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
3
.
1
. Cloning and sequence analyses of three grape6ine 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. Se- quence 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 ob- tain 3- and 5- ends of the three cDNAs using
RACE techniques [20]. For each cDNA, at least three clones from each ending region were
analysed. Sequencing of these clones showed that each of PCR products contained the expected
overlaps respectively 154 and 205 bp consistent with the primer position. The composite and con-
tiguous sequences was also confirmed by generat- ing
and sequencing
full-length cDNA
amplification products using primer pairs, EF, GH, or IJ. Thus, three complete and distinct
cDNA sequences, named VvAdh1, VvAdh2, and VvAdh3 were obtained.
Nucleotide sequence
comparisons of
these cDNAs Table 2 showed high identity within the
translated regions ranging from 77.9 to 80.1,
Table 2 Comparison of the VvAdh cDNA sequences
Region VvAdh1 vs.VvAdh3
VvAdh1 vs.VvAdh2 VvAdh2 vs.VvAdh3
41.0 37.3
39.7 5-Untranslated region
77.9 Translated region
77.9 80.1
51.0 49.0
46.1 3-Untranslated region
84.0 85.3
Residue identity 87.6
VvAdh1 and VvAdh2 displaying the highest degree of similarity to one another. In contrast, their
untranslated regions UTR were significantly di- vergent only 37.3 – 41.0 identity for the 5-ends
and 46.1 – 51.0 for the 3-ends. When compared to known grapevine Adh genes, VvAdh1 and
VvAdh2 were respectively identical to the corre- sponding regions of Adh1 [17], and Adh2 Verrie`s,
unpublished results, whereas VvAdh3 was unre- lated to presently known sequences. The single
complete ORFs of VvAdh1 and VvAdh2 1140 bp each encode a 380 amino acid polypeptide,
whereas VvAdh3 1146 bp encodes a polypeptide of 382 residues. Multiple alignment of the deduced
amino acid sequences of VvAdhs is shown in Fig. 1. Many of the nucleotide differences observed in
the coding region of the cDNAs did not alter the polypeptide sequence encoded by the three genes.
However, VvAdh3 has a six-base insertion near the 3-end, which resulted in two additional
residues in the encoded polypeptide. The catalytic domains, together with the coenzyme and sub-
strate binding sites, are conserved within this fam- ily. Thus, changes in residues observed between
the three polypeptides are not predicted to greatly affect regions, important for the function of the
proteins. Comparison of predicted polypeptides Table 2 showed that identity between VvAdh1
and VvAdh2 87.6 was higher than with VvAdh3 84 and 85 identity respectively. Pre-
dicted molecular mass of encoded polypeptides was almost the same around 41 kDa, but compu-
tation of the theoretical isoelectric point pI pre- dicted values ranging from 5.73 to 6.78 Table 3.
Fig. 1. Multiple alignment of the deduced amino acid sequences of cDNA clones encoding grapevine ADH polypeptides. Amino acids conserved between any two sequences are indicated in reverse contrast, those with chemical similarities are indicated in grey
shaded areas, and those quite divergent are presented without shaded areas. A hyphen indicates an amino acid missing.
3
.
2
. Quantitati6e expression analysis The high homology between the VvAdh cDNA
coding regions Table 2 make it impossible to discriminate between the expression of the individ-
ual isogenes when using these regions to design primers or probes, whereas divergence of the
cDNA 5 and 3-ends renders such discrimination possible. We therefore used primers and probes
from these regions for gene-specific expression analysis Section 2. The specificity of these probes
was confirmed by successive hybridisation of the three VvAdh cDNAs to each probe, as no cross-
hybridisation could be detected data not shown. Northern blot hybridisation was previously used
to investigate the general expression pattern of the Adh gene family in different grapevine organs and
in particular in berries [17]. But with specific probes, this technique was not sensitive enough to
detect accumulation of the individual VvAdh iso- genes during fruit development. Thus, quantitative
RT-PCR was used as an alternative to analyse the individual expression of VvAdhs, whatever their
mRNA abundance in the fruit might be. We there- fore designed specific primers corresponding to the
5 and 3-ends of the cDNAs for the differentiation of the RT-PCR products. Amplification of the
VvAdh transcripts was found to be effectively lin- ear for VvAdh1 between 15 and 40 cycles, 5 – 20
cycles VvAdh2, and 10 – 30 cycles for VvAdh3 data not shown. From these results, the number
of PCR cycles chosen for further quantitative RT- PCR experiments was respectively 30, 15 and 25
for VvAdh1, VvAdh2 and VvAdh3. These data indicated that relative abundance of grapevine
Adhs transcripts among total RNA was quite dif- ferent, VvAdh2 being the predominant isogene
expressed in berries.
3
.
3
. Adh gene expression in de6eloping grape berries
To examine VvAdh isogene expression, total
Fig. 2. Adh isogene expressions during grape berry develop- ment from cv. Danuta performed by quantitative RT-PCR.
A Refractive index and pH. B ADH activity per berry and per g FW. C Blots obtained after linear amplification
of full-length VvAdh1, VvAdh2 and VvAdh3 cDNAs hy- bridised with specific probes, and partial tubulin cDNA as
control. D Comparison of relative hybridisation signal of VvAdh2 arbitrary units and enzyme activity, expressed
per berry.
Table 3 Properties of the predicted polypeptides deduced
VvAdh2 VvAdh1
Property VvAdh3
380 380
Residues 382
41.24 41.19
41.05 Molecular mass kDa
6.78 5.73
pI 6.14
RNA was isolated from grape berries at five differ- ent stages along the development of the fruit 2 –
14 weeks postflowering and quantitative RT-PCR was performed. Developing grape berries show a
characteristic rapid increase in sugar content eval-
uated by refractive index and in pH Fig. 2A at 8 weeks post flowering, which continues to rise
throughout ripening. The ADH activity level, ex- pressed per weight or per berry Fig. 2B, was low
during the first developmental stages, but sharply increased after ve´raison, as earlier reported [15,16].
Results of VvAdh expression obtained by quan- titative PCR and amplification of a beta-tubulin
fragment, used as a control of the reaction effi- ciency reverse transcriptase and polymerase and
gel loading are presented in Fig. 2C. Each VvAdh isogene showed a different and characteristic pat-
tern of expression. VvAdh1 expression was de- tected in the first phase of fruit development
reaching a maximum at 5 weeks post-flowering, and being faintly detectable after 8 weeks. Expres-
sion of VvAdh3, which was higher than VvAdh1, was also detected from immature stage up to the
onset of ripening 8 weeks, declining thereafter. A quite different pattern was obtained for VvAdh2,
showing a very low expression at 2 and 5 weeks after flowering and a transcript accumulation that
started at ve´raison. As VvAdh2 is the most tran- scribed isogene in ripening berry, we compared its
expression pattern with ADH activity Fig. 2D. Hybridisation signal was calculated by berry and
relative data were compared to enzyme activity. The pattern of VvAdh2 is in accordance with the
observed trend for ADH activity per berry and is consistent with expression being controlled at the
transcriptional level. The increases in ADH en- zyme activity and in VvAdh2 expression, both
occurred simultaneously with changes in sugar and acidity contents Fig. 2B. The fact that 14 weeks
after flowering, the relative expression by berry decreased, while relative enzyme activity continued
to increase suggests that this ADH is a stable protein that exhibits relatively low turnover.
3
.
4
. Purification and kinetic parameters of two acti6e recombinant ADHs
To get information on some properties of the VvAdh encoded enzymes, analysis was focused on
the two most transcribed isogenes in berry, i.e. VvAdh2 and VvAdh3. Recombinant pET-3a
VvAdh2 and pET-3aVvAdh3 were therefore ex- pressed as soluble, refolding active proteins in E.
coli BL21DE3. Preliminary induction trials were performed at 28 and 37°C to yield proteins that
were expressed mainly in the soluble fraction. The ADH expression level was measured in extracts of
transformed strains with or without control re- combinant plasmid before and after 2 h induction
with 0.4 mM IPTG. For pET-3aVvAdh3, induc- tion at 37°C resulted in a 196-fold increase of
ADH activity, whereas highest activities were ob- tained at 28°C for pET-3aVvAdh2 with a 20-fold
increase data not shown. These active VvADHs were then purified on QAE-cellulose and eluted
with a NaCl gradient Section 2. Elution profiles of VvADH activity were different for the two
recombinant proteins, VvADH2 being eluted at 0.1 M NaCl whereas VvADH3 was less retained
on the column data not shown. After column purification step, recovery of total enzyme activity
was 17 for VvADH2 and 34 for VvADH3, corresponding respectively to a 10- and 5-fold
purification Table 4. The overproduction of VvADHs in strains containing the recombinant
plasmids pET-3aVvAdh2 and pET-3aVvAdh3 was analysed by SDS-PAGE Fig. 3A and B and
compared to the non-recombinant control pET-3a lanes 1. For both pET-3aVvAdh constructs the
presence of an additional band in soluble extracts was revealed by silver staining lanes 2. Larger
amount of this band was obtained after fractiona- tion on QAE column lanes 3. The nature of the
expression product as ADH was confirmed by western blotting lanes 4 using anti-ADH anti-
Fig. 3. Electrophoresis profiles of E. coli cells with or without recombinant plasmids as observed on SDS-PAGE 10:
purification and immunoblotting of V. 6inifera L. ADHs. Protein detection was performed by silver staining. A: pET-
3aAdh2, B: pET-3aAdh3. Lane 1: total cell proteins from E. coli pET-3a control, lane 2: soluble extract of pET-3aAdhs,
lane 3: purified ADHs, lane 4: western blotting after transfer of lane 3 to nitrocellulose membrane detected with specific
rice anti-ADH antibody [6]. The numbers on the left indicate molecular weight in kDa of the marker proteins.
C .
Tesnie `r
e, C
. Verrie
`s Plant
Science
157 2000
77 –
88
Table 4 Purification of VvADHs overexpressed in E. coli from pET-3aVvAdh2 and pET-3aVvAdh3 constructs, and main kinetic parameters of the purified ADHs
a
Step Specific activity
Total activity Recovery
Purification Total protein
K
m
mM fold
mmolmin per mg mg
mmolmin Acetaldehyde
NADH Ethanol NAD
100 –
VvADH2 2.508
98 39
crude extract
10 0.45
0.02 10.3
17 0.03
386 0.044
17 QAE ion
exchange 0.423
943 100
– VvADH3
399 crude
extract 4323
34 5
9.00 0.03
2.00 0.04
QAE ion 0.031
134 exchange
a
The parameters were calculated by fitting the Michaelis–Menten equation on initial rate of experimental data Sigmaplot 2.0, Jandel Corp.. Reactions were carried out in 50 mM sodium phosphate buffer pH 5.8 forward reaction or in 50 mM glycine–NaOH buffer pH 9.4 reverse reaction. Each value is the mean of three independent
measurements on two different clones.
body from rice [6]. The VvADHs overproduced from recombinants pET-3aVvAdh2 and pET-3a
VvAdh3 presented the same size of 48 kDa. Dis- crepancy observed between measured molecular
mass and that predicted from VvAdh cDNAs 41 kDa is likely an artefact, as addition of glycerol in
extracts used to maintain enzyme activity, could have slow down migration of the polypeptides.
Determination of kinetic parameters of these two VvADHs was performed for acetaldehyde and
ethanol, and K
m
values obtained from hyperbolic saturation curves are listed in Table 4. Overex-
pressed enzyme from pET-3aVvAdh2 displayed the lowest K
m
for acetaldehyde 0.45 mM with the highest V
m
300 mmolmin per mg protein, com- pared to the parameters obtained for the reverse
reaction K
m
= 10.3 mM and V
m
= 22 mmolmin
per mg protein for ethanol. VvADH3 had a very high K
m
for acetaldehyde 9 mM, but also a very high V
m
5900 mmolmin per mg protein, whereas parameters for ethanol were lower K
m
= 2.00 mM
and V
m
= 230 mmolmin per mg protein. Both
enzymes showed low K
m
for NADHNAD coen- zymes between 0.02 and 0.04 mM. These results
suggested that VvADH2 and VvADH3 enzymes function in forward and reverse directions, and
that reduction of acetaldehyde to ethanol is the preferential reaction for VvADH2. ADH activity
of both proteins was also measured with other NADPNADPH coenzymes. NADPH-linked ac-
tivity for VvADH2 and VvADH3 represented re- spectively 4 and 23 of the NADH-linked activity
at the same pH value data not shown, and no NADP-linked activity could be detected for either
of the isogene products.
4. Discussion
