Molecular cloning and expression of cDNAs encoding alcohol
dehydrogenases from
Vitis
6
inifera
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 (Vitis6iniferaL.) 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 characterisedAdhgenes 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 threeAdhisogenes 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 theAdhmRNAs 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 overexpressingE. 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 multipleAdhexpressed in berries is discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Alcohol dehydrogenase; Fruit development; Grape berry;Vitis6iniferaL.
www.elsevier.com/locate/plantsci
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 multi-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 induceAdhgene expression in a variety of plants [1,8,9]. The versatility of tran-scription fromAdh 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 ripeVitis6iniferaL. 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:[email protected] (C. Tesnie`re).
C.Tesnie`re,C.Verrie`s/Plant Science157 (2000) 77 – 88
78
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 divergentAdh -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
6inifera 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 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 min at 4°C and supernatant was filtered through Miracloth. CsCl was then added to the filtrate (0.2 g/ml) 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 5mg of total RNA using a poly(T)15as primer and
Super-script II reverse tranSuper-scriptase (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-209a
B Plant Adhs E8R 904-CRTCNTGRACACAYTCRAA-876a
VvAdhs
C E4R 395-ATCCTGAGGAGGTCACACA-377a
D VvAdhs E7F 751-AAGAARTTTGGYGTSACYG-766a
1UTR5F 6-AGGAGCACCATATCTTTGGAG-26
Plant Tub TUBF 614-TTGTTGAGCCATACAATGC-632b
K
L Plant Tub TUBR 1273-AGTACCAATGCAAGAAAGC-1255b
1UTR3F 1193-GATGAGAGAGTCTAATTGAAT-1213
VvAdh1 M
1212-GATTTGCCTATTCCAGTCG-1230
N VvAdh2 2UTR3F
VvAdh3
O 3UTR3F 1193-ATGAGTGAAGTGTATGTTAGATG-1215
P VvAdh2 2NF 58-TTATACATATGTCAAGCACAGCTG-73c
VvAdh2 2BR
Q 1200-ATAGGATCCTTATGCATCCATGCG-1186c
VvAdh3 3NF
R 34-TTATACATATGTCTAATACAGCTGGTC-52c
VvAdh3 3BR 1182-TAGGATCCCTAGGCTCCCATGC-1169c
S
aReferred to positions of VvAdh1.
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 poly(C) region. The 3%-end fragments were obtained using an hybrid
NN-oligo(dT15)-adapter primer [20]. The
subse-quent PCR amplifications were carried out as described above, using either the anchor primer (lacking poly(C) region) and the antisense consen-sus primer C for the 5%- ends, or the consensus
sense primer D and the adapter primer (lacking poly(T) 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) orpGemT-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.fr/services/menuserv.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
C.Tesnie`re,C.Verrie`s/Plant Science157 (2000) 77 – 88
80
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-3a/VvAdh2 and pET-3a/
VvAdh3. These constructs were transformed into the host strain E. coli BL21(DE3) for isopropyl-b -thiogalactopyranoside (IPTG)-induced expression.
E. coliBL21 (DE3) cells transformed with either
pET-3a (as a control), pET-3a/Adh2 or pET-3a/
Adh3 were grown at 37°C in LB medium
contain-ing 50 mg/ml 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% (v/v) glycerol). After successive treat-ments with Triton X-100 (1%) plus lysozyme (100
mg/ml) during 30 min at 37°C, and DNAse (1
mg/ml) 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 NADH/NAD
(from 0.03 to 0.24 mM) or acetaldehyde/ethanol (from 0.25 to 10 mM) with 0.5 – 1 mg of purified
enzymes were used for Km 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 SDS/PAGE 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 berryAdh 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
Table 2
Comparison of the VvAdhcDNA sequences
Region VvAdh1 vs.VvAdh2 VvAdh1 vs.VvAdh3 VvAdh2 vs.VvAdh3
41.0
37.3 39.7
5%-Untranslated region
77.9
Translated region 80.1 77.9
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).
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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
(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-3a/VvAdh3 were therefore ex-pressed as soluble, refolding active proteins in E.
coli BL21(DE3). 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-3a/VvAdh3, 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-3a/VvAdh2 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-3a/VvAdh2 and pET-3a/VvAdh3 was analysed by SDS-PAGE (Fig. 3A and B) and
compared to the non-recombinant controlpET-3a
(lanes 1). For both pET-3a/VvAdh 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 ADH
anti-Fig. 3. Electrophoresis profiles ofE.colicells with or without recombinant plasmids as observed on SDS-PAGE (10%): purification and immunoblotting of V. 6inifera L. ADHs.
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Table 4
Purification of VvADHs overexpressed inE.colifrom pET-3a/VvAdh2 andpET-3a/VvAdh3 constructs, and main kinetic parameters of the purified ADHsa
Step Total protein Total activity Specific activity Recovery (%) Purification Km (mM)
(fold) (mmol/min per mg)
(mg) (mmol/min)
Acetaldehyde NADH Ethanol NAD
100 –
VvADH2 2.508 98 39
crude extract
10 0.45 0.02 10.3
17 386 0.03
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
aThe parameters were calculated by fitting the Michaelis–Menten equation on initial rate of experimental data (Sigmaplot 2.0, Jandel Corp.). Reactions were carried out
body from rice [6]. The VvADHs overproduced from recombinants pET-3a/VvAdh2 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 Km values obtained from hyperbolic
saturation curves are listed in Table 4. Overex-pressed enzyme from pET-3a/VvAdh2 displayed the lowestKmfor acetaldehyde (0.45 mM) with the
highest Vm (300 mmol/min per mg protein),
com-pared to the parameters obtained for the reverse reaction (Km=10.3 mM and Vm=22 mmol/min
per mg protein for ethanol). VvADH3 had a very highKm for acetaldehyde (9 mM), but also a very
highVm (5900 mmol/min per mg protein), whereas
parameters for ethanol were lower (Km=2.00 mM
and Vm=230 mmol/min per mg protein). Both
enzymes showed low Km for NADH/NAD
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
NADP/NADPH 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
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 molecu-lar level. Full-length grapevine cDNAs that
en-code 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 toAdh from some other species [17]
than to VvAdh3. On the other hand, amino acid
identity between VvAdh3 and Adhs from other
species was lower than between VvAdh2 and
VvAdh3 (data not shown). However, each of these sequences are more closely related to other dicots than to micro-organisms, excluding the possibility
that one or more of theAdh cDNAs resulted from
yeast or fungal contamination. These results added
to our knowledge on the multigenicity of Adh in
V. 6inifera [17,18] with the description of the cod-ing region of two additional genes (VvAdh2 and VvAdh3), leading to four the number ofAdhgenes found in cv. Danuta genome, one of them [18] being likely not functional. This diversity is
consis-tent with the number of Adh genes found to be
expressed in plant species such as tomato
[3,11,27,28]. In grapevine, no data is up to now available on the dispersion of the Adhgenes in the
V. 6inifera genome, and in particular whether
some of these genes could be clustered as a tandem, as it has been reported for tomato [11]. Individual isogene expression was investigated during fruit development using RT-PCR and gene-specific probes to ensure gene-specificity and sensitivity of the analyses. In particular, quantitative PCR was chosen due to large anticipated differences in
abundance of Adh isogene mRNAs, as it has been
reported in other small-multigene families [29]. This great difference in mRNA abundance was confirmed by the number of PCR cycles, which were needed to obtain readable blots for each isogene. These results indicated that VvAdh1 and
VvAdh3 represent a small proportion of total
RNAs from berry. On the contrary, VvAdh2 is the
most abundant grapevine Adh among isogenes
expressed in the fruit, and represented likely the major form detected by northern blot of RNA extracted from berry when using a non-isogene-specific probe. Use of non-isogene-specific probes allowed us to precise the individual pattern of VvAdh isogenes. VvAdh1 and VvAdh3 are expressed up to ve´raison and downregulated thereafter, whereas VvAdh2 is upregulated during ripening. In a previous study [17], northern blot from developing berries hy-bridised with a non-specific Adh probe, reflects
hybridisation with all the Adh mRNAs. However,
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86
is ripening-related. Expression patterns for sugar transporters recently reported in a berry develop-mental series [30] presented similarities to those of VvAdhs. Pattern of VVSUC27 transcripts is close
to that of VvAdh1, and VvSUC11 and VvSUC12
mRNAs, which could be involved in ripening-re-lated sucrose import, have patterns similar to VvAdh2. Although the physiological significance is not known, it could indicate that common factors might control transcription of sucrose transporters and Adh genes in berries.
Data presented here indicated that VvAdh2
likely contributed to changes in ADH activity during grape berry ripening. In higher plants, ac-tive ADHs are homo- and heterodimeric enzymes of two subunits of approximately the same size. In grape, dimeric structure of the active proteins ex-pressed during fruit development should be inves-tigated. However, from this study, it is likely that
VvAdh2 and VvAdh3 cDNAs encode
homod-imeric enzymes with different properties, suggest-ing different roles in berry development. VvAdh2 encoded for a protein that expected pI (5.73) is close to data obtained for isoelectric focusing of ADH purified from ripe grape berries [31]. In addition, main kinetic parameters of the pET-3a/
VvAdh2 overexpressed protein are in agreement
with the characteristics of the purified enzyme from ripe grape berries [23]. On the contrary,
polypeptides encoded by other VvAdh cDNAs
ex-hibit quite different predicted charge. For
in-stance, the computation of the theoretical
isoelectric point (pI) predicted 6.78 value for
VvAdh3 polypeptide. The higher retention on
QAE cellulose column observed for the recombi-nant VvADH2 protein compared to VvADH3 is coherent with the difference of one unit expected between the two protein charges. Kinetic parame-ters of the overexpressed protein from pET-3a/
VvAdh3 were different from those obtained from
pET-3a/VvAdh2 or in vivo proteins [31], in partic-ular at the acetaldehyde affinity level. Km for this
substrate was 20-fold higher for VvADH3 than for
VvADH2. However, maximum velocity of
VvADH3 was also increased, resulting in a similar catalytic efficiencies (Vm/Km) for acetaldehyde of
both enzymes. Compared to NADH-linked activ-ity, VvADH3 displayed also a higher NADPH-linked activity than VvADH2. Thus, despite overall structural similarity, different kinetic parameters of enzymes were observed.
As in grapevine, encoded Adh2 and Adh3
polypeptides from tomato exhibit a difference in predicted charge (0.85 unit between pI) [3,11]. Also similar to grape berry, the tomato isogene encoding the more acidic form (pI5.93 for Adh2) is the one expressed in the fruit, but at extremely ripe stage [3]. This isogene is also induced under anaerobiosis in tomato fruit, as well as in other organs [32,33]. In grape, previous study presented evidence that Adh transcripts did not change sig-nificantly in ripe berries submitted to hypoxia, suggesting that the high Adh expression level in the fruit have somehow precluded the response to anaerobisosis [14]. On the contrary, cultured grape cells under the same conditions transiently induced
Adh gene expression. Further experiments should
be performed to investigate if VvAdh2 is the up-regulated isogene in response to hypoxia, and also to know whether any of the three reported cDNA are expressed elsewhere during plant development. Events occurring in berry at the onset of ripen-ing result in the requirement for decreased and increased expression of Adh isogenes to cope with ripening. The high Km value for acetaldehyde of
In this paper, we demonstrated that three grapevine Adh isogenes are expressed during dif-ferent periods in grape berry development and ripening. The presence of three isoforms that are differentially expressed suggests that they may play distinct metabolic roles. The results obtained on biochemical properties of VvADHs expressed in an heterologous system support the idea that various isogene products meet demands of differ-ent physiological conditions. It could reflect a tight adaptation of the fruit to the developmental events. Taken as a whole, our results suggested that evolution of VvAdh expression during fruit
development could be the consequence of
a complex interplay among transcriptional
activa-tion of VvAdh genes, changes in mRNAs
half-life, and differential synthesis of VvAdh subunits with consequent modulation of enzyme character-istics.
During the final editing processing of this
manuscript, Or et al. [37] registered two Adh
cDNA clones from developing grape berry (cv Perlette). At the nucleotide level, one of them (Acc. No AF195 867) is highly homologous to
V6Adh2 cDNA between part of exon 4 up to
3%-end, whereas 5%-end is quite different. The other one (G6ADH, Acc. No. AF195 866) is also ho-mologous to the totality ofV6Adh2 cDNA from 5%
to 3% ends. Furthermore at the amino acid level,
residues of G6ADH6 are 99% identical to those of VvAdh2. These results confirm the predominance
of V6Adh2 among the small Adh gene family
expressed in berries, as the nucleotide sequence
differences between V6Adh2 and G6ADH may
originate from the cloning of the same gene from different V. 6inifera cultivars.’’
Acknowledgements
The nucleotide sequence data reported will ap-pear in the EMBL, GenBank and DDBJ Nucle-otide 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. Kad-owaki 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.
References
[1] D.P. Matton, P. Constabel, N. Brisson, Alcohol dehy-drogenase gene expression in potato following elicitor and stress treatment, Plant Mol. Biol. 14 (1990) 775 – 783.
[2] R.G. Gregerson, L. Cameron, M. McLean, P. Dennis, J. Strommer, Structure, expression, chromosomal location and product of the gene encoding Adh2 in Petunia, Genetics 133 (1993) 999 – 1007.
[3] T. Longhurst, E. Lee, R. Hinde, C. Brady, J. Speirs, Structure of the tomatoAdh2 gene andAdh2 pseudoge-nes, and a study ofAdh2 gene expression in fruit, Plant Mol. Biol. 26 (1994) 1073 – 1084.
[4] B.R. Morton, B.S. Gaut, M.T. Clegg, Evolution of alco-hol dehydrogenase genes in the palm and grass families, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 11735 – 11739.
[5] C. Chang, E.M. Meyerowitz, Molecular cloning and DNA sequence of theArabidopsis thalianaalcohol dehy-drogenase gene, Proc. Natl. Acad. Sci. U.S.A. 83 (1986) 1408 – 1412.
[6] H.I. Kadowaki, M. Matsuoka, N. Murai, K. Harada, Induction of two alcohol dehydrogenase polypeptides in rice roots during anaerobiosis, Plant Sci. 54 (1988) 29 – 36.
[7] A.A. Millar, M.R. Olive, E.S. Dennis, The expression and anaerobic induction of alcohol dehydrogenase in cotton, Biochem. Genet. 32 (1994) 279 – 300.
[8] P.J. Christie, M. Hahn, V. Walbot, Low-temperature accumulation of alcohol dehydrogenase-1 mRNA and protein activity in maize and rice seedlings, Plant Phys-iol. 95 (1991) 699 – 706.
[9] A.A. Millar, E.S. Dennis, The alcohol dehydrogenase genes of cotton, Plant Mol. Biol. 31 (1996) 897 – 904. [10] R. Dolferus, M. Jacobs, W.J. Peacock, E.S. Dennis,
Differential interactions of promoter elements in stress responses of theArabidopsis Adhgene, Plant Physiol. 105 (1994) 1075 – 1087.
[11] J.C. Ingersoll, M. Rothenberg, B.E. Liedl, K. Folkerts, D. Garvin, M.R. Hanson, J.J. Doyle, M.A. Mutschler, A novel anther-expressedAdh-homologous gene in Lycop-ersicon esculentum, Plant Mol. Biol. 26 (1994) 1875 – 1891.
[12] G.J. van Eldik, R.K. Ruiter, M.M.A. van Herpen, J.A.M. Schrauwen, G.J. Wullems, Induced ADH gene expression and enzyme activity in pollinated pistils of Solanum tuberosum, Sex Plant Reprod. 10 (1997) 107 – 109.
[13] M. Tadege, C. Kuhlemeier, Aerobic fermentation during tobacco pollen development, Plant Mol. Biol. 35 (1997) 343 – 354.
[14] C. Tesnie`re, C. Romieu, M.E. Vayda, Changes in gene expression of grapes in response to hypoxia, Am. J. Enol. Vitic. 44 (1993) 445 – 451.
C.Tesnie`re,C.Verrie`s/Plant Science157 (2000) 77 – 88
88
[16] N. Terrier, Aspects bioe´nerge´tiques et mole´culaires du stockage des acides organiques dans la baie de raisin (Vitis6iniferaL.), Thesis ENSA, Montpellier, 1997.
[17] P. Sarni-Manchado, C. Verrie`s, C. Tesnie`re, Molecular characterization and structural analysis of one alcohol dehydrogenase gene (GV-Adh1) expressed during ripen-ing of grapevine (Vitis6inifera L.) berry, Plant Sci. 125
(1997) 177 – 187.
[18] C. Verrie`s, C. Be`s, P. This, C. Tesnie`re, Cloning and characterization of Vine-1, a LTR-retrotransposon like element in Vitis 6inifera L. and other Vitis species, Genome 43 (2000) 366 – 379.
[19] C. Tesnie`re, M.E.V. Vayda, Method for the isolation of high quality RNA from grape berry tissues without contaminating tannins or carbohydrates, Plant Mol. Biol. Rep. 9 (1991) 242 – 251.
[20] M.A. Frohman, M.K. Dush, G.R. Martin, Rapid pro-duction of full-length cDNAs from rare transcripts: am-plification using a single gene-specific oligonucleotide primer, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 8998 – 9002.
[21] B. Bjellqvist, G.J. Hughes, Ch. Pasquali, N. Paquet, F. Ravier, J.C. Sanchez, S. Frutiger, D.F. Hochstrasser, The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences, Electrophoresis 14 (1993) 1023 – 1031. [22] M.M. Bradford, A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding, Anal. Biochem. 72 (1976) 248 – 254.
[23] I. Molina, C. Salles, M. Nicolas, J. Crouzet, Grape alcohol dehydrogenase. II Kinetics studies: mechanism, substrate and coenzyme specificity, Am. J. Enol. Vitic. 38 (1987) 60 – 64.
[24] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head bacteriophage T4, Nature 227 (1970) 680 – 685.
[25] H. Blum, H. Beier, H.J. Gross, Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels, Electrophoresis 8 (1987) 93 – 99.
[26] W.L. Gerlach, A.J. Pryor, E.S. Dennis, R.J. Ferl, M.M. Sachs, W.J. Peacock, cDNA cloning and induction of the alcohol dehydrogenase gene (adh1) of maize, Proc. Natl. Acad. Sci. U.S.A. 79 (1982) 2981 – 2985.
[27] S.D. Tanksley, Linkage, chromosomal association, and expression of Adh-1 and Pgm-2 in tomato, Biochem. Genet. 17 (1979) 1159 – 1167.
[28] S.D. Tanksley, R.A. Jones, Effects of O2 stress on
tomato alcohol dehydrogenase activity: description of a second ADH coding genes, Biochem. Genet. 19 (1981) 397 – 409.
[29] C.S. Barry, B. Blume, M. Bouzayen, W. Cooper, A.J. Hamilton, D. Grierson, Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato, Plant J. 9 (1996) 525 – 535.
[30] C. Davies, T. Wolf, S.P. Robinson, Three putative su-crose transporters are differentially expressed in grapevine tissues, Plant Sci. 147 (1999) 93 – 100. [31] I. Molina, M. Nicolas, J. Crouzet, Grape alcohol
dehy-drogenase. I Isolation and characterization, Am. J. Enol. Vitic. 37 (1986) 169 – 173.
[32] D. van der Straeten, R.A. Rodrigues Pousada, J. Gielen, M. van Montagu, Tomato alcohol dehydrogenase. Ex-pression during fruit ripening and under hypoxic condi-tions, FEBS Lett. 295 (1991) 39 – 42.
[33] A.R.C. Chen, T.J. Chase, Alcohol dehydrogenase 2 and pyruvate decarboxylase induction in ripening and hy-poxic tomato fruit, Plant Physiol. Biochem. 31 (1993) 875 – 885.
[34] P. Perata, A. Alpi, Ethanol-induced injuries to carrot cells. The role of acetaldehyde, Plant Physiol. 95 (1991) 748 – 752.
[35] J.K. Roberts, J. Callis, D. Wemmer, V. Walbot, O. Jardetzky, Mechanisms of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia, Proc. Natl. Acad. Sci. U.S.A. 81 (1984) 3379 – 3383.
[36] M. Tadege, I. Dupuis, C. Kuhlemeier, Ethanolic fermen-tation: new functions for an old pathway, Trends Plant Sci. 4 (1999) 320 – 325.
[37] E. Or, J. Baybik, S. Lavee, A. Sadka, A. Ogredovitch, Isolation and characterization of two cDNA clones (Accession No. AF195 866 and AF195 867) encoding alcohol dehydrogenase in grape (Vitis 6inifera cv.
Perlette) developing fruits, Plant Physiol. 122 (2000) 619.
