High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1

Abstract

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.

Figure 1.

Scheme of the Flavonoid Biosynthesis Pathway. Only the flavonoid classes relevant to this article are shown (in boxes). ANS, anthocyanidin synthase; C, cyanidin; CHI, chalcone isomerase; CHS, chalcone synthase; D, delphinidin; DFR, dihydroflavonol reductase; DK, dihydrokaempferol; DM, dihydromyricetin; DQ, dihydroquercetin; F3H, flavanone-3-hydroxylase; F3′H, flavanone-3′-hydroxylase; F3′5′H, flavanone-3′5′-hydroxylase; FLS, flavonol synthase; K, kaempferol; M, myricetin; P, pelargonidin; PAL, Phe-ammonia lyase; Q, quercetin; 4CL, 4-coumarate:coenzyme A ligase; C4H, cinnamic acid 4-hydroxylase.

Figure 2.

Constructs Used in this Study. Fusion constructs consisted of the following gene fusions: pBBC10, 35S-C1; pBBC20, E8-LC⁻; pBBC30, E8-LC⁺; pBBC200, 35S-C1/E8-LC⁻; pBBC300, 35S-C1/E8-LC⁺; pBBC250, E8-C1/E8-LC⁻; and pBBC350, E8-C1/E8-LC⁺. C1, maize C1 cDNA; LC, maize LC cDNA (LC⁻, version minus the 5′ leader; LC⁺, version plus the 5′ leader); Pd35s, double 35S promoter of CaMV; Pe8, tomato E8 promoter; Tnos, Agrobacterium nopaline synthase terminator, Trbc: pea ribulose bisphosphate carboxylase small subunit terminator.

Figure 3.

HPLC Results, Recorded at 370 nm, of Hydrolyzed Extracts from the Flesh of Red Fruit of Wild-Type and Transgenic (LC/C1) Tomato Plants. q, quercetin; k, kaempferol; AU, absorption units.

Figure 4.

HPLC Results, Recorded at 280 nm, of Nonhydrolyzed Extracts from the Flesh of Red Fruit of Wild-Type and Transgenic (LC/C1) Tomato Plants. AU, absorption units; K, kaempferol; N, naringenin; WT, wild type.

Figure 5.

HPLC Results, Recorded at 370 nm, of Hydrolyzed Extracts from Whole Red Fruit of Tomato Plants. Samples were from wild-type (A), LC (B), C1 (C), and LC/C1 (D) tomato plants. AU, absorption units; k, kaempferol; q, quercetin.

Figure 6.

Quantification of Flavonoids in Hydrolyzed Extracts of Whole Red Fruit Obtained from Plants Transformed with LC/C1 Constructs. (A) Kaempferol, quercetin, and naringenin levels are shown for plants transformed with 35S-C1/E8-LC⁺ (3000 series). WT, untransformed control wild-type fruit (n = 10). (B) Kaempferol levels are shown in plants transformed with constructs 35S-C1/E8-LC⁺ (3000 series), 35S-C1/E8-LC⁻ (2000 series), and E8-C1/E8-LC⁻ (2500 series).

Figure 7.

Stability of the High-Kaempferol Phenotype. Red fruit were harvested from segregating T1 (A) and homozygous T2 (B) populations of three independent transgenic lines. Three fruit of each plant were pooled, hydrolyzed extracts were prepared, and quercetin and kaempferol levels were determined. The results shown represent mean values obtained for 5 to 10 transgenic plants for each transgenic (+) and azygous (−) line. For comparison, analysis of hydrolyzed extracts from fruit of FM6203 parent plants that did not go through the tissue culture procedure is shown. DW, dry weight.

Figure 8.

Flavonol Levels in Leaves of Homozygous LC/C1 T2 Lines and T0 Lines Transformed with LC or C1 Alone. Leaves were harvested from homozygous T2 populations of three independent transgenic lines. Three leaves of each plant were pooled, hydrolyzed extracts were prepared, and quercetin (A) and kaempferol and naringenin (B) levels were determined. The results shown represent mean values obtained for 5 to 10 transgenic plants for each transgenic (+) and azygous (−) line. Similarly, kaempferol levels were determined in T0 lines transformed with the single-gene constructs pBBC10, pBBC20, and pBBC30 (C). DW, dry weight.

Figure 9.

Phenotypical Analysis of Leaves and Green and Red Fruit of LC/C1 Plants. In plants containing 35S-C1/E8-LC, anthocyanins accumulate in older leaves. In plants expressing both C1 and LC under the control of the fruit-specific E8 promoter, this effect is not seen. Fruit of either type of LC/C1 plants do not accumulate anthocyanins.

Figure 10.

TaqMan Analysis of LC and C1 Gene Expression in Red Fruit and Leaves of Wild-Type and Transgenic Tomato Plants. Results are shown separately for red fruit (A) and leaves (B). Total RNA was reverse transcribed, and aliquots were amplified using primer pairs specific for LC, C1, and CYP (cyclophylin). RNA levels for each gene were expressed relative to the amount of CYP RNA, as described in Methods. The mean value for CYP expression (determined as Ct values) was 20.44 ± 0.34 for all fruit samples (mean ± sd; n = 13) and 18.09 ± 0.22 for all leaf samples (n = 6).

Figure 11.

SYBR-Green RT-PCR Analysis of Flavonoid Pathway Gene Expression in Fruit Peel and Flesh during Ripening of Wild-Type and LC/C1 Tomato Fruit. Total RNA was reverse transcribed, and aliquots were amplified using primer pairs specific for PAL, CHS, CHI, F3H, F3′H, F3′5′H, FLS, DFR, ANS, GT, RT, and the internal control ASR1. RNA levels for each gene were expressed relative to the amount of ASR1 RNA, as described in Methods, and multiplied by 1000. The mean value for ASR1 expression (determined as Ct values) of all fruit samples was 15.69 ± 0.33 (mean ± sd; n = 6). WT, wild type.

Figure 12.

Comparative Analysis of Flavonoid Pathway Gene Expression in Leaves and Fruit of WT and LC/C1 Tomatoes. (A) SYBR-Green RT-PCR analysis of flavonoid pathway gene expression in green wild-type and purple LC/C1 tomato leaves, as described in Figure 11. RNA levels for each gene were expressed relative to the amount of ASR1 RNA and multiplied by 10. Because the ASR1 gene has a 100-fold lower expression in leaves than in fruit, multiplication of the ratios by 10 instead of 1000 (as for fruit) results in values that can be compared directly with those given for fruit. The raw Ct values for ASR1 expression in leaves were 22.67 (wild type) and 22.47 (LC/C1). WT, wild type. (B) Comparison of RNA expression levels of F3H, F3′H, F3′5′H, FLS, DFR, and ANS in turning LC/C1 peel and flesh relative to the levels in LC/C1 leaves. Expression levels in leaves were extrapolated to the levels found in fruit, based on the constitutive expression of CYP in leaves and green fruit (mean ± sd of Ct values is 19.47 ± 0.27; n = 4) and of ASR1 in the three ripening stages tested (mean ± sd of Ct values is 15.69 ± 0.33; n = 6). Expression of each gene is given as a percentage of the expression in leaves. Experimental details are described in Methods.