Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes

Abstract

Tomatoes are a principal dietary source of carotenoids and flavonoids, both of which are highly beneficial for human health. Overexpression of genes encoding biosynthetic enzymes or transcription factors have resulted in tomatoes with improved carotenoid or flavonoid content, but never with both. We attempted to increase tomato fruit nutritional value by suppressing an endogenous photomorphogenesis regulatory gene, DET1, using fruit-specific promoters combined with RNA interference (RNAi) technology. Molecular analysis indicated that DET1 transcripts were indeed specifically degraded in transgenic fruits. Both carotenoid and flavonoid contents were increased significantly, whereas other parameters of fruit quality were largely unchanged. These results demonstrate that manipulation of a plant regulatory gene can simultaneously influence the production of several phytonutrients generated from independent biosynthetic pathways, and provide a novel example of the use of organ-specific gene silencing to improve the nutritional value of plant-derived products.

Figure 1

Fruit-specific phenotypes of T₂ generation transgenic plants containing a TDET1 inverted-repeat transgene driven by different promoters. (a) Immature fruits from wild-type (T56) plants. (b) Immature fruits from plant containing P119 promoter construct. (c) Immature fruits from plant containing 2A11 promoter construct. (d) Immature fruits from plant containing TFM7 promoter construct. (e) Fully red-ripe fruits from wild-type plants and transgenic plants containing the three promoter constructs. (f) Cross-sections of fruits shown in e.

Figure 2

Analysis of TDET1 RNA in transgenic plants containing fruit-specific promoter constructs. (a) Real time RT-PCR analysis of leaves (L) and mature green fruits (F) from T56 (wild type) and T₃ generation transgenic plants. TDET1 mRNA abundance is shown relative to mRNA levels in leaves of wild-type plants. Each bar represents three repetitions from each RNA sample (derived from pools of three fruits or leaves per plant, and from two individual plants for each genotype). Error bars representing standard deviations are shown in each case. (b) Semi-quantitative RT-PCR analysis of TDET1 expression in leaves (L) and mature green fruits (F) from T₃ generation plants transformed with fruit-specific promoter constructs. Full length TDET1 was synthesized from RNA extracted from leaves and fruits from the same plants. The HY5 gene was used as a control for cDNA synthesis. (c) Semi-quantitative RT-PCR analysis of TDET1 mRNA levels in different tissues from wild-type (T56) plants. IF, immature green fruit; BR, breaker stage fruit; S, stem; YL, young leaf; OL, old leaf. The HY5 gene was used as control for cDNA synthesis. (d) siRNA analysis of mature green fruits (F) and leaves (L) from transgenic plants containing fruit-specific promoter constructs. TDET1-derived siRNAs migrated the same as a 25-nucleotide DNA oligonucleotide marker. 5S rRNA is shown as loading control. In each panel, the results from three independent T₃ plants are shown.

Figure 3

Quantification of carotenoid and flavonoid contents in red-ripe fruits from T56 (wild type, WT) and different lines of T₂ generation plants containing fruit-specific promoter constructs. (a) Lycopene content. (b) β-carotene content. (c) Flavonoid content. In a and b the data represent mean values and s.e.m. and are derived from samples of three fruits taken from three to five sibling T₂ plants per line (except 2A11-011 (n = 2), TFM7-012 (n = 2) and WT (n = 6)). In c the data represent mean values and s.e.m. and are derived from three to five fruits per plant. Statistical analysis of differences in transgenic lines with respect to WT was performed using Student's t-test.