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
2
.
1
. RNA isolation and RT-PCR reactions Total RNA was extracted from grape Vitis
6 inifera L. berries from the seedless cultivar
Danuta cross between Dattier de Beyrouth x Sul- tana Moscata as described by Tesnie`re 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 Na
2
- EDTA, 1 wv sodium deoxycholate, 1.5 wv
SDS, 1 mM ATA, 5 mM thiourea, 1 vv NP-40 and 10 mM DTT and homogenised. The extracts
were centrifuged at 12 000 × g for 15 min at 4°C and supernatant was filtered through Miracloth.
CsCl was then added to the filtrate 0.2 gml at room temperature and the resulting homogeneous
solution was layered on a 10 ml cushion contain- ing 5.7 M CsCl in 10 mM Tris – HCl pH 7.5.
Ultracentrifugation was performed for 24 h at 20°C at 110 000 × g in a swinging rotor SW-28,
Beckman. RNA pellets were treated with LiCl and sodium acetate as previously described, but
the selective precipitation of viscous components by ethanol was omitted.
First strand cDNA was synthesised from 5 mg of total RNA using a polyT
15
as primer and Super- script II reverse transcriptase Gibco-BRL, ac-
cording to the manufacturers instructions. After the reaction was stopped by heating at 70°C for 10
min, and treated with RNAse H, PCR amplifica- tions were carried out using degenerated or spe-
Table 1 Oligonucleotide primers used in this study
Region and sense cDNA
Name Positions and 5-3 DNA sequences
Plant Adhs A
E2F 190-GAYGTNTAYTTYTGGGARGC-209
a
B Plant Adhs
E8R 904-CRTCNTGRACACAYTCRAA-876
a
VvAdhs C
E4R 395-ATCCTGAGGAGGTCACACA-377
a
D 751-AAGAARTTTGGYGTSACYG-766
a
E7F VvAdhs
1UTR5F 6-AGGAGCACCATATCTTTGGAG-26
VvAdh1 E
1320-GGAATGAGTTTTGACCTCC-1339 1UTR3R
VvAdh1 F
VvAdh2 G
32-AGTGTGAGAAGGAATATAGCC-52 2UTR5F
2UTR3R VvAdh2
1380-CTGTTATATATTGCAACTGC-1361 H
3UTR5F VvAdh3
9-TTCTCTCAGAGTTGTTAAAGC-29 I
1361-AATTTCCACAAGCGAGTG-1344 3UTR3R
J VvAdh3
Plant Tub TUBF
614-TTGTTGAGCCATACAATGC-632
b
K L
1273-AGTACCAATGCAAGAAAGC-1255
b
TUBR Plant Tub
1UTR3F 1193-GATGAGAGAGTCTAATTGAAT-1213
VvAdh1 M
1212-GATTTGCCTATTCCAGTCG-1230 N
VvAdh2 2UTR3F
VvAdh3 O
3UTR3F 1193-ATGAGTGAAGTGTATGTTAGATG-1215
P VvAdh2
2NF 58-TTATACATATGTCAAGCACAGCTG-73
c
VvAdh2 2BR
Q 1200-ATAGGATCCTTATGCATCCATGCG-1186
c
VvAdh3 3NF
R 34-TTATACATATGTCTAATACAGCTGGTC-52
c
VvAdh3 3BR
1182-TAGGATCCCTAGGCTCCCATGC-1169
c
S
a
Referred to positions of VvAdh1.
b
Referred to positions of beta-tubulin from Solanum tuberosum TUBST2 accession number STBETTUB2.
c
BamHI and NdeI restriction sites are underlined.
cific primers Table 1 as follows: each reaction 25 ml final volume contained 1 ml of RT reaction
product, 0.4 mM of each primer, 200 mM of each dNTP, 0.75 unit of Taq DNA polymerase
Promega and 1 × PCR buffer. The reactions were cycled 32 times at 94°C for 1 min 4 min for
the first cycle, 49°C for 1 min and 72°C for 1 min 15 min for the final cycle.
2
.
2
. Cloning and characterisation of full-length Adh cDNAs from grape berry
Full-length Adh cDNAs were obtained using total RNA isolated from berries at ve´raison. In a
first step, cDNA amplification was carried out using the degenerate primers A and B Table 1
designed from highly conserved regions of the plant Adh genes from exon 2 to exon 8 by
Gregerson et al. [2]. Grapevine consensus primers C derived from exon 4 and D derived from exon
7 were then designed Table 1 and used with RACE PCR [20] to obtain full-length nucleotide
sequences. The 5-end fragments were obtained with the 5 RACE system Gibco-BRL, including
an anchor primer with a polyC region. The 3-end fragments were obtained using an hybrid
NN-oligodT
15
-adapter primer [20]. The subse- quent PCR amplifications were carried out as
described above, using either the anchor primer lacking polyC region and the antisense consen-
sus primer C for the 5- ends, or the consensus sense primer D and the adapter primer lacking
polyT region for the 3- ends. Finally, full length cDNA products around 1350 bp were obtained
using the primer pairs EF, GH and IJ Table 1, which correspond to unique sequences in the 5-
and 3-UTRs of the grape Adh isogenes, VvAdh1, VvAdh2 and VvAdh3, respectively.
After purification from low-melting agarose gels using Jet-Sorb kit Bioprobe, the PCR products
were ligated into pTag Novagen or pGemT-Easy vectors Promega. The ligation products were
transformed into E. coli strain DH5a. DNA was sequenced on the two strands using the Applied
Biosystems
PRISM™ Ready
Reaction DyeDeoxy™ Terminator Cycle Sequencing kit,
following the manufacturers instructions. Samples were run in an Applied Biosystems automatic
sequencer model 373A Foster City, CA. Nucle- otide and deduced amino acid sequence compari-
sons against data bases were done using the Infobiogen
Network service
software http:
www.infobiogen.frservicesmenuserv.html. Se-
quence alignment
was performed
using the
CLUSTAL-W program. Predicted polypeptide molecular mass and isoelectric points were ob-
tained using the ExPASy Molecular Biology Server [21].
2
.
3
. Quantitati6e RT-PCR RT were performed using total RNA extracted
from berries of homogeneous size collected from 2 to 14 weeks postflowering. PCR reactions were
run with the specific primers EF, GH and IJ as described above to amplify respectively VvAdh1,
VvAdh2 and VvAdh3 cDNAs. As control, a tubu- lin fragment 660 bp was amplified in parallel
with primers K and L Table 1, designed from conserved coding regions of beta-tubulin se-
quences
from plants
Arabidospis thaliana,
Hordeum 6ulgare, Oriza sati6a, Solanum tubero- sum. Test for linearity of amplification was per-
formed for each cDNA with different number of cycles. Aliquots of the PCR reactions were re-
solved by electrophoresis on 1 agarose gel and products were blotted on a nylon membrane. Hy-
bridisation was performed using VvAdh gene-spe- cific probes. These probes were generated between
primers MF, NH and OJ Table 1, respectively, designed from the 3-UTRs of VvAdh1 147 bp,
VvAdh2 169 bp and VvAdh3 169 bp. Primer annealing temperature was 53°C with 20 cycles of
PCR amplification. The fragments generated were cloned and sequenced, and specificity of the
probes was confirmed by hybridisation. Filter hy- bridisation and washing were as described by
Sarni-Manchado et al. [17]. The hybridisation sig- nals were quantified by direct scanning of the
membrane, using a phosphor imager STORM, Molecular Dynamics.
2
.
4
. Heterologous protein expression analysis The grapevine Adh cDNAs were modified by
specific PCR amplification primers to introduce a 5 NdeI site at the translational start codon, and a
3 BamHI site at the stop codon. The sets of forward and reverse primers used for PCR amplifi-
cations were the following: PQ for VvAdh2, and RS for VvAdh3 Table 1. The PCR parameters
were as described above paragraph 2.1. except
that primer annealing temperature was 50°C with 30 cycles. The resulting PCR products were first
cloned into pGemT-Easy vector Promega and sequenced. Fragments were recovered by NdeI
BamHI digestion, and ligated into the NdeI BamHI restricted vector pET-3a Novagen to
give respectively pET-3aVvAdh2 and pET-3a VvAdh3. These constructs were transformed into
the host strain E. coli BL21DE3 for isopropyl-b- thiogalactopyranoside IPTG-induced expression.
E. coli BL21 DE3 cells transformed with either pET-3a as a control, pET-3aAdh2 or pET-3a
Adh3 were grown at 37°C in LB medium contain- ing 50 mgml ampicillin and induced with 0.4 mM
IPTG. After an induction period of 2 h at either 28 or 37°C, the cells were harvested, washed and
suspended in buffer A 50 mM Tris – HCl extrac- tion buffer, pH 7.5, 0.5 mM DTT, 1 mM PMSF
and 20 vv glycerol. After successive treat- ments with Triton X-100 1 plus lysozyme 100
mgml during 30 min at 37°C, and DNAse 1 mgml during 1 h at 37°C, extracts were cleared
by centrifugation 12 000 × g, 15 min at 4°C. ADH enzymes were purified by anion exchange
chromatography
on QAE-cellulose
column Sigma using a linear gradient of 0 – 400 mM
NaCl in buffer A. Protein content was determined with Bradford’s dye method [22], using bovine
serum albumin as standard. The purified enzymes were used for determination of kinetic parameters
of the recombinant ADHs. Experiments were per- formed at least twice for each construct, using
independent clones.
2
.
5
. Enzyme acti6ity, kinetics and electrophoresis methods
Extracts for ADH activity assays were prepared from berries as previously described [16]. Determi-
nation of ADH activity was performed by measur- ing either the reduction rate of acetaldehyde
forward reaction or the oxidation rate of ethanol reverse reaction at 340 nm according to Molina
et al. [23]. For the forward reaction, the assay mixture contained 50 mM sodium phosphate
buffer pH 5.8, 0.24 mM NADH, 5 mM acetalde- hyde. The reverse reaction was carried out in a
mixture containing 50 mM glycine – NaOH buffer pH 9.4, 0.24 mM NAD and 5 mM ethanol.
Reactions containing 5 – 50 ml of extract were started with the addition of the substrate, and
background activity without substrate was sub- tracted. Various concentrations of NADHNAD
from 0.03 to 0.24 mM or acetaldehydeethanol from 0.25 to 10 mM with 0.5 – 1 mg of purified
enzymes were used for K
m
determinations. The steady-state parameters were determined by filling
initial rate values on the Michaelis – Menten equa- tion with the help of the SigmaPlot 2.0 software
Jandel Corp..
The relative mass and purity of overexpressed ADHs was monitored by SDSPAGE on 10
acrylamide gels as described [24]. Proteins were stained with silver nitrate [25]. Proteins were blot-
ted onto nitrocellulose filters in a Mini Trans-Blot system Bio-Rad according to the manufacturer
protocol. ADH was detected by incubation with a specific rice anti-ADH antibody [6] and developed
with alkaline phosphatase-coupled goat anti-rabbit antibodies Sigma.
3. Results
