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American Society of Plant Biologists High-Flavonol Tomatoes Resulting from the Heterologous Expression of the Maize Transcription Factor Genes LC and C1
a Plant Research International, Droevendaalsesteeg 1, P.O. Box 16, 6700 AA Wageningen, The Netherlands 1 To whom correspondence should be addressed. E-mail a.g.bovy{at}plant.wag-ur.nl; fax 31-317-418094
Flavonoids are a group of polyphenolic plant secondary metabolites important for plant biology and human nutrition. In particular flavonols are potent antioxidants, and their dietary intake is correlated with a reduced risk of cardiovascular diseases. Tomato fruit contain only in their peel small amounts of flavonoids, mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. To increase flavonoid levels in tomato, we expressed the maize transcription factor genes LC and C1 in the fruit of genetically modified tomato plants. Expression of both genes was required and sufficient to upregulate the flavonoid pathway in tomato fruit flesh, a tissue that normally does not produce any flavonoids. These fruit accumulated high levels of the flavonol kaempferol and, to a lesser extent, the flavanone naringenin in their flesh. All flavonoids detected were present as glycosides. Anthocyanins, previously reported to accumulate upon LC expression in several plant species, were present in LC/C1 tomato leaves but could not be detected in ripe LC/C1 fruit. RNA expression analysis of ripening fruit revealed that, with the exception of chalcone isomerase, all of the structural genes required for the production of kaempferol-type flavonols and pelargonidin-type anthocyanins were induced strongly by the LC/C1 transcription factors. Expression of the genes encoding flavanone-3'-hydroxylase and flavanone-3'5'-hydroxylase, which are required for the modification of B-ring hydroxylation patterns, was not affected by LC/C1. Comparison of flavonoid profiles and gene expression data between tomato leaves and fruit indicates that the absence of anthocyanins in LC/C1 fruit is attributable primarily to an insufficient expression of the gene encoding flavanone-3'5'-hydroxylase, in combination with a strong preference of the tomato dihydroflavonol reductase enzyme to use the flavanone-3'5'-hydroxylase reaction product dihydromyricetin as a substrate.
Flavonoids form a large group of polyphenolic compounds that occur naturally in plants. Based on their core structure, the aglycone, they can be grouped into different classes, such as chalcones, flavanones, dihydroflavonols, flavonols, and anthocyanins (Figure 1) . To date, >4000 different flavonoids have been identified. This large diversity is attributable to single or combinatorial modifications of the aglycone, such as glycosylation, methylation, and acylation. As a group, flavonoids are involved in many aspects of plant growth and development, such as pathogen resistance, pigment production, UV light protection, pollen growth, and seed coat development (Harborne, 1986  ).
There is increasing evidence to suggest that flavonoids, in particular those belonging to the class of flavonols (such as kaempferol and quercetin), are potentially health-protecting components in the human diet as a result of their high antioxidant capacity (Rice Evans et al., 1995 , 1997; Sugihara et al., 1999; Dugas et al., 2000; Duthie and Crozier, 2000; Ng et al., 2000) and their ability, in vitro, to induce human protective enzyme systems (Cook and Samman, 1996; Manach et al., 1996; Janssen et al., 1998; Choi et al., 1999; Frankel, 1999; Hollman and Katan, 1999; Shih et al., 2000). Based on these findings, it was postulated that flavonoids may offer protection against major diseases such as coronary heart diseases and cancer (Hertog and Hollman, 1996; Steinmetz and Potter, 1996; Trevisanato and Kim, 2000). In addition, several epidemiological studies have suggested a direct relationship between cardioprotection and consumption of flavonols from dietary sources such as onion, apple, and tea (Hertog et al., 1993; Keli et al., 1996). These studies suggest that a systematic increase in the daily intake of certain flavonoids could lead to a 30 to 40% reduction in death by coronary heart diseases.
Based on studies of this type, there is growing interest in the development of food crops enriched with health-protective flavonoids. An excellent candidate for such an approach is tomato, one of the most important food crops worldwide. In tomato, the main flavonol is rutin (quercetin-3-O-rutinoside) (Woeldecke and Herrmann, 1974
Previously, we had characterized the flavonoid content of fruit of the processing tomato variety FM6203 (Muir et al., 2001
In the past decade, a number of regulatory genes involved in the production of flavonoids have been isolated. In general, these genes belong to one of two families of transcription factors, the MYB-type C1 family and the basic helix-loop-helix, MYC-type R family (Dooner et al., 1991 We have expressed the maize LC and C1 genes specifically in the fruit of transgenic tomatoes. In this article, we show that the expression of both genes is required and sufficient to upregulate the flavonoid pathway in tomato flesh, a tissue that normally does not produce any flavonoids. This resulted in a strong accumulation of flavonols and a more modest increase in flavanones, but remarkably, not in the accumulation of anthocyanins. We provide a mechanism to explain the accumulation of these types of flavonoids compared with the anthocyanins reported in other plants.
Approach, Gene Constructs, and Plant Transformation Because we had shown previously (Muir et al., 2001 ) that the flavonoid biosynthesis pathway is not active in tomato fruit flesh, based on both metabolite and gene expression studies, we aimed to upregulate the entire flavonoid pathway in this tissue by expressing the maize transcription factor genes LC and C1 in transgenic tomato plants.
The LC gene was expressed under the control of the fruit-specific tomato E8 promoter, whereas the C1 gene was expressed under the control of either the E8 promoter or the constitutive double 35S promoter of Cauliflower mosaic virus (CaMV). LC and C1 are thought to act in a coordinated manner (Mol et al., 1998
The constructs referred to above were used to transform FM6203 tomato tissue by Agrobacterium tumefaciens–mediated transformation. Transgenic plants obtained after transformation with construct pBBC10, pBBC20, pBBC30, pBBC200, pBBC250, pBBC300, or pBBC350 were numbered from 100, 200, 300, 2000, 2500, 3000, and 3500 onward, respectively.
Analysis of Flavonoid Levels in the Fruit of Transgenic Plants
To determine whether both LC and C1 were required to upregulate the flavonoid pathway, hydrolyzed extracts from whole red fruit of plants transformed with either C1 or LC alone and wild-type plants were analyzed by HPLC. As shown in Figure 5 , the chromatograms of plants transformed with only one of these two transcription factor genes showed no differences compared with untransformed wild-type plants. By contrast, whole fruit extracts of plants transformed with both LC and C1 revealed a clear accumulation of kaempferol. Detailed spectral analysis of the chromatogram peaks in nonhydrolyzed extracts confirmed that in LC/C1 plants, both kaempferol and naringenin glycosides were increased, and that in LC or C1 single-gene transgenic plants, no significant difference in the levels of any tested flavonoids was observed compared with the wild type (data not shown).
Hydrolyzed extracts of whole red fruit were used to quantify the levels of quercetin, kaempferol, and naringenin in LC/C1 plants. Figure 6A shows only the results obtained for 35S-C1/E8-LC+ plants (3000 numbers), because this series was analyzed most extensively. In fruit of transformed plants, kaempferol levels were increased up to 60-fold relative to the mean level in wild-type fruit, whereas quercetin was increased slightly (up to 3-fold) in fruit of only a few plants. When expressed on a whole-fruit basis, the level of naringenin was increased up to twofold in only a few plants (Figure 6A), although all fruit accumulated naringenin in the flesh (Figure 4). This apparent discrepancy is explained by the fact that the naringenin level measured in the whole LC/C1 fruit represents the increased amount of naringenin present in flesh plus the amount of naringenin formed during hydrolysis from naringenin chalcone that is always present in the peel, the latter being highly variable between fruit samples. From the data in Figure 6A, it can be concluded that the accumulation of kaempferol accounted predominantly for the increase in total flavonols (quercetin plus kaempferol) in the fruit (up to 20-fold).
A significant accumulation of kaempferol also was observed for the other three types of LC/C1 plants (data not shown). To compare the effect of the LC leader and the type of promoter controlling C1 gene expression, we determined the levels of kaempferol in hydrolyzed red fruit extracts of 35S-C1/E8-LC- (2000 series), 35S-C1/E8-LC+ (3000 series), and E8-C1/E8-LC- (2500 series). Unfortunately, for E8-C1/E8-LC+ plants (3500 series), only three transformants were obtained; therefore, these plants were not used in this comparison. As shown in Figure 6B, all three types of constructs tested resulted in plants with kaempferol levels ranging from 2 mg/kg fresh weight (equal to control levels) to 40 to 80 mg/kg fresh weight. Apparently, neither the LC leader (LC- versus LC+) nor the type of promoter driving C1 expression (35S versus E8) was a critical determinant for the extent of flavonoid induction in tomato fruit. Nevertheless, the highest kaempferol levels were found in plants transformed with E8-C1/E8-LC- (2500 series). However, we cannot yet conclude that this is attributable to a significant promoter effect, because there was a rather large (twofold to threefold) variation in kaempferol levels within fruit of a single transgenic plant during the year and between clones of the same line (data not shown).
Anthocyanin formation, which has been reported to be induced by LC in various plant species, such as tomato (Goldsbrough et al., 1996 In summary, these results show that both regulatory genes (LC and C1) are required and sufficient to induce the biosynthesis of flavonoids in tomato fruit. This leads to a marked accumulation of kaempferol and naringenin glycosides in the flesh, a tissue that normally does not produce significant levels of any flavonoids.
Stability of the High-Flavonol Phenotype
Flavonoids in Leaves of Transgenic Plants The flavonoid content in mature leaves of homozygous T2 populations of the above-mentioned LC/C1 lines was determined by HPLC analysis of hydrolyzed extracts. As shown in Figure 8 , the level of the main flavonol in leaves, quercetin, was not altered significantly in transgenic leaves (+) compared with azygous leaves (-). However, there was a clear increase in the levels of both kaempferol and naringenin in transgenic leaves compared with their azygous relatives. In leaves of primary transformants containing either LC or C1 alone, there was no significant increase in the level of kaempferol. This finding indicates that, as in fruit, the expression of both LC and C1 is required and sufficient to upregulate the flavonoid biosynthesis pathway in leaves. In all plants analyzed for flavonoids in leaves, the C1 gene was expressed under the control of the constitutive double 35S promoter of CaMV, whereas the LC gene was expressed under the control of the fruit-specific tomato E8 promoter. The requirement for LC in addition to C1 expression to upregulate the flavonoid pathway in leaves suggests that the activity of the E8 promoter is not confined strictly to the fruit but also is significant in leaves. Leaves of LC/C1 plants expressing both LC and C1 under the control of the E8 promoter were not analyzed in this experiment.
Analysis of Anthocyanins in Tomato Leaves Although all transgenic plants were phenotypically similar to the wild type, we observed a clearly increased reddish-purple anthocyanin pigmentation in nodes and old leaves of some LC/C1 plants containing 35S-C1/E8-LC compared with LC/C1 plants containing E8-C1/E8-LC, plants expressing LC or C1 alone, or wild-type plants (Figure 9) .
To compare the anthocyanins produced in LC/C1 leaves with those accumulating in wild-type tomato leaves in response to abiotic stress, the anthocyanin content and composition in extracts of 3-week-old, light-stressed LC/C1 and wild-type seedlings was determined using HPLC/photodiode array/mass spectrometry. In both extracts, the same three major anthocyanin peaks could be identified, corresponding to petunidin 3-(caffeoyl)rutinoside-5-glucoside, petunidin 3-(p-coumaroyl)rutinoside-5-glucoside, and malvidin 3-(p-coumaroyl)rutinoside-5-glucoside (Table 1). Quantification of these peaks, using malvidin 3-glucoside as a standard, revealed that light-stressed LC/C1 seedlings contained threefold to fourfold higher levels of these anthocyanins than light-stressed wild-type seedlings. In agreement with published data (Bongue-Bartelsman and Philips, 1995 ), our HPLC/photodiode array/mass spectrometry measurements revealed that all anthocyanins detected in these colored leaves were of the petunidin and malvidin type and thus were derived solely from the precursor dihydromyricetin. In contrast to our results with mature leaves, in which we observed a clear increase of kaempferol and, occasionally, anthocyanins in LC/C1 plants relative to wild-type plants, these light-stressed LC/C1 and wild-type seedlings contained similarly high levels of both quercetin and kaempferol. This finding suggests that the LC/C1 effect observed in mature leaves and fruit mimics an integral part of the natural induction of the flavonoid pathway in response to abiotic stress.
Analysis of LC and C1 Gene Expression The expression of the introduced LC and C1 genes in transgenic plants was analyzed by real-time quantitative reverse transcriptase–mediated (RT) PCR. For these experiments, we made use of the fluorogenic TaqMan assay on the ABI PRISM7700 sequence detection system. Using this technology, we analyzed transgene expression in fruit of single-gene plants (LC or C1 alone) and double-gene plants (LC and C1 together). As an internal control, we used the tomato cyclophylin gene (CYP), which is expressed constitutively in turning and red tomato fruit, as observed by RNA gel blot analysis (data not shown). Total RNA was isolated from red fruit of the tomato plants mentioned above, and first-strand cDNA was synthesized by reverse transcription. Aliquots of 100 ng of cDNA were used in three TaqMan PCRs with probes for LC, C1, and CYP, respectively. LC and C1 mRNA expression levels were quantified relative to the amount of CYP mRNA. The absolute amount of CYP mRNA varied by <2.5-fold between the different transgenic plants (mean ± SD of Ct values [see Methods] was 20.44 ± 0.34; n = 13), indicating that this gene can be used as an authentic internal control. As shown in Figure 10 , the control plant 004 failed to give a signal with either the C1 probe or the LC probe. By contrast, all transgenic plants tested showed clear expression of the introduced transgenes at the transcriptional level. The C1 single-gene plants (104, 109, and 117) showed only C1 gene expression, and the LC single-gene plants (201, 204, 209, 302, and 305) showed only LC gene expression, whereas all tested LC/C1 double-gene plants (2509, 2511, 2519, and 3031) showed expression of both LC and C1 in their fruit. Although the expression levels of the individual LC and C1 genes in some single-gene transgenic plants were higher than the levels found in some double-gene plants, there was no detectable increase in kaempferol levels in hydrolyzed extracts of any single-gene plant compared with the wild type (Figure 8). These results confirm our previous conclusion that the expression of both LC and C1 together is required to upregulate the flavonoid pathway in tomato fruit.
In the same way, LC and C1 mRNA levels were determined in mature leaves of transgenic and wild-type plants (Figure 10). In the control plant 003, there was no detectable expression of either LC or C1. In the leaves of the C1 single-gene plant 104, there was high C1 expression and, as expected, no LC expression. In leaves of LC/C1 plants containing 35S-C1/E8-LC, high C1 expression and low, but significant, LC expression were observed. These results are consistent with our previous observation that in leaves, as in fruit, the expression of both LC and C1 is required to upregulate the flavonoid pathway. Although the LC gene is expressed under the control of the E8 promoter, its expression in leaves remains sufficient to induce the biosynthesis of flavonoids, provided that C1 is expressed simultaneously by the 35S promoter.
Effect of LC/C1 on the Expression of Flavonoid Pathway Genes
As shown in Figure 11
, there was a clear expression of PAL, F3H, F3'H, FLS, GT, and RT in the peel of wild-type green fruit. Transcript levels of these genes increased during ripening, reaching a maximum level in the turning stage, and then decreased again in the red stage. Expression levels for CHS and CHI were low in green fruit peel and thus may be responsible for the relatively low flavonoid levels found in green fruit (Muir et al., 2001
Because mature leaves of some LC/C1 plants contain both kaempferol- and quercetin-type flavonols and delphinidin-type anthocyanins (Figure 1), this tissue is expected to produce adequate transcript levels of all of the flavonoid structural genes involved. Expression levels in green wild-type and purple LC/C1 mature leaves of all genes tested are shown in Figure 12A . Compared with that in fruit, the LC/C1 effect on flavonoid gene expression is much more modest in leaves (ranging from twofold to sixfold). This is most likely the result of the fact that the flavonoid pathway is already active in wild-type leaves. In addition, we observed much less variation in the transcript levels between genes in leaves (Figure 12A) and fruit (Figure 11), suggesting that all of the genes are expressed at a sufficient level to produce the flavonols and anthocyanins found. To compare gene expression levels in leaves and fruit, we determined the RNA levels of the genes encoding enzymes acting at and around the branch point between the formation of flavonols and anthocyanins (Figure 1) in peel and flesh of turning LC/C1 fruit relative to the levels found in LC/C1 leaves (Figure 12B). This was possible because the two internal controls used showed constant expression levels in leaves and green fruit (CYP) and in the three ripening stages tested (ASR1). In LC/C1 flesh, expression levels of F3H, FLS, DFR, and ANS were in the same range as in leaves, whereas expression levels of F3'H and F3'5'H were 10- to 100-fold lower than those in LC/C1 leaves. This finding suggests that the expression of the latter two genes may be limiting steps in the partitioning of the pathway toward dihydroquercetin and dihydromyricetin in LC/C1 flesh.
In LC/C1 peel, we also observed a low expression level of F3'5'H but a very strong expression of F3'H and FLS compared with that in leaves. This finding suggests that in LC/C1 peel, the production of dihydromyricetin is limited because of the low F3'5'H expression and that there is a strong pull of the pathway toward the production of dihydroquercetin, quercetin, and kaempferol because of the high F3'H and FLS expression. In combination with the apparent substrate preference of the DFR enzyme for dihydromyricetin and of the FLS enzyme for dihydrokaempferol and dihydroquercetin, these expression data suggest that F3'5'H expression is a key factor in the accumulation of anthocyanins in LC/C1 leaves and their absence in fruit.
In this article, we show that the simultaneous expression of the maize transcription factor genes LC, a member of the MYC-type R gene family, and C1, a member of the MYB-type C1 gene family, in fruit of transgenic tomato plants leads to a strong induction of flavonoid biosynthesis in the flesh of the fruit, a tissue that normally lacks significant levels of any flavonoids. On a whole-fruit basis, this resulted in an up to 60-fold increase in the level of the flavonol kaempferol, which was conjugated with different sugars. In addition to these kaempferol glycosides, a significant increase was observed in the level of several naringenin glycosides in the flesh. Both LC and C1 were required and sufficient to induce the biosynthesis of these flavonoids, because plants containing either LC or C1 alone did not differ from control plants with respect to flavonoid levels.
Two versions of the LC cDNA were used in our gene constructs: one with and one without the 5' untranslated region (UTR). The LC 5' UTR contains a small upstream open reading frame that is involved in the translational control of LC expression. It was shown by transient expression studies in maize kernels that the removal of this 5' UTR results in 25- to 30-fold enhanced LC protein expression (Damiani and Wessler, 1993 We aimed to induce the flavonoid biosynthesis pathway specifically in the fruit by expressing either the LC gene or both LC and C1 under the control of the fruit-specific E8 promoter to prevent the possible confounding effects of increased flavonoid levels during tissue culture or subsequent plant growth. Nevertheless, in leaves of E8-LC/35S-C1 plants, a small but clear increase in flavonoids was detectable, whereas in leaves of plants expressing E8-LC or 35S-C1 alone, no increase of flavonoids was detected compared with control leaves. This suggests that in leaves, as in fruit, the expression of both LC and C1 is required to induce the biosynthesis of certain flavonoids. TaqMan gene expression analysis showed that a low but significant expression of the LC gene was detectable in E8-LC/35S-C1 leaves, indicating that the E8 promoter is somewhat leaky or not restricted fully to fruit.
Genes that encode homologs of the maize R and C1 transcription factor families have been expressed ectopically in maize cell lines (Grotewold et al., 1998
To provide an explanation for the accumulation of flavonols and the lack of anthocyanins in our LC/C1 fruit, we investigated the partitioning of the flavonoid pathway toward flavonols and anthocyanins in more detail by combining metabolic information with gene expression data. As shown in Figure 1, dihydrokaempferol (DK) is produced as a product of the F3H enzyme. Using DK as a substrate, the action of the two cytochrome P450 hydroxylases F3'H and F3'5'H leads to the production of two other dihydroflavonols, dihydroquercetin (DQ) and dihydromyricetin (DM). These three types of dihydroflavonols (DK, DQ, and DM) serve as common precursors for the formation of the three types of flavonols (kaempferol, quercetin, and myricetin) through the action of FLS as well as the three types of anthocyanins (pelargonidin, cyanidin, and delphinidin) through the action of DFR. In LC/C1 leaves, all three types of dihydroflavonols (DK, DQ, and DM) must be present, because significant amounts of the derived reaction products kaempferol, quercetin (both flavonols), and delphinidin (anthocyanin) are present. This is in agreement with our observations that in leaves, all genes required for the biosynthesis of DK, DQ, and DM, including F3'H and F3'5'H, are expressed. However, and in accordance with previous findings (Bongue-Bartelsman and Philips, 1995 In LC/C1 fruit peel, at least two types of dihydroflavonols (DK and DQ) must be formed, because their respective reaction products (kaempferol and quercetin) were present in this tissue. This is in agreement with our gene expression data showing that all genes, except CHI, required for the production of DK and DQ, plus the flavonols and anthocyanins derived from them, were expressed. The lack of anthocyanins in fruit peel can be explained by the observation that DFR cannot use DK and DQ as substrates and that the only substrate that can be used by DFR (DM) is not produced, as a result of a very low F3'5'H expression in fruit peel compared with leaves. A similar situation was found in LC/C1 fruit flesh. Despite the induction of DFR, ANS, and FLS, only kaempferol was detected in this tissue. This finding suggests that of the dihydroflavonols, only DK is produced in fruit flesh. The absence of DQ and DM, and thus their respective reaction products quercetin (from FLS activity) and delphinidin (from DFR activity), most likely is attributable to the very low, and LC/C1-insensitive, expression of both F3'H and F3'5'H in fruit flesh compared with leaves. Together, these results lead to the conclusion that expression of the LC and C1 transcription factors in tomato leads to the induction of all of the genes required for the production of flavonols and anthocyanins, except for CHI and the two B-ring hydroxylases F3'H and F3'5'H. The endogenous expression pattern of the F3'5'H gene (a 10- to 100-fold higher level in leaves than in fruit), in combination with the substrate specificity of the tomato DFR enzyme (i.e., DM), are the primary factors determining the absence of anthocyanins in fruit and their presence in leaves of these LC/C1 tomato lines. The observation of anthocyanins in some young green LC/C1 fruit suggests that, at least in these fruit, all flavonoid pathway genes, including F3'5'H, were expressed sufficiently to enable anthocyanin formation at early stages of fruit development. Our results also indicate that the types of flavonols found in fruit peel and flesh and in leaves are determined mainly by the expression of the F3'H gene, in combination with the substrate specificity of the FLS enzyme. In addition to the low F3'5'H gene expression, a strong pull of the pathway toward DQ, quercetin, and kaempferol by F3'H and FLS may be of significance for the absence of anthocyanins in fruit peel as well.
In contrast to our results with E8-LC and E8-LC/35S-C1 plants, Goldsbrough et al. (1996)
Other explanations also are possible. For example, we cannot exclude the possibility that the F3'5'H gene is expressed in ripe cherry tomato fruit as a result of genotypical or physiological differences. Alternatively, the DFR enzyme in cherry tomatoes may have different substrate specificity than the one in our tomato line, such that it is able to use DK or DQ as substrate. It is noteworthy, in this respect, that alteration of a single amino acid in the substrate binding domain of the petunia DFR enzyme was sufficient to alter its specificity for the three dihydroflavonol substrates (Johnson et al., 2001 In conclusion, these results clearly show that ectopic expression of combinations of transcription factor genes is a powerful way to influence metabolic pathways. However, the effects on metabolite content are not easy to predict and can vary considerably between plant species and varieties as a result of gene dosage effects, the endogenous and transgene-induced expression profiles of structural genes, and the substrate specificities of the enzymes involved, which may differ from plant to plant.
Plant Growth Conditions Tomato (Lycopersicon esculentum) line FM6203 and transgenic plants were grown in a containment greenhouse with a 16-h photoperiod and 21/17°C day/night temperatures.
Cloning Procedures and Plasmid Constructions In general, all promoters, structural genes, and terminators used were amplified by PCR with primers containing the desired restriction enzyme site attached to their 5' ends. Restriction sites were chosen in such a way that all promoters were cloned as KpnI-BamHI fragments, all structural genes were cloned as BamHI-SalI fragments, and all terminators were cloned as SalI-ClaI fragments, unless stated otherwise.
Plasmid pFLAP10 (P35S-C1-Tnos) was constructed as follows. First, the Agrobacterium tumefaciens nopaline synthase (NOS) terminator was amplified from plasmid pBI121 (Jefferson et al., 1987
To construct plasmids pFLAP20 (Pe8-LC--Trbc) and pFLAP30 (Pe8-LC+-Trbc), the LC gene and the pea ribulose bisphosphate carboxylase terminator (Trbc) were isolated from plasmid pAL144 (LC-) and pAL65 (LC+), respectively (Lloyd et al., 1992
To make the two-gene constructs pFLAP200 (P35S-C1-Tnos/Pe8-LC--Trbc) and pFLAP300 (P35S-C1-Tnos/Pe8-LC+-Trbc), the inserts of plasmids pFLAP10 and either pFLAP20 or pFLAP30 were cloned behind each other in plasmid pUCM3, a derivative of plasmid pUCAP (van Engelen et al., 1995 To make the two-gene constructs pFLAP250 (Pe8-C1-Tnos/Pe8-LC--Trbc) and pFLAP300 (Pe8-C1-Tnos/Pe8-LC+-Trbc), the inserts of plasmids pFLAP15 and either pFLAP20 or pFLAP30 were cloned behind each other in plasmid pUCM3. First, the insert of pFLAP15 was cloned as a KpnI-ClaI fragment in pUCM3. This resulted in plasmid pFLAP150. Then, the inserts of plasmids pFLAP20 and pFLAP30 were cloned as a NotI-AscI fragment behind the C1 gene in plasmid pFLAP150, resulting in plasmids pFLAP250 and pFLAP350, respectively.
For plant transformation, we made use of the binary vector pBBC3, a derivative of plasmid pGPTV-KAN (Becker et al., 1992
Plant Transformation
Tomato variety FM6203 was transformed by Agrobacterium-mediated transformation of cotyledons (Fillatti et al., 1987
Harvest of Plant Material
Flavonoid Extraction and HPLC Analyses
Absorbance spectra and retention times of eluting peaks were compared with those of commercially available flavonoid standards (Apin Chemicals, Abingdon, UK). It was established that during the hydrolysis step, 100% of aglycones were released from their respective glycosides, whereas >95% of naringenin chalcone was converted chemically into naringenin. Recoveries of quercetin, kaempferol, and naringenin standards added to peel or flesh extracts just before hydrolysis were >90%. The lowest detection limit for flavonoids in the tomato extracts was Anthocyanins in seedlings were extracted in acid methanol (95% methanol plus 5% 1 N HCl; 0.5 g fresh weight in 10 mL). After sonication and centrifugation at 3500g, the residue was extracted twice. The supernatants were combined, methanol was evaporated after adding 5 mL of water, and the remaining water extract was washed with hexane to remove lipid-soluble compounds. Analysis of anthocyanins in the extracts was performed by reverse-phase HPLC (Hewlett-Packard 1100 HPLC, equipped with a Phenomenex Aqua C18 4.6 x 150-mm column, 5 µm, at 35°C; Phenomenex, Torrence, CA) with photodiode array and mass detection. The mass spectrometer was a Finnigan LCQ (ThermoQuest, San Jose, CA) with an electrospray ionization interface. Both the auxiliary and the sheath gas were a mixture of nitrogen and helium. The capillary voltage was 3 V, and the capillary temperature was 195°C. Spectra were recorded in positive ion mode between mass-charge ratios of 120 and 1500. The mass spectrometer was programmed to perform a series of three scans: a full mass spectrum, an MS2 of the most abundant ion in the first scan using a collision energy of 30, and an MS3 of the most abundant ion in the second scan using a collision energy of 30. An aliquot of the aqueous anthocyanin extract was subjected to hydrolysis by mixing (1:1) with 6 N HCl and heating at 100°C in a closed vial purged with nitrogen for 45 min. Separation of this hydrolyzed extract was on a Waters Spherisorb ODS2 column (3 µm; 4.6 x 150 mm; 35°C) using a gradient of acetonitrile in 4.5% formic acid. Quantification of anthocyanins was made from the areas of their peaks recorded at 520 nm compared with an external standard of malvidin 3-glucoside.
TaqMan Analysis
Total RNA was isolated from red fruit and leaves of tomato plants as described (Bovy et al., 1995
SYBR-Green Analysis As an alternative to the use of TaqMan probes, the fluorescent intercalating dye SYBR-Green can be used to monitor the RT-PCR on line with the ABI PRISM7700 sequence detection system. This dye gives a specific fluorescent signal when bound to double-stranded DNA, so fluorescence increases with the formation of PCR product. Although the sensitivity of SYBR-Green is at least as good as that for TaqMan probes, it is necessary that the PCR be specific, because the dye will bind equally well to aspecific PCR amplification products. Therefore, for each primer combination, the specificity of the amplification must be confirmed on an agarose gel. As for TaqMan probes, quantitation of target mRNA levels can be performed relative to an endogenous control RNA using the comparative Ct method. Using sequence information from randomly chosen or specifically isolated tomato cDNAs (data not shown) or from ESTs and cDNAs present in the public domain databases, gene-specific primers were developed for tomato homologs of the genes encoding Phe-ammonia lyase, the flavonoid pathway genes chalcone synthase, chalcone isomerase, flavanone-3-hydroxylase, flavanone-3'-hydroxylase, flavanone-3'5'-hydroxylase, flavonol synthase, dihydroflavonol reductase, and anthocyanidin synthase, and the flavonol glycosyltransferases flavonol-3-glucosyltransferase and flavonol-3-glucoside-rhamnosyltransferase. All of these tomato homologs showed >90% amino acid identity to the established petunia genes. The sequences of all of the primers used are shown in Table 2.
Total RNA was isolated from turning fruit of tomato plants as described previously (Bovy et al., 1995
To compare the expression levels in leaves with those found in fruit, we made use of the constitutive expression of our internal controls in leaves and green fruit (CYP) and in the three ripening stages tested (ASR1). First, expression levels of each gene in turning fruit were expressed relative to their expression in green fruit using ASR1 as a control, according to the equation Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
We thank Ronald Davis for the kind provision of constructs pAL69, pAL144, and pAL71; Bob Cowper for organizing and performing the T1 and T2 trials; Carl Jarman for PCR screening of T1 and T2 seeds for segregation analysis; and Dirk Bosch for critically reading the manuscript.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.004218. Received April 30, 2002; accepted June 26, 2002.
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Abstract
INTRODUCTION
, were performed as described previously (Sambrook et al., 1989
0.1 µg/mL, corresponding to 1 mg/kg fresh weight. Variation between replicate extractions and injections was <5%. 
Ct, were calculated to normalize for the differences in the total amount of cDNA present in each reaction and the efficiency of the RT step. Finally, the target RNA expression level was expressed as a percentage of the control RNA expression level using the equation 2 EXP 
-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907.
