Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol157.Issue1.2000:

Plant Science 157 2000 77 – 88 Molecular cloning and expression of cDNAs encoding alcohol dehydrogenases from Vitis 6inifera L. during berry development Catherine Tesnie`re , Clotilde Verrie`s Unite´ de Recherche de Biochimie Me´tabolique et Technologie, INRA, ISVV M, IPV, 2 , Place Viala, Montpellier cedex 1 , France Accepted 31 March 2000 Abstract Three full-length cDNAs VvAdh1, VvAdh2, and VvAdh3 encoding alcohol dehydrogenases EC 1.1.1.1 were obtained from grape berries Vitis 6inifera 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 ve´raison. 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. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Alcohol dehydrogenase; Fruit development; Grape berry; Vitis 6inifera L. www.elsevier.comlocateplantsci

1. Introduction

Alcohol dehydrogenase genes encode glycolytic enzyme ADH, EC 1.1.1.1 that have been charac- terised at the molecular level in a wide range of flowering plants. Adh genes belong to small multi- gene families generally composed by two or three members [1 – 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 anaer- obiosis [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 tran- scription from Adh promoter to respond to differ- ent stresses has been studied in details in Arabidopsis [10]. In addition, tissue specific and developmentally regulated Adh gene expression have been recently reported [11 – 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 6inifera L. berries [14]. ADH activity is induced at the onset of ripening, i.e. ve´raison and coordinated with berry development [15,16]. The ADH induction in berries is apparently regulated at the transcrip- Corresponding author. Tel.: + 33-4-99612531; fax: + 33-4- 99612857. E-mail address : tesniereensam.inra.fr C. Tesnie`re. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 2 7 4 - 0 tional level as transcript abundance in other or- gans is rather limited [17]. Genomic analyses indi- cated 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-cli- macteric fruit and to further elucidate the role of Adh in developing grape berries, the Adh isogenes involved in the berry ripening process were investi- gated. 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 bio- chemical properties. Thus, Adh gene expression during berry development is complex with three ADH gene products likely playing distinct metabolic roles.

2. Materials and methods