Fruit constitutes a major component of our diet, providing fiber, vitamins, minerals, and many other phytonutrients that promote good health. Fleshy fruits, such as tomatoes, already contain high levels of several of these ingredients. Nevertheless, efforts have been invested in increasing and diversifying the content of phytonutrients, such as carotenoids and flavonoids, in tomato fruits. Increasing levels of phytonutrients, such as lycopene, is highly justified from the perspective of the lycopene extraction industry due to cost effectiveness reasons. Diversifying phytonutrients, in particular those that contribute to fruit color, could potentially provide an array of attractive colors to our diet. Our major goal was to devise a novel strategy for developing tomato fruits with enhanced levels of phytochemicals known to promote good health with special emphasis on lycopene content. A further important goal was to analyze global gene expression of selected genetic lines produced throughout this study in order is to dissect the molecular mechanisms regulating phytonutrients accumulation in the tomato fruit. To achieve these goals we proposed to: 1. combine, by classical breeding, engineered polyamine metabolism with photomorphogenic high pigment mutants in order generate tomato plant with exceptionally high levels of phytonutrients; 2. use gene transfer technology for genetic introduction of key genes that promote phytonutrient accumulation in the tomato fruit, 3. Analyze accumulation patterns of the phytonutrients in the tomato fruit during ripening; 4. Analyze global gene expression during fruit ripening in selected genotypes identified in objectives 1 and 2, and 5. Identify and analyze regulatory mechanisms of chloroplast disassembly and chromoplast formation. During the 3 years research period we have carried out most of the research activities laid out in the original proposal and our key conclusions are as follows: 1. the engineered polyamine metabolism strategy proposed by the US collaborators can not increase lycopene content either on its own or in combination with an hp mutant (hp-2ᵈᵍ); 2. The hp-2ᵈᵍ affects strongly the transcriptional profile of the tomato fruit showing a strong tendency for up- rather than down-regulation of genes, 3. Ontology assignment of these miss-regulated genes revealed a consistent up-regulation of genes related to chloroplast biogenesis and photosynthesis in hp-2ᵈᵍ mutants throughout fruit development; 4. A tendency for up-regulation was also usually observed in structural genes involved in phytonutrientbiosynthesis; however this up-regulation was not as consistent. 5. Microscopic observations revealed a significantly higher number of chloroplasts in pericarp cells of mature-green hp-2ᵈᵍ/hp-2ᵈᵍ fruits in comparison to their normal fully isogenic counterparts. 6. The relative abundance of chloroplasts could be observed from early stages of fruit development. Cumulatively these results suggest that: 1. the overproduction of secondary metabolites, characterizing hp-2ᵈᵍ/hp-2ᵈᵍ fruits, is more due to chloroplast number rather then to transcriptional activation of structural genes of the relevant metabolic pathways, and 2. The molecular trigger increasing metabolite levels in hp-2ᵈᵍ mutant fruits should be traced at early stage of fruit development.
Modulating phytonutrient content in tomatoes combining engineered polyamine metabolism with photomorphogenic mutants