Elsevier

Plant Science

Volume 157, Issue 1, 8 August 2000, Pages 77-88
Plant Science

Molecular cloning and expression of cDNAs encoding alcohol dehydrogenases from Vitis vinifera L. during berry development

https://doi.org/10.1016/S0168-9452(00)00274-0Get rights and content

Abstract

Three full-length cDNAs (VvAdh1, VvAdh2, and VvAdh3) encoding alcohol dehydrogenases (EC 1.1.1.1) were obtained from grape berries (Vitis vinifera L.) by means of PCR and RACE. Pairwise comparisons at the nucleotide level showed that the three cDNAs displayed strong homology in the coding region, but were highly divergent in the 5′ and 3′ untranslated regions. VvAdh1 and VvAdh2 corresponded to the two previously characterised Adh genes from grapevine, but VvAdh3 was unrelated to known grapevine Adh sequences. The two first cDNAs presented a single ORF of 380 amino acids, whereas the last one has two additional residues. Moreover, the three encoded polypeptides possessed the 22 residues strictly conserved between Adh from different kingdoms. Expression pattern of the individual isogenes was investigated during fruit development. Specific primers were designed, and quantitative RT-PCR experiments were performed to increase the sensitivity of detecting isogenes with a low expression level. Results presented here revealed different developmental regulation of the three Adh isogenes during fruit ripening. VvAdh1 and VvAdh3 transcripts were temporarily accumulated in young, developing berry, whereas VvAdh2 was overexpressed later in fruit development, from the onset of ripening (véraison). Expression analysis also indicated that VvAdh2 accounted for most of the Adh mRNAs present in berries during development. The increased ADH activity detected in berries correlated with the expression pattern of VvAdh2 transcripts. The VvAdh2 and VvAdh3 encoded enzymes were purified from overexpressing E. coli cells. Comparison of kinetic properties of the two ADH enzymes showed a difference in affinity with either ethanol or acetaldehyde as substrates. Significance of multiple Adh expressed in berries is discussed.

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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