perennial plant species, especially at the molecular level. Full-length grapevine cDNAs that encode Adhs have been obtained and molecularly analysed. Sequence alignments gave clear evidence of three distinct cDNAs, highly divergent in the non-coding regions and encoding three different Adh genes. VvAdh1 sequence appears to be more closely related
Acknowledgements
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AF194 173, AF194 174, AF194 175 and AF196 485, for VvAdh1, VvAdh2, VvAdh3 and VvTub, respectively. We are grateful to H. Kadowaki for providing anti-ADH antibody from rice and to V. Lullien, C. Romieu, F.X Sauvage and M.E. Vayda for helpful discussions. This work was supported by the AIP Matural program grants from INRA.
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