JXB Advance Access originally published online on June 23, 2006
Journal of Experimental Botany 2006 57(10):2445-2453; doi:10.1093/jxb/erl008
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RESEARCH PAPER |
Up- and down-regulation of Fragariaxananassa O-methyltransferase: impacts on furanone and phenylpropanoid metabolism
1Technical University Muenchen, Biomolecular Food Technology, Lise-Meitner-Str. 34, D-85354 Freising, Germany
2Plant Research International, Business Unit Genetics and Breeding, PO Box 16, 6700 AA Wageningen, The Netherlands
*To whom correspondence should be addressed. E-mail: schwab{at}wzw.tum.de
Received 7 March 2006; Accepted 30 March 2006
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Abstract |
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A complex mixture of hundreds of substances determines strawberry (Fragariaxananassa) aroma, but only
15 volatiles are considered as key flavour compounds. Of these, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is regarded as the most important, but it is methylated further by FaOMT (Fragariaxananassa O-methyltransferase) to 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF) during the ripening process. It is shown here that transformation of strawberry with the FaOMT sequence in sense and antisense orientation, under the control of the cauliflower mosaic virus 35S promoter, resulted in a near total loss of DMMF, whereas the levels of the other volatiles remained unchanged. FaOMT repression also affected the ratio of feruloyl 1-O-ß-D-glucose and caffeoyl 1-O-ß-D-glucose, indicating a dual function of the enzyme in planta. Thus, FaOMT is involved in at least two different biochemical pathways in ripe strawberry fruit. Key words: Fragariaxananassa, fruit ripening, O-methyltransferase, strawberry, volatiles
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Introduction |
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Combined biochemical and molecular analyses of volatile components released by fruit have demonstrated that their biogenesis forms an integral part of the ripening programme. Biomolecular work on fruit ripening has been performed mainly on tomato [Lycopersicon esculentum (=Solanum lycopersicum)] although in recent years there has been a dramatic increase in the investigations of other fruit species including melon (Hadfield et al., 2000), grape (Davies and Robinson, 2000), citrus (Alonso and Garnel, 1995), raspberry (Jones et al., 2000), pear (Itai et al., 2000), banana (Clendennen and May, 1997), and strawberry (Wilkinson et al., 1995; Aharoni et al., 2000, 2004; Aharoni and Vorst, 2002). The majority of the studies identified genes with elevated expression during ripening and correlated their putative identity with a specific ripening process.
Strawberry (Fragariaxananassa), a member of the rose family (Rosaceae), has emerged as a suitable model plant for studying non-climacteric fruit ripening (Giovannoni, 2001). The fruit are very popular, are cultivated almost worldwide, are consumed fresh or preserved, or are processed into various products (Hancock, 1999). Although strawberry fruit is derived from the flower receptacle, it has the same development and ripening characteristics of true fruit, including the degradation of chlorophyll, the accumulation of anthocyanins, the softening which is partially mediated by cell wall-hydrolysing enzymes, the metabolism of sugars and organic acids, and the production of flavour compounds.
Compounds contributing to the flavour of strawberries have been extensively studied. More than 360 volatiles have been identified (Nijssen, 1996) but only 15–20 of them are believed to be essential for the sensory quality of strawberries, together with the non-volatile sugars and organic acids (Schieberle and Hofmann, 1997). The contribution of volatiles to the flavour of a foodstuff can be determined using the ‘aroma value’ concept which calculates the ratio of concentration to odour threshold of the corresponding volatile molecule. This concept, recently applied to strawberry juice (Schieberle and Hofmann, 1997), showed that (Z)-3-hexenal (grass-like), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF, Furaneol®) (caramel-like, sweet), methyl butanoate (fruity), ethyl butanoate (fruity), ethyl 2-methylpropanoate (fruity), and diacetyl (buttery) are the key flavour compounds in the typical odour of strawberry juice. Among these, HDMF (Fig. 1) is the most important because of its high concentration in strawberry fruit (up to 55 mg kg–1 fresh weight) (Larsen, 1992) and low odour threshold (10 ppb in water) (Schieberle and Hofmann, 1997).
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HDMF was first isolated from pineapples (Rodin, 1965), and a few years later from strawberries (Ohloff, 1969), but, since then, it has been identified in a number of different fruit (Schwab and Roscher, 1997). HDMF is frequently accompanied by its methyl ether 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF, mesifuran) (Fig. 1), first reported in 1965 (Willhalm, 1965), and HDMF-glucosides (Mayerl et al., 1989; Roscher et al., 1996). The furanones were isolated from fruit but not from roots, stems, leaves, flowers, or other parts of the plant (Schwab and Roscher, 1997). HDMF and DMMF content in strawberries varies remarkably among the different cultivars and varieties (Douillard and Guichard, 1989, 1990). Racemization of HDMF (Fig. 1) takes place after the enantioselective biosynthesis (Raab et al., 2003), thus HDMF and DMMF occur as racemates in the different fruits (Bruche et al., 1991).
Several efforts have been made to clarify the biosynthesis of HDMF and its derivatives (Bood and Zabetakis, 2002). Even though the biogenesis in fruit is not fully understood, all studies showed that HDMF is derived from sugar metabolism (Wein et al., 2001). The quantification of HDMF and DMMF during fruit ripening indicated a rapid conversion of HDMF to DMMF and HDMF-glucoside (Pérez et al., 1996), and in vivo feeding experiments demonstrated the incorporation of 14C label into DMMF after the application of both S-[methyl-14C]-adenosyl-L-methionine ([14C]SAM) and [14C]HDMF (Roscher et al., 1997). Finally, an O-methyltransferase (Fragariaxananassa O-methyltransferase, FaOMT) cDNA was obtained by screening a strawberry cDNA library, cloned, and heterologously expressed in Escherichia coli. The FaOMT protein catalyses the transfer of the methyl group from SAM, not only to HDMF but also to caffeic acid, thereby forming the corresponding O-methyl ethers (Fig. 1) (Wein et al., 2002). Due to the expression pattern of FaOMT and the enzymatic activity in the different stages of fruit ripening, it was proposed that FaOMT is involved in phenylpropanoid metabolism and in the biosynthesis of the strawberry volatile DMMF.
Apart from changes in texture, maturing strawberry fruit undergo dramatic changes in the levels of different types of phenolic compounds, which provide it with colour, flavour, and the resistance to pathogenic attack and environmental conditions such as UV exposure. During early stages, non-tannin flavonoids and mainly condensed tannins accumulate to high levels and give strawberry an astringent flavour (Cheng and Breen, 1991). Later, when fruit start to ripen, other flavonoids such as anthocyanins (mainly pelargonidin glucoside), flavonols, and cinnamoyl ß-D-glucose accumulate to high levels (Latza et al., 1996; Manning, 1998; Moyano et al., 1998; Aharoni et al., 2000; Deng and Davis, 2001). Free hydroxycinnamic acids are rarely detected in ripe strawberries, but conjugated forms such as sugar ester have frequently been reported (Määttä-Riihinen et al., 2004). Hydroxycinnamic acids are also precursors of lignin, and in grasses, ferulate moieties play an important role in other cell wall polymerization processes, such as cross-linking between polysaccharides. Thus, many published caffeic acid O-methyltransferases have been associated with lignin biosynthesis (Joshi and Chiang, 1998).
We are interested in the role of FaOMT in the biosynthesis of the odorous compound DMMF as it is disliked by flavourists. Heterologously expressed FaOMT shows a preference (100:1) for caffeic acid over HDMF (Wein et al., 2002), but only DMMF can be detected in appreciable amounts in fruit. This suggests, but does not prove, that FaOMT may be involved preferentially in the formation of DMMF.
Most of the important molecular insights into fruit ripening have been obtained using overexpression and antisense technology in transgenic fruit (Speirs et al., 1998; Brummell et al., 1999; D'Aoust et al., 1999; Lu et al., 2001; Jiménéz-Bermúdez et al., 2002). In this report, by up- and down-regulating FaOMT using the cauliflower mosaic virus (CaMV) 35S promoter, the function of the FaOMT enzyme is assessed in planta. After selecting transgenic plants with a higher or lower content of FaOMT transcripts using the quantitative real-time polymerase chain reaction (PCR) approach, these results were correlated with the HDMF to DMMF, and caffeoyl ß-D-glucose to feruloyl ß-D-glucose ratios determined by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography-UV-electrospray ionization-tandem mass spectrometry (LC-UV-ESI-MSn), respectively. Our results suggest that, in accordance with the in vitro activity, FaOMT is not only the sole enzyme involved in the methylation of HDMF, but it also participates in the formation of ferulic acid in the fruit.
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Materials and methods |
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Plant material
Strawberry (Fragariaxananassa) cv. Calypso plants were grown under standard greenhouse conditions with a 16 h photoperiod. Tissues harvested for RNA extraction or chemical analysis were immediately frozen in liquid nitrogen and stored at –80 °C until use. Homogenization was carried out with liquid nitrogen.
Chemicals
Except when noted, all chemicals, salts, solvents, and phenolic compounds were purchased from Sigma (Deisenhofen, Germany), Aldrich (Deisenhofen), Fluka (Deisenhofen), and Roth (Karlsruhe, Germany).
Plant transformation
For plant transformation, the strawberry (Fragariaxananassa) cv. Calypso was used because of its high transformation frequency and high fruit yield all year round. Under greenhouse conditions, the ‘everbearing’ cultivar Calypso continuously produces fruit which allows multiple harvests and the analysis of the fruit over the year. Plant transformation was carried out as described in detail elsewhere (Schaart et al., 2002). Young folded leaves were collected and surface-sterilized using a 2% (w/v) sodium hypochlorite solution. Segmented (4–6 mm) leaflets were transfected using supervirulent Agrobacterium tumefaciens strain AGL0 containing the FaOMT gene in a derivative of the binary pBINPLUS plasmid under the control of a CaMV 35S promoter. After co-cultivation for 4 d in the dark at 25 °C, the leaf explants were transferred to selection medium. Regenerated shoots were multiplied on proliferation medium and subsequently primary transgenic plants were transferred to the greenhouse. Several ripe fruits (n=3) were harvested from each transgenic line and analysed for FaOMT mRNA level by quantitative PCR. The following year, different batches of ripe fruit were harvested from selected transgenic lines at different dates for further analysis of metabolites. In most plant species, a generative progeny is produced for the selection of transgenic plants that are used for further studies. In the case of octoploid strawberry, the high genetic background variation hinders the analysis of a generative progeny. If a primary transgenic octoploid strawberry is selfed, the offspring are genetically diverse with respect to the genetic background, and the cultivar characteristics are lost (Mathews et al., 1998). However, an advantage of a vegetatively propagated crop such as strawberry is that the selected transgenic lines with their specific expression levels of the target gene can be maintained forever in the same genetic background, but transgenic instability and somaclonal variation have to be considered (Bhat and Srinivasan, 2002; Labra et al., 2004). In our transformation system, the ‘everbearing’ cultivar Calypso was used that can produce up to eight harvests per year under greenhouse conditions. This allowed the detection of chimerism and transgene instability of primary transgenics which were taken out.
TaqMan® quantitative PCR assay
TaqMan® quantitative PCR was carried out as described elsewhere (Schaart et al., 2002). Briefly, the TaqMan quantitative PCR was set up in three replicates of 20 µl volumes containing first-strand cDNA from 10 ng of total RNA, 800 nM forward OMT-primer (5'-ACC GGC GAG ACT CAG ATG AC-3') and 800 nM reverse OMT-primer (5'-GCG AAG AGG TTG GCT TCC TC-3') for the sense plants, or 200 nM forward OMT-primer (5'-AGT TGG ATT TTG GAT GCT GTC AT-3') and 100 nM reverse OMT-primer (5'-AGA TCA GCA CAG CAT CTC AAA AAC-3') for the antisense plants, 10x TaqMan buffer A, including a passive internal reference dye, 5.5 mM MgCl2, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 400 µM dUTP, 0.01 U µl–1 AmpErase uracil-n-glycosylase, and 0.025 U µl–1 AmpliTaq Gold DNA polymerase. All reagents were supplied with the TaqMan PCR Core Reagent Kit (Perkin-Elmer, Applied Biosystems). Cycling parameters for the PCR were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. As an endogenous control, a cDNA from strawberry was selected that showed strong homology to a putative DNA-binding protein (DBP) gene from Arabidopsis thaliana. The DBP from strawberry exhibited a similar level of expression in a northern blot for several strawberry tissues (Schaart et al., 2002). Levels of FaOMT mRNA isolated from fruit (n=3) of sense, antisense, and control plants were standardized by means of DBP expression. Finally, the concentrations of FaOMT mRNA of the different transgenic clones were calculated as n-fold difference from FaOMT mRNA in samples of the different control plants. For standardization, mRNA was isolated from leaves and fruit of control plants, respectively. All PCRs were incubated in the ABI Prism 7700 Sequence Detection System (Perkin Elmer, Applied Biosystems). For the real-time analysis, PCR products were directly detected by monitoring the increase in fluorescence from the dye-labelled FaOMT- or DBP-specific DNA probe. The amplification was plotted as the normalized reporter signal 
Rn (the reporter dye was normalized to the internal reference dye and corrected for the baseline value) against the number of PCR cycles. For each reaction, the threshold cycle, CT, which is defined as the PCR cycle at which a statistically significant increase of Rn is first detected, was determined. The final relative quantification was done using the comparative CT method (User bulletin no. 2, ABI PRISM 7700 Sequence Detection System, December 1997; Perkin-Elmer, Applied Biosystems) in which the differences in the CT for the FaOMT amplicon and the CT for the endogenous control DBP, called CT, were calculated to normalize for the differences in the total amount of cDNA present in each reaction and the efficiency of the reverse transcription step. To compare two samples, the CT values were subtracted from each other, giving CT. The relative amount of FaOMT mRNA copies was finally given by 2–CT.
Identification of metabolites: XAD® solid-phase extraction of strawberry fruit
Amberlite XAD-2 polymeric adsorbent (20–60 mesh; Aldrich, Deisenhofen, Germany) used to fill a glass column (50 cmx2.5 cm) was washed with methanol (100 ml) and conditioned with distilled water (200 ml). For the analysis of volatile and glycosidically bound compounds in strawberry fruit, 2 g of frozen ripe material was homogenized with 20 ml of water using an Ultra Turrax® (T18 basic, IKA® Works Inc., Wilmington, NC, USA) and centrifuged (3500 g, 10 min). The supernatant was loaded onto the XAD column and the solid residue was re-extracted twice. After rinsing the column with 100 ml of distilled water, volatiles were eluted with 50 ml of diethyl ether followed by glycosides with 80 ml of methanol. The water phase from the diethyl ether extract was added to the methanolic extract, which was concentrated in vacuo to 1 ml and used directly for LC-UV-ESI-MSn analysis. The diethyl ether extract was dried over anhydrous sodium sulphate and concentrated using a Vigreux column to 1 ml, pipetted into a GC vial, and the residual organic solvent was removed with a stream of nitrogen until the volume reached 50 µl. For GC quantification, phenol (0.1 mg ml–1) was added as an internal standard.
Liquid chromatography-UV-electrospray ionization-tandem mass spectrometry (LC-UV-ESI-MSn)
The system used for LC-UV-ESI-MSn analysis was a Bruker esquire 3000 plus mass spectrometer, equipped with an Agilent 1100 HPLC system, composed of an Agilent 1100 quaternary pump and an Agilent 1100 variable wavelength detector. The column was a Eurospher C18 column, particle size 5 µm, 10 cmx2 mm (Grom Analytik & HPLC GmbH, Rottenburg, Germany). The ionization parameters were as follows. The voltage of the capillary was 3074 V and the end plate was set to –500 V. The capillary exit was –109.8 V and the Octopole RF amplitude 120 Vpp. The temperature of the dry gas (N2) was 300 °C at a flow of 10 l min–1. The full scan mass spectra of the glycosides were measured from m/z 50–500 until the ion charge control (ICC) target reached 20 000 or 200 ms, whichever was reached first. Tandem mass spectrometry was performed using helium as the collision gas, and the collision energy was set at 1.0 V. All mass spectra were acquired in the negative ionization mode. Auto-tandem mass spectrometry was used to break down the most abundant [M-H]– or the [M+HCOO]– ion of the different compounds of the strawberry extracts. Identification of the glycosylated compounds was achieved using enzymatically produced reference compounds. The LC parameters were from 0% acetonitrile and 100% water (acidified with 0.05% formic acid) to 50% acetonitrile and 50% acidic water in 35 min, then in 2.5 min to 100% acetonitrile, and kept for 2.5 min at these conditions, finally back to 100% water and 0% acetonitrile in 5 min at a flow rate of 0.2 ml min–1. The detection wavelength was 280 nm.
Capillary gas chromatography–mass spectrometry (GC–MS)
GC–MS analysis was performed with a Thermo Finnigan Trace DSQ mass spectrometer coupled to a Thermo Finnigan Trace GC with a split injector (1:20) equipped with Xcalibur software (version 1.4). The GC was equipped with a BPX5 20 M fused silica capillary column (30 mx0.25 mm inner diameter; thickness of the film=0.25 µm). The GC parameters were as follows: initial temperature of 40 °C for 3 min, then increased to 250 °C at 5 °C min–1 intervals. The helium gas flow rate was 3 ml min–1. The EI-MS ionization voltage was 70 eV (electron impact ionization) and the ion source and interface temperature were kept at 230 °C and 240 °C, respectively. Compounds were identified by comparing their mass spectra and retention indices with the NIST mass spectra library and reference compounds.
Quantification via LC-UV-ESI-MSn and statistical analysis
Fruit harvested from the transgenic lines were pooled regardless of harvest date and were independently purified at least three times. Control fruit from several Calypso plants were individually analysed throughout the course of the experiment. Each purified sample was measured once with LC-UV-ESI-MSn. Due to the lack of other reference compounds, a single natural log-transformed reference curve was generated (not shown) using synthesized cinnamoyl-glucose (Plusquellec et al., 1986) and this was used to quantify all compounds as cinnamoyl-glucose equivalents by integration of their signals in the MS ion traces. Statistical analysis of differences between the mean metabolite levels of individual plants (excepting controls, which were grouped together) was performed using a one-way analysis of variance (ANOVA). Significant differences between different groups were determined by t-test using SigmaPlot 8.0 at a level of P <0.01.
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Results |
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Generation of transgenic strawberry plants with altered expression of FaOMT
To obtain up- or down-regulation of FaOMT in strawberry fruit, Fragariaxananassa cv. Calypso was transformed with FaOMT sense (S) and antisense (AS) constructs. The primary transgenic, kanamycin-resistant lines, 12 FaOMT AS-lines and 11 FaOMT S-lines, were transferred to the greenhouse. In several transgenic lines, phenotypic changes were observed such as delayed flowering (FaOMT S2) and a slightly changed fruit colour in the first round of fruit production (FaOMT S1). These changes occurred at a low frequency (3%) and most probably can be attributed to somaclonal variation. Plants with reduced growth occurred at a higher frequency (22%) (FaOMT AS2, -AS4, -AS11, -S8, and -S9).
Quantitative RNA analysis of transgenic strawberry plants
To determine up- and/or down-regulation of FaOMT expression in the transgenic lines, the relative FaOMT mRNA levels in transgenic and control lines were quantified using TaqMan® quantitative real-time PCR (Table 1a, b). Previously, it has been shown that FaOMT is mainly expressed in ripening fruit and is only present at low levels in other tissues (Wein et al., 2002). Therefore, overexpression of the FaOMT gene was measured in the leaves of S-lines whereas down-regulation of FaOMT expression was measured in ripe fruit samples of FaOMT AS-lines. Five of the six FaOMT S-lines selected for TaqMan® analysis contained higher levels of FaOMT transcripts in the leaf tissue than the control plants. One line (FaOMT S9) showed a lower expression level (24% of the control level) resulting from a known phenomenon called co-suppression (van der Krol et al., 1990; Hamilton and Baulcombe, 1999). This line also possessed a severely altered phenotype, in that the plant and leaf sizes were significantly reduced in comparison with the control plants. As expected, the five antisense lines that were analysed contained lower levels of FaOMT mRNA ranging from 74% to 3% of the control level (Table 1b).
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Quantification of metabolites in transgenic and control plants
Enzyme activity tests performed with recombinant FaOMT protein have already demonstrated its ability to catalyse the methyl transfer from SAM to HDMF and caffeic acid in vitro (Fig. 1) (Wein et al., 2002). To determine the in planta function of the protein, its potential substrates and products were isolated by solid phase extraction from ripe fruit of control and transformed lines harvested in the second year.
Levels of HDMF and DMMF as well as concentrations of caffeoyl ß-D-glucose and feruloyl ß-D-glucose were quantified by GC–MS and LC-UV-ESI-MSn, respectively in the same samples (Fig. 2). Since previous studies have shown that hydroxycinnamic acids occur in strawberry fruit exclusively as the conjugated forms (Määttä-Riihinen et al., 2004), the glucose esters were used to calculate the amounts of ferulic and caffeic acid. Vanillin, another product of the methylation reaction catalysed by FaOMT, was also detected by GC–MS, but levels were too low for accurate quantification. Like Watson et al. (2002), substantial fruit-to-fruit and harvest-to-harvest variation was observed here among the absolute metabolite levels within each line (Table 2). However, when the concentration of HDMF was normalized to the total concentration of furanones (HDMF and DMMF), little variation was seen among strawberries from control plants (Table 2). Feruloyl ß-D-glucose levels, normalized to the combined level of feruloyl ß-D-glucose and caffeoyl ß-D-glucose, also showed little variation among controls (data not shown). Thus, we used these relative values were used as the basis for our metabolite comparison, the results of which are shown in Figs 3 and 4. Here, the white bars show the normalized concentration of the FaOMT substrates (respectively HDMF and caffeoyl ß-D-glucose as a percentage), whereas the grey bars represent the normalized levels of the FaOMT products (respectively DMMF and feruloyl ß-D-glucose as a percentage).
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Strawberries from four of the 11 transgenic lines (FaOMT AS2, FaOMT AS4, FaOMT AS11, and FaOMT S9) produced significantly lower levels of the methylated product DMMF as compared with the substrate HDMF, resulting in a higher proportion of HDMF relative to the total amount of furanones (Fig. 3). The ratio of HDMF to DMMF of 68:32 in the control fruit increased to 99:1 in transgenic fruit from the AS-lines FaOMT AS4, FaOMT AS11, FaOMT AS2, and the co-suppressor FaOMT S9, indicating a near total loss of DMMF (Fig. 2). FaOMT transcript levels in fruit correlated fairly well with HDMF:DMMF ratios (Fig. 3). Significant losses of DMMF in fruit tissue were only observed when FaOMT transcript levels were reduced to <8% of the control (as measured in fruit tissue of the AS-lines) whereas a change in transcript level to 45% of the control was insufficient to affect the furanone ratios (Fig. 3). The normalized levels of HDMF in the fruit picked from the other transgenic lines were not significantly different from the controls. Five of the six plants containing a FaOMT construct in the sense orientation showed overexpression of the transcript as measured in leaves, but the increase did not affect the relative levels of HDMF and DMMF in the fruit tissue.
Next, several of the transgenic lines (FaOMT AS4, FaOMT AS11, FaOMT S9, FaOMT S1, FaOMT S8, FaOMT AS3, and FaOMT AS9) were analysed by LC-UV-ESI-MSn to determine the level of the other substrate and product of FaOMT (caffeic acid and ferulic acid) (Fig. 4). As with the furanones, only the transgenic lines with strongly reduced levels of FaOMT mRNA (FaOMT AS4, FaOMT AS11, and FaOMT S9) contained significantly lower levels of the FaOMT product (ferulic acid) compared with the substrate of FaOMT (caffeic acid), quantified as glucose esters. However, in none of the transgenic plants did the reduction of the transcripts result in a near total loss of feruloyl ß-D-glucose as was observed for the production of DMMF. In fruit of the line FaOMT S1, a significantly higher concentration of the methylated product, feruloyl ß-D-glucose, was detected. However, this higher concentration was not confirmed by a higher FaOMT mRNA level in fruit tissue (not determined) or a higher DMMF level.
Since the recovery of transformants via seed is not a viable option in vegetatively propagated species, it is imperative to confirm the presence of uniformly and stably transformed plants. Therefore, a number of fruit over the year was analysed and harvest-to-harvest variation for FaOMT transcripts or metabolite levels was monitored. Figure 5 shows the results of the metabolite analyses of additional fruit samples harvested at the end of the second year. The normalized metabolite levels agree very well with data obtained in the first series of experiments (Figs 3, 4), confirming the presence of true transformants for lines FaOMT AS4, FaOMT AS11, and FaOMT S9.
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To address the in planta function of FaOMT, it was decided to up- and down-regulate FaOMT by sense and antisense approaches, respectively, utilizing the CaMV 35S promoter. Primary transgenic lines were produced and transferred to the greenhouse. Somaclonal variation was observed at a relatively low frequency (3%) in our transformation system. A similar frequency of somaclonal variants was found in cv. Chandler (Kaushal et al., 2004). Plants with reduced growth occurred at a higher frequency (22%) (FaOMT AS2, FaOMT AS4, FaOMT AS11, FaOMT S8, and FaOMT S9) and phenotypically resembled transgenic strawberry plants in which the cinnamoyl CoA reductase (CCR) gene, acting in lignin biosynthesis, was down-regulated (data not shown). In transgenic alfalfa and maize, the strong down-regulation of a caffeic acid 3-O-methyltransferase resulted in decreased lignin content (Guo et al., 2001; He et al., 2003). In some cases, such as in transgenic tobacco (Humphreys and Chapple, 2002), reduced lignin contents were related to stunted plant growth. So, although not conclusively proven, the FaOMT phenotype observed might be the effect of changed hydroxycinnamic acid levels leading to altered lignin content or composition (Tsai et al., 1998).
GC–MS and LC-UV-ESI-MSn analyses were performed from the same samples to evaluate the profiles of potential substrates and products. As only trace amounts of free hydroxycinnamic acids were detected at levels 103-fold less than the detected levels of the glucose ester (data not shown), it is assumed that caffeic acid and ferulic acid are almost completely metabolized and that the relative glucose ester levels reflect the levels of their corresponding precursor acids.
Down-regulation of the FaOMT mRNA level in fruit tissue to 45% of the control levels was not enough to cause a significant effect on the metabolites involved. Only reductions to <8% of the control level caused significant effects on metabolite concentrations. Transgenic lines with strikingly reduced amounts of FaOMT transcripts contain the lowest proportion of the methylated products DMMF and feruloyl ß-D-glucose. Thus, FaOMT is involved in both pathways despite a much higher preference of the recombinant enzyme (100-fold) for caffeic acid in in vitro studies (Wein et al., 2002). In accordance with this observation, in vivo changes in FaOMT mRNA levels affect the substrate-to-product ratios of the catalysed reactions differently. Although the reduction of FaOMT mRNA leads to a near total absence of DMMF production, this dramatic effect does not necessarily apply for feruloyl ß-D-glucose. An explanation for these differences in effect could be that caffeic acid is the preferred substrate of FaOMT. Thus, decreased levels of FaOMT would affect the DMMF concentration to a larger extent when HDMF and caffeic acid are provided simultaneously as substrates. Alternatively, the expression of an additional O-methyltransferase, either more specific for the hydroxycinnamic acid or co-localized with the acid, could also account for this observation. In wild strawberries (Fragaria vesca L.), mRNAs that are differentially expressed during ripening have been characterized (Nam et al., 1999). One of these sequences encodes a putative caffeoyl-CoA 3-O-methyltransferase (CCOMT), but a comparison revealed only a distant structural similarity between FaOMT and CCOMT.
In addition, it appears that the up-regulation of FaOMT transcription such as determined for plant FaOMT S1 and FaOMT S8 also affects DMMF and feruloyl ß-D-glucose production differently, although only two lines showed the effect (Figs 3, 4). In line with the argument for the impact of FaOMT down-regulation on the relative metabolite levels, a likely explanation could be the preference of the enzyme. Thus, enhanced levels of FaOMT would affect ferulic acid concentration relative to the substrate levels to a larger extent when HDMF and caffeic acid are provided simultaneously as substrates. Other possible explanations for the unaffected furanone ratio would be that DMMF and ferulic acid formation proceed in different cell types within the fruit or cell compartments. However, FaOMT does not bear a known signal sequence. However, formation of large amounts of ferulic acid could also proceed in leaves where high levels of FaOMT have been determined.
Only recently has a cDNA encoding a UDP-Glc:cinnamate glucosyltransferase [Fragariaxananassa glucosyltransferase (FaGT)] been isolated from ripe strawberry cv. Elsanta that catalyses the formation of 1-O-acyl-glucose esters of cinnamic acid, benzoic acid, and their derivatives in vitro (Lunkenbein et al., 2006). Quantitative analysis of metabolite levels in transgenic lines containing an antisense construct of FaGT demonstrated that the enzyme is involved in the formation of cinnamoyl glucose and p-coumaroyl glucose during ripening. FaGT catalyses two reactions in strawberry fruit similarly to FaOMT.
In conclusion, the ratio of the odorous furanones in strawberry fruit was successfully modified using a transgenic approach. As a second effect of the reduced levels of FaOMT transcripts, we observed the reduction of feruloyl ß-D-glucose was observed, confirming the dual function of FaOMT in strawberry fruits.
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Acknowledgements |
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We thank Degussa for financial support.
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Abbreviations |
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CaMV, cauliflower mosaic virus; DBP, DNA-binding protein; DMMF, 2,5-dimethyl-4-methoxy-3(2H)-furanone; FaGT, Fragariaxananassa glucosyltransferase; FaOMT, Fragariaxananassa O-methyltransferase; GC–MS, gas chromatography–mass spectrometry; HDMF, 4-hydroxy-2,5-dimethyl-3(2H)-furanone; LC-UV-ESI-MSn, liquid chromatography-UV-electrospray ionization-tandem mass spectrometry; SAM, S-adenosyl-L-methionine.
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References |
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