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Transgenic tomato plants altered in the expression of biosynthetic genes involved in flavonoid biosynthesis

During the funding period major objectives have been the creation and validation of genetic variability in flavonoid accumulation in tomato fruits, the biochemical and molecular characterisation of flavonoid-enriched tomatoes and feeding studies to validate possible health promoting effects of high-flavonoid tomatoes. To design high-flavonoid tomato fruits three general strategies, aimed at overcoming possible bottlenecks in biosynthetic capacity, were followed.

These included (i) increased synthesis of precursors, (ii) inhibition of competing pathways and (iii) ectopic expression of regulatory proteins. To increase carbon flux towards flavonoid biosynthesis it was planned to increase the amount of phosphoenolpyruvat (PEP) and erythrose-4-phosphate in plastids of transgenic tomato plants.

To increase PEP availability in plastids two novel PEP-synthesising pathways based on expression of PGM/enolase (partner 1) and PPDK (partner 3), respectively, and overexpression of the plastidic PEP-translocator (Partner 3) were achieved. PGM/enolase as well as PPDK expression lead to increased PEP levels in leaf plastids of transgenic plants.

To increase plastidic erythrose-4-phosphat content in transgenic tomato plants, over-expression of transketolase (partner 7) and of XPT (partner 3) was attempted. To increase transketolase activity, respective coding regions were isolated from E. coli, spinach, P. patens and tested for functionality in E. coli, P patens and transgenic tomato plants. Activity could be demonstrated in E. coli, however, transgenic tomato plants did not show any increase in transketolase activity. XPT expression in transgenic tomato plants was verified by northernblotting.

Besides attempts to increase PEP and E4P levels, enhancement of the first committed step in shikimate biosynthesis, the condensation of phosphoenoylpyruvate and erythrose-4-phosphate by the action of 3-deoxy-arabino-heptulosonate 7-phosphate synthase, was planned to be increased (partner 7). However, despite several attempts to increase activity of DAHPS in transgenic tomato plants, none of the regenerated plants revealed increased enzyme activities. Since plant and non-plant sources have been tested as gene donor in combination with various promoters, this result can only be explained by rigorous post-translational regulation of and DAHPS activity.

In addition to the discussed approaches, transgenic tomato plants with ectopic expression of flavonol synthase, dehydroflavonol reductase (DFR), isoflavone synthase (IFS) and hydroxycinnamoyl CoA quinate transferase (HQT) have been developed.

In conclusion, numerous transgenic lines altered in expression of individual pathway genes have been created and are available for further analysis.

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Reported by

Friedrich-Alexander Universtity Erlangen-Nuremberg
Staudtstrasse 5
91058 Erlangen
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