JXB Advance Access originally published online on November 16, 2005
Journal of Experimental Botany 2006 57(1):91-99; doi:10.1093/jxb/erj007
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RESEARCH PAPER |
Effects of genetic manipulation of alcohol dehydrogenase levels on the response to stress and the synthesis of secondary metabolites in grapevine leaves
1UMR 1083, Sciences Pour l'Oenologie, Centre INRA/Agro-M, 2 Place Viala, F-34060 Montpellier Cedex 01, France
2UMR 1098, Biologie des Espèces Pérennes Cultivées, Centre INRA/Agro-M, 2 Place Viala, F-34060 Montpellier Cedex 01, France
3UMR1212, Ingénierie des Réactions Biologiques—Bioproductions, Université Montpellier II, Place Eugène Bataillon, F-34095 Montpellier Cedex 05, France
* To whom correspondence should be addressed. Fax: +33 4 99 61 28 57. E-mail: tesniere{at}ensam.inra.fr
Received 20 July 2005; Accepted 28 September 2005
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Abstract |
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The functional role of Adh in regulating susceptibility to abiotic stress and the synthesis of secondary metabolites was investigated in transgenic grapevine plants over- and underexpressing alcohol dehydrogenase (Adh). Plants were transformed with gene constructs containing a sense or antisense orientated grapevine VvAdh2 cDNA under the constitutive cauliflower mosaic virus 35S promoter. Plants transformed with either antisense orientation or the Adh-less construct displayed a low but detectable constitutive ADH activity, whereas plants transformed with the sense-expressed transgene showed a significantly higher (100-fold) ADH activity than the control. Compared with the control, the sense transgene induced an overexpression of VvAdh2 transcripts, whereas a reduced VvAdh2 expression was detected in antisense transformants. Grapevine plants overexpressing Adh displayed a lower sucrose content, a higher degree of polymerization of proanthocyanidins, and a generally increased content of volatile compounds, mainly in carotenoid- and shikimate-derived volatiles. In general, no significant differences between sense/antisense transformants were observed with regard to carotenoid and chlorophyll contents, suggesting a strong metabolic regulation of the synthesis of these compounds.
Key words: Abiotic stress, alcohol dehydrogenase, carotenoids, grapevine, phenolic compounds, sugar content, transformants, volatile compounds
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The expression of the alcohol dehydlrogenase (Adh) gene is known to be regulated developmentally and to be induced by environmental stresses (Matton et al., 1990
; Christie et al., 1991; Ingersoll et al., 1994; Bucher et al., 1995). Several approaches have been undertaken to assess the functional role of Adh in development, stress response, and metabolite synthesis. Among these, studies of Arabidopsis mutants with defective Adh expression showed depressed seed germination in Adh-inductive conditions, i.e. anoxia or hypoxia, as well as defective responses to cold and osmotic stresses (Conley et al., 1999). Complementation of a tobacco Adh-deficient mutant did not modify the original phenotype, but regulation of ADH expression was recovered at the transcriptional level in a tissue-specific and developmental fashion (Rousselin et al., 1994). In Arabidopsis thaliana, Adh overexpression improved the tolerance of hairy roots to low oxygen conditions and was effective in improving root growth (Shiao et al., 2002). However, it had no effect on flooding survival (Ismond et al., 2003). With respect to secondary metabolites, ADH is involved in the interconversion of volatile compounds such as aldehydes and alcohols (Bicsak et al., 1982; Molina et al., 1986; Longhurst et al., 1990), and Adh overexpression in tomato has been shown to modify the balance between C6 aldehydes and alcohols in ripe fruits (Speirs et al., 1998).
In grapevine, Adh genes belong to a small multigene family which has been well characterized (Or et al., 2000; Tesniere and Verries, 2001, Verries et al., 2004; Tesniere et al., 2005). Among these genes, VvAdh2 has been described as a berry ripening-related isogene (Tesniere and Verries, 2000). In addition, recent data suggested that regulation of the expression of this isogene could partly be related to the ethylene signalling pathway (Tesniere et al., 2004). In the present work, sense and antisense strategies were used to investigate the role of VvAdh2 in V. vinifera further. After the initial characterization of transformed plants, the effect of Adh up- and down-regulation on the response to abiotic stress and on the content of sugars and some secondary metabolites was investigated in leaves.
To study the putative role of alcohol dehydrogenase in plant development and response to stress, a number of transgenic grapevine transformants were produced with modified levels of ADH activity. The expression of the VvAdh isogenes and the content of sugars, phenolic and volatile compounds have been investigated. Analysis of transformants with normal, sense or antisense constructs indicated changes in the content of some phenolic compounds and volatile secondary metabolites of V. vinifera, belonging to the classes of monoterpenes, C13-norisoprenoids and shikimates.
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Materials and methods |
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Construction of Adh transgenes
The grapevine VvAdh2 cDNA (Tesniere and Verries, 2000
), previously cloned into pGemT-Easy (Promega, France) was used for the construction of the Adh transgenes. The VvAdh2 cDNA was EcoRI digested and cloned into pRT101 (Toepfer et al., 1987), between the CaMV 35S promoter and the Nos terminator (Fig. 1). The 1.39 kb cDNA included a 5' untranslated region 26 bp upstream of the ATG start codon and a 183 bp 3' untranslated region of the TAA stop codon. Sense and antisense orientations were controlled using BglII/BamHI digestions. The cassettes were then PstI excised and inserted into the pCambia 1305.1 binary vector (http://www.cambia.org). The native binary vector was used directly to obtain control transformed plants.
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Grapevine transformations
The constructs were transferred from E. coli DH5
to Agrobacterium tumefaciens EHA 105 (Hood et al., 1993) by electroporation (2 kV, 90 
, 25 µF) with chimeric constructs or native binary vector. Plant transformations were performed by a somatic embryogenesis-based method (Torregrosa et al., 2002a) for grapevine (Vitis vinifera) cv. Portan, with hygromycin selection. The presence of the cassettes in the in vitro primary transformants was PCR checked and GUS staining using the marker gene allowed further identification of the transformants. Transformants were vegetatively propagated by in vitro microcuttings through standard in vitro culture methods to maintain four vitroplants per line. Vitroplants were acclimated to greenhouse conditions.
Genomic Southern blot analyses
Genomic DNA was extracted from young leaves with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Three micrograms each of the genomic DNAs were digested with EcoRI and fractionated by electrophoresis on 0.7% agarose-TBE (90 mM Tris/borate and 2 mM EDTA, pH 8.0) gels. The DNA was transferred onto Hybond N (Amersham, France) nylon membranes as described by the manufacturer. The filter was hybridized with a 32P-labelled probe corresponding to the XhoI-NheI 3.87 kb fragment of the native pCambia 1305.1 plasmid. Hybridization was performed at 65 °C for 20 h according to the manufacturer's recommendations. The filters were washed once with 2x SSC, 0.1% SDS at room temperature for 10 min, followed by two washes with 0.2x SSC, 0.1% SDS at 65 °C. The membranes were blotted by capillarity, dried, and analysed by phosphorus imaging. The number of copy inserts was estimated from the number of hybridization bands divided by two, as digestion with EcoRI excised the introduced VvAdh2 cDNA from the inserted gene constructs leaving left and right portions of the inserted T-DNA on separate EcoRI fragments.
Northern blot analyses
Total RNA was extracted from 0.5–1 g of ground, frozen young leaves according to Tesniere and Vayda (1991) with slight modifications as described by Sarni-Manchado et al. (1997). 10 µg RNA were fractionated through a 1.2% agarose-formaldehyde gel, and transferred to a nylon membrane (Hybond N, Amersham). Prehybridization and hybridization were performed as described in Tesniere and Verries (2000), using isogene-specific VvAdh probes and a 18S ribosomal probe for normalization. The hybridization signals were quantified by direct scanning, using a phosphor imager (STORM, Molecular Dynamics). Three replicates were performed.
Abiotic stress treatment
Abiotic stress corresponding to hypoxia was assessed by flushing Petri dishes with nitrogen. Hypoxic treatments were applied on intact detached grapevine leaves incubated in 55 mm diameter Petri dishes on semi-solid MS/2 medium. Two replications were performed with four boxes of five leaves each per transformed line. After 3 d of treatment, leaves were sampled for an ADH activity evaluation.
Protein and ADH enzyme activity measurements
Leaf tissues were extracted and aliquots of the supernatants stored at –80 °C until assayed, according to Torregrosa et al. (2002b). Protein concentration was measured using the Bradford method (Bradford, 1976). ADH enzyme activity was assayed by measuring the reduction rate of acetaldehyde at 340 nm as previously described (Molina et al., 1987; Tesniere and Verries, 2000).
Metabolite analysis
For metabolite analysis, young leaves from grapevine transformants were collected on greenhouse-grown transformants. For each control, sense and antisense conditions, samples from the different plants of the same line were pooled, washed, and quickly dried with tissue paper. Leaves were then frozen in liquid nitrogen, ground to powder and stored at –80 °C until use.
For sugar extraction, 10 ml boiling water were added to 2 g of frozen powder. The mixture was boiled for 5 min. After centrifugation at 10 000 g for 5 min, an aliquot of supernatant was used for sugar content determination. This was performed by enzymatic analysis under continuous flux, using the glucose/fructose kit from Enzytec (Humeau Laboratory, France). Sucrose content was evaluated as the difference of sugars before and after hydrolysis.
Phenolic compounds were extracted from 200 mg of ground frozen leaves with 2 ml of acetone:water:trifluoroacetic acid (600:400:0.5, by vol.) containing p-hydoxybenzoic acid methyl ester (200 mg l–1) as the internal standard. After agitation for 2 h, extracts were centrifuged at 13 000 g for 10 min at 4 °C. Two aliquots of 200 µl were evaporated. One was diluted with an equal volume of water:methanol:HCl (800:190:10, by vol.) prior to HPLC analysis of phenolic acids and flavonols. Depolymerization was carried out on a second aliquot according to Kennedy and Jones (2001), followed by an HPLC analysis on a C18 (Atlantis dc18, Waters) column (4.6x250 mm; i.d. 5 µm), with a flow rate of 1 ml min–1. Two solvents were used: solvent A (water containing 2% formic acid) and solvent B [acetonitrile:water:formic acid (800:180:20, by vol.)]. Elution was started with 100% of solvent A for 5 min followed by a linear gradient from 100% to 10% of A over 30 min, then 40% for 30 min, ending with washing and re-equilibration steps. UV detection was recorded at 280 nm and the fluorescence output signal was monitored (excitation wavelength 275 nm, emission wavelength 322 nm). For the identification of the new compounds, LC-MS analysis was performed on a mass spectrometer equipped with an ESI source and an ion trap mass analyser. The mass spectrometer was operating in the negative ion mode. Each sample was analysed in triplicate.
The carotenoids and the chlorophylls were extracted overnight in the dark at –20 °C with 250 µl of acetone from about 10 mg powdered and frozen leaves from grapevine transformants. The supernatant was diluted with an equal volume of equilibration mixture [acetonitrile:methanol:triethylamine:acetic acid (600:400:1:0.5, by vol.)] and filtered prior to HPLC analysis according to Steghens et al. (1997) with the only change that the elution was carried out with a 0–30% linear gradient of dichloromethane instead of a step gradient. Pigments were identified according to their absorption spectra using a photodiode-array detector and quantified by integration of peak areas at 450 nm.
For the extraction of volatile compounds approximately 10 g frozen powdered leaves from grapevine were suspended in 50 ml of phosphate-citrate buffer (0.1 M, pH 5.0) containing gluconolactone (50 mM). The mixture was agitated under nitrogen (30 min, 4 °C), following by centrifugation (10 000 g, 15 min, 4 °C). The supernatant was filtered through a cellulose nitrate membrane filter (5 µm), and 32 µg of 4-nonanol was added as internal standard (solubilized in ethanol). This was extracted with 3x20 ml of pentane/dichloromethane mixture (2:1; v/v) The organic layer was concentrated at 35 °C to c. 500 µl by distillation through Vigreux and then a Dufton column. The extracts containing free volatiles were stored at – 20 °C until analysis by GC-FID and GC-MS. The remaining aqueous phase was loaded on a SPE RP18 column (LiChrolut, Merck) for the adsorption of glycosidically bound volatiles (Schneider et al., 2004). These were eluted with 25 ml methanol. After solvent elimination under vacuum, the residue was taken into 300 µl of phosphate–citrate buffer above and washed with pentane/dichloromethane (2:1, v/v) (5x1 ml) to remove trace amounts of volatiles. 100 µl of a glycosidase preparation (AR-2000, 70 mg ml–1, Gist-Brocades, Seclin, France) was added and the mixture was incubated at 40 °C for 16 h (Günata et al., 1985). Released volatiles from glycosides were extracted with pentane/dichloromethane (2:1, v/v) (5x1 ml). The organic extract was added with 32 µg of 4-nonanol, concentrated to c. 500 µl as above and stored at –20 °C until analysed by GC-FID and GC-MS.
GC-FID and GC-MS analysis conditions
Free and enzymatically liberated volatiles were analysed using a Varian 3800 gas chromatograph equipped with a DB-Wax (J&W Scientific, Folson, CA) capillary column (30 mx0.25 mm; i.d. 0.25 µm film thickness). The hydrogen carrier gas flow rate was 1 ml min–1. The oven temperature program was: 60 °C for 3 min, from 60 °C to 245 °C at 3 °C min–1, then 245 °C for 20 min. The FID and injector temperatures were maintained at 250 °C. 1.5 µl of organic extracts were injected in splitless mode. The amounts of compounds were calculated as 4-nonanol equivalents.
A Varian 3800 gas chromatograph coupled to a Saturn ion-trap mass spectrometer was used for the GC/MS identification of volatiles. The capillary column used and the oven temperature were as above. Mass spectra were recorded in electron impact (EI) ionization mode. The ion trap, the manifold and the transfer line temperatures were set respectively at 150 °C, 45 °C, and 250 °C. Mass spectra were scanned in the range of m/z 29–350 amu at 1 s intervals. Identification was carried out by comparison of linear retention indices and EI mass spectra with published data or with data from authentic compounds (Wirth et al., 2001).
The analyses of free and bound compounds were performed in triplicate. The means of three concentrations and the standard deviations are reported in the tables together with analysis of variance.
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Results |
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Transformation with VvAdh2 constructs and initial characterization
Grapevine were transformed with three types of cassettes as presented in Fig. 1. One construct (control C) had no VvAdh2 cDNA (Fig. 1A), whereas the two others (Fig. 1B, C) included the VvAdh2 cDNA, respectively, in a sense (S) or in an antisense (R) orientation relative to the promoter. In all constructs, the CaMV 35S promoter was used to provide constitutive expression of the cDNA. Putatively transformed embryos were selected for their resistance to hygromycin. The presence, in the regenerated lines, of both plasmid and VvAdh2 gene was respectively checked by GUS staining and by polymerase chain reaction (data not shown). For grapevine three control lines (VvC1, VvC2, VvC3), two sense lines of (VvS1, VvS2) and three antisense lines (VvR1, VvR2, VvR3) were recovered. Further confirmation of cassette integration was achieved by Southern blot analysis of young leaf DNA.
Southern blot analysis of young leaf DNA was used to estimate the number of htpII and VvAdh2 inserts integrated into each plant. When transformant DNA was digested with EcoRI, two to four bands corresponding to fragments containing either the htpII or the uiAd gene were detected in all plants (Fig. 2), indicating that the plants had integrated one to two copies into their genome. Dissimilar hybridization patterns in the analysed plants confirmed they arose from independent transformation events.
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Controls and transformants integrating the VvAdh2 gene were propagated and maintained in vitro and transferred to soil to acquire adult traits. Expression of the different VvAdh genes was assessed in leaves from transformed plants in order to specify the molecular frame encompassing the VvAdh2 expression levels modification. For VvAdh1 and VvAdh3, a similarly low expression was detected in the C, S, and R lines (Fig. 3). A higher expression was detected in the control for VvAdh2. Compared with control, this expression was significantly enhanced in S lines, whereas it was significantly lowered in R lines. These results clearly indicated that transformation resulted in VvAdh2 over-expression in sense transformants and in VvAdh2 under-expression in antisense transformants at the transcript level.
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Transformed plants were assessed for their phenotypic characteristics. Measurements were performed using four to six transformants per line, with three independent repetitions. Under normal growth, no obvious phenotypic differences in vegetative development could be observed between any of the C, S, and R grapevine lines (data not shown).
Response to stress of transgenic grapevine leaves
The specific activity of ADH in leaves was assayed under aerobic and anaerobic conditions (Fig. 4). Under aerobic conditions, no significant differences in ADH activity were observed between C and R lines, whereas this activity was about 100 times higher in the S lines as compared to the C lines. Thus, S transformants displayed a high ectopic ADH expression driven by the 35S constitutive promoter. Under anaerobiosis, the increase of ADH activity observed in the S transformants was not significant (1.5 times higher), whereas C and R transformants both showed significantly enhanced (5–8-fold) levels of ADH activity.
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Sugar and phenolic compound content in leaves of transgenic grapevine lines
Glucose and fructose content (around 8 g kg–1 FW) was similar in C, S, and R lines (Fig. 5). Sucrose content, around 5 g kg–1 FW in C lines was significantly lower in other transformants, with 0.5 and 3.5 g kg–1 FW, respectively, for S and R lines.
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Among the phenolic compounds, the hydroxycinnamic acid content was generally lower in the R lines than in C and S lines, trans-caftaric acid being the more affected form (Fig. 6A). As to flavonols identified by LC-MS, variation found between the lines was different: quercetin 3-glucoside, which is the main flavonol in the leaves, was higher in the C line than in the R and S lines, whereas the level of quercetol-3-glucuronide was four times higher in the R line than in the other lines (Fig. 6B). Proanthocyanidins have been studied after depolymerization, allowing different characteristics to be calculated from the results of these reactions according to Souquet et al. (2004)
. For instance, the mean degree of polymerization (mDP), i.e. the number of units in the polymer, was significantly higher in the S line than in C line, whereas it was significantly lower in the R line (Fig. 6C). The concentration of proanthocyanidins in the C line was more important than in the two other lines. Compared with the control, S and R lines contained proanthocyanidins with, respectively, more and less upper units and the inverse for terminal units as epicatechin and epigallocatechin (data not shown). However, the level of gallate units and the ratio between trihydroxylated and dihydroxylated units were similar in all the lines (data not shown).
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Carotenoid and chlorophyll contents in leaves of transgenic grapevine lines
Carotenoid and chlorophyll levels in different samples were evaluated by reverse phase HPLC and spectrometric detection. Overall 17 compounds were identified from their retention indices and by comparison with reference substances from the literature and known spectra (Table 1). Among the grape leaf carotenoids, the most abundant are ß-carotene, neoxanthin, and lutein, representing nearly 80% of the total amount. The contents of carotenoids were very similar for the C and R lines. Compared with the C line, these compounds were generally at higher levels in the S line, but only cis-violaxanthin and lutein isomer-2 levels were significantly higher. Contents of chlorophylls a' and b' were also significantly higher in the S and R lines.
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Volatile composition in leaves of transgenic grapevine lines
The levels of C6 compounds, terpenes, shikimate derivatives, and C13-norisoprenoids in leaves obtained from different transgenic grapevine lines were determined relatively to internal standard 4-nonanol. No significant differences were observed for free volatiles (data not shown). Twenty-seven glycosidically bound volatiles were identified and quantified (Table 2). For bound fractions, C and R lines contents were very similar, whereas compounds of all classes were generally 5–10-fold higher in the S line than in the C line. In the C and R lines, C13-norisoprenoids represented the major class of compounds (around 73%), whereas shikimate derivatives and C13-norisoprenoids were equally represented in the S line (around 48%). In the shikimate derivatives class, benzyl alcohol represented the major compound in all lines. In addition, the level of this compound exhibited the highest variation with a 53-fold increase in the S line compared with the C line. Of the C13-norisoprenoids, megastigmane-3,9-diol is among the most abundant compound, as previously shown in leaves from the Shiraz cultivar (Skouroumounis et al., 2000
; Wirth et al., 2001), reaching 600 µg kg–1 in the C line. Also in the C13-norisoprenoid class, vomifoliol, among the least abundant compound in the C line, exhibited a 141-fold increase, becoming the major compound of this class in the S line. By contrast, 3,6-dihydroxy-megastigm-7-ene-9-one was the only compound that decreased in the S line (5-fold), and to an even greater extent (70-fold) in the R line.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In grapevine, dramatic molecular and biochemical changes influence various aspects of the berry development such as secondary metabolism (Boss and Davies, 2001
; Shoseyov and Bravdo, 2001), growth regulators balance (Düring et al., 1978), and orientation of the primary metabolism towards anaerobic metabolism (Tesniere and Verries, 2000). Among the genes that show changes in expression profiles in berries, VvAdh expression varies strongly during berry development, with the VvAdh2 isogene being specifically expressed during ripening (Tesniere and Verries, 2000).
A number of previous works developed with model plants, showed that Adh expression is stress-related (Matton et al., 1990; Christie et al., 1991; Ingersoll et al., 1994; Bucher et al., 1995) and linked to secondary metabolism changes (Bicsak et al., 1982; Longhurst et al., 1990; Speirs et al., 1998).
The purpose of this study was to investigate the putative involvement of VvAdh2 in the development of some processes related to abiotic stress resistance and/or secondary metabolism. Therefore, VvAdh2 was introduced into grapevine to express sense and antisense VvAdh2 ectopically and the effect of ADH2 overexpression or silencing was monitored.
In grapevine plants, overexpression of the Adh gene driven by the CaMV 35S promoter resulted in high levels of constitutive ADH enzyme activity which was not significantly enhanced by hypoxia. This is corroborated by a higher expression of VvAdh2 transcripts in S lines compared with the control, whereas no change in the expression of the other VvAdh transcripts was observed. Thus, no gene silencing or co-suppression was encountered in any of the sense lines recovered in this work, as it has been sometimes described (Jorgensen, 1990). The grapevine antisense lines displayed no visible changes in ADH activity in air or under oxygen deprivation with respect to the controls. No changes in enzyme activity were evident. However, in antisense plant leaves, expression of VvAdh2 was limited in leaves, compared with the control. The other effects of antisense transformation on secondary metabolites were also rather limited and, in most cases, contents were found that were similar to control plants. This could indicate that some compensatory reaction has occurred in these transformed plants.
Although glucose and fructose content were unchanged in leaves overexpressing ADH, sucrose content was drastically reduced by almost 90%, compared with the control. Plants possess two alternative biochemical pathways for sucrose degradation. One involves hydrolysis by invertase; the other route is catalysed by sucrose synthase. Interestingly, overexpression of both activities led to a strong induction of ADH activity in growing transgenic tubers (Bologa et al., 2003). Thus, some link could exist between the increase in ADH activity and the activities of enzymes related to sucrose catabolism.
Concerning phenolic compounds, the composition of hydroxycinnamic acids in leaves was comparable to that in grape berries and the content was the same as that described in leaves from other varieties (Boursiquot et al., 1986). Among the phenolic compounds analysed, significant changes were observed between the lines for trans-caftaric acid, quercetin 3-glucoside, quercetol-3-glucuronide, and the proanthocyanidin mDP. For instance, a higher mDP level was observed in the S line compared with the C line. One hypothesis could be that a polycondensation reaction had somehow occurred between proanthocyanidin and acetaldehyde (Fulcrand et al., 1996) at a higher rate in transformants overexpressing ADH, leading to a decrease in free units and to a decrease in the number of terminal units. The inverse was also observed for the R line with an increase in free units and in terminal units.
The carotenoid content from each of the C, S, and R lines was also investigated. In the S line, cis-violaxanthin, but also lutein isomer-2, significantly exceeded the amount observed in the C line. The absence of significant changes in the other carotenoids and the chlorophylls a and b between transformed lines suggested that their contents are under strong regulatory control.
Other metabolites derived from the carotenoids such as C13 derivatives (Schwartz et al., 2001; Baumes et al., 2002) exhibited an important and significant variation between the transformed lines. In grapevine, the free fraction of volatile compounds did not vary between the lines (data not shown), but the glycosidically bound fraction was generally found to be higher in the overexpressing line compared with the other lines. The glycosylation of secondary metabolites is generally considered to protect the cells from any form of toxicity exhibited by the free secondary metabolites (Hösel, 1981). The accumulation of benzyl alcohol to high levels in the S line may be related to the substrate specificity of the ADH enzyme. Indeed, Molina et al. (1986) have shown that benzaldehyde, but not benzyl alcohol, was a substrate for grapevine ADH. This could explain why only the latter is detected in C and R lines and is accumulated in the S line.
In the C13-norisoprenoid class, vomifoliol and its derivative 3,6-dihydroxy-megastigm-7-ene-9-one exhibited the largest variations (in opposite directions) in both S and R lines compared with the C line. It was unexpected that a similar modification of these two compounds should be observed (although not to the same extent) in both S and R lines. Interestingly, both compounds show structural similarity with abscisic acid (ABA). For instance, vomifoliol has been used for its effects on stomatal aperture similar to ABA (Stuart and Coke, 1975; Artsaenko et al., 1995; Fuchs et al., 1999). Furthermore, this compound could be generated from an oxidative enzymatic cleavage of ABA (Winterhalter and Schreier, 1995). Carotenoid dioxygenases were shown to intervene in the formation of C13-norisoprenoids (Schwartz et al., 2001) and ABA (Schwartz et al., 1997). The enhancement in relevant activity could have occurred in the S line. In addition, the levels of monoterpenes were higher in the S line than those of the C and R lines. This may be correlated with the increase in levels of C13-norisoprenoid compounds in the S line, since monoterpene and C13-norisoprenoid compounds share common biosynthetic pathways. Analysis of the expression of these biosynthetic gene would definitely help to interprete these results. However, the synthesis of secondary metabolites such as C13-norisoprenoids, monoterpenes, and shikimate derivatives involves several pathways and the majority of the relevant genes are still unknown.
From the results presented here, it cannot be excluded that the alcohol/acetaldehyde or NADH/NAD ratios could be altered in Adh-overexpressing plants resulting in changes in downstream reactions. Further evaluation in Adh-overexpressing lines of short-chain alcohol dehydrogenase activity, which usually catalyses alcohol-ketone/aldehyde substrate transitions, would elucidate this point.
Experiments are underway to characterize the metabolic changes induced by the transformation of the Adh function in developing berries and to discover whether they compare with the changes observed in grapevine leaves in terms of secondary metabolites.
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Acknowledgements |
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We thank Dr J Speirs for critical reading of the manuscript. We are grateful to Dr JP Boutin for conducting the carotenoid analysis. We acknowledge the technical assistance of G Lopez and C Brachet for their care of the plants. We also thank Dr F Dosba (AgroM/UMR BEPC) and Dr G Albagnac (INRA/UMR SPO) for supporting our research effort, and the Languedoc-Roussillon Region for the financial support of the GC-MS equipment.
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Footnotes |
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Abbreviations: ABA, abscisic acid; ABRE, abscisic acid responsive element; ADH, alcohol dehydrogenase; CaMV, cauliflower mosaic virus.
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References |
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