Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato

Abstract

Fruit constitutes a major component of human diets, providing fiber, vitamins, and phytonutrients. Carotenoids are a major class of compounds found in many fruits, providing nutritional benefits as precursors to essential vitamins and as antioxidants. Although recent gene isolation efforts and metabolic engineering have primarily targeted genes involved in carotenoid biosynthesis, factors that regulate flux through the carotenoid pathway remain largely unknown. Characterization of the tomato high-pigment mutations (hp1 and hp2) suggests the manipulation of light signal transduction machinery may be an effective approach toward practical manipulation of plant carotenoids. We demonstrate here that hp1 alleles represent mutations in a tomato UV-DAMAGED DNA-BINDING PROTEIN 1 (DDB1) homolog. We further demonstrate that two tomato light signal transduction genes, LeHY5 and LeCOP1LIKE, are positive and negative regulators of fruit pigmentation, respectively. Down-regulated LeHY5 plants exhibit defects in light responses, including inhibited seedling photomorphogenesis, loss of thylakoid organization, and reduced carotenoid accumulation. In contrast, repression of LeCOP1LIKE expression results in plants with exaggerated photomorphogenesis, dark green leaves, and elevated fruit carotenoid levels. These results suggest genes encoding components of light signal transduction machinery also influence fruit pigmentation and represent genetic tools for manipulation of fruit quality and nutritional value.

Fig. 1.

Genetic mapping of HP1 candidates. (Left) Mapping on the L. pennellii introgression lines of chromosome 2. The chromosome is drawn as an open bar and introgressed segments as solid bars to the right. The bins defined by the introgressions are designated by capital letters to the left. The hp1 locus and bin locations of candidate gene sequences are indicated to the right. (Right) Fine mapping of the hp1 locus and candidate genes in an F₂ population of 7,850 individuals derived from the cross L. esculentum hp1/hp1 × L. pennellii introgression line IL2-1 (Hp1/Hp1); the number of recombinants in the F₂ population between indicated loci is shown on the left.

Fig. 2.

Point mutations at the hp1 locus.

Fig. 3.

Normal, mutant, and transgenic plant phenotypes. R represents the presence of the relevant RNAi construct. (A) Representative 6-day (5-dark-grown days followed by 1 day in light) seedlings showing the additive effect of the hp1 hp2 double mutant. (B) Representative field-grown plants of LeHY5-deficient (LeHY5-Ra) and a nontransgenic segregant (WTa) 15 days after field transplant. (C) Mature leaves from representative field-grown plants from a LeCOP1LIKE-deficient line (LeCL-Ra and a nontransgenic segregant (WTa). (D) Mature green and red ripe fruits from field-grown plants of hp1, LeCOP1LIKE-deficient (LeCL-Ra), WT Ailsa Craig (WT), and LeHY5- deficient (LeHY5-Ra) lines. (E) Seedlings of LeHY5-deficient (Ra), WT Ailsa Craig (WT), and two LeCOP1LIKE-deficient (Ra and Rb) lines grown in white light. (F) Field-grown mature plants of LeHY5-deficient (Ra) and a nontransgenic segregant (WTa). (G) Representative mature green (MG), breaker (BR), and red ripe (RR) fruits, sun-exposed side forward, from field-grown plants of WT Ailsa Craig (WT) and a LeHY5-deficient (Ra) line.

Fig. 4.

Effects of LeHY5 (Left) and LeCOP1LIKE (Right) RNAi on gene expression. RNA gel-blots for LeHY5 and LeCOP1LIKE RNAi T₁ progeny are shown. R represents the presence of the indicated transgene, whereas a, b, and c indicate progeny derived from independent transformation events. ³²P-labeled probes to LeHY5 and LeCOP1LIKE were derived from sequences not used in the RNAi constructs to prevent cross-hybridization with transgene RNA. LeCOP1 is the most similar known homolog of LeCOP1LIKE and was used to demonstrate specificity of LeCOP1LIKE RNAi repression. All filters were stripped and reprobed with an 18S rRNA gene to control for RNA loading.

Fig. 5.

Quantitation of LeHY5 and LeCOP1LIKE repression effects. Bars indicate SE. R indicates the presence of the indicated transgene. (A) Seedling hypocotyl lengths of WT Ailsa Craig (WT), three LeHY5-deficient (HY5-Ra, -b, and -c), and two LeCOP1LIKE-deficient (CL-Ra and -b) lines grown in white light or darkness. (B) Chlorophyll content in mature leaves of three LeHY5 (HY5-Ra,-b, and -c) and two LeCOP1LIKE (CL-Ra and b) RNAi lines. FW, fresh weight. (C) Chlorophyll content in mature green fruits of the same lines indicated in B. L and S designate pericarp tissues taken from the side of the fruit exposed to natural light and shade, respectively. (D) Total carotenoids in red ripe fruits of the same lines indicated in B. AU, arbitrary units.

Fig. 6.

Transmission electron microscopy of tomato fruit pericarp chloroplasts. Typical thylakoids including grana stacks can be observed in WT (cv. Ailsa Craig) (A) and hp1 (B). (D-F) LeHY5 RNAi lines showing deficiencies in both organization and abundance of thylakoids and accumulation of plastoglobuli. Samples shown are from LeHY5-Ra.(C) Relative plastid numbers in these same lines.