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Transcript profiles of transgenic tomato plants indicative of high-flavonoid varieties

Miroarrays have been used to monitor changes in gene expression
- During normal ripening of Microtom fruits and
- In fruits of transgenic plants with altered phenolic profiles.

The following experiments have been carried out:
- Microtom fruit ripening. Microtom fruits, harvested at various stages of ripening were profiled for gene expression changes using microarrays. Based on multivariate analyses it was concluded that the green fruits were most deviating from the coloured fruit stages. Most dramatic gene expression changes occur in the transition from green to turning: groups of “green” genes are down-regulated, whereas many “ripening”-related genes are induced. In the over-ripe stage, a number of genes are down-regulated relative to ripe fruits. This experiment provided us with a good overview of the gene expression changes that occur during normal fruit ripening and the results confirmed the current insight in the molecular and biochemical changes which occur during tomato fruit development.

- Gene expression changes in Ros and Ros/Del fruits (result 33971). Microarrays have been used to find target genes in microtom fruits over-expressing the Antirrhinum transcription factor genes Rosea (Ros) alone or both Ros and the Delila (Del) together. Metabolic analyses revealed that Ros fruits contained increased levels of phenylpropanoids and simple phenolic acids, whereas Ros/Del fruits contained increased levels of both phenylpropanoids, simple phenolic acids and strongly increased levels of delphinidin-type anthocyanins. Microarray analysis confirmed the metabolite analyses and revealed a strong up-regulation of phenylpropanoid and flavonoid genes in Ros and Ros/Del plants. Microarray results were confirmed and extended by real-time SYBR Green RT-PCR analyses. The gene expression results showed that Ros alone leads to up-regulation of phenylpropanoid genes and early flavonoid genes, but not the late flavonoid genes required for the production of anthocyanins. Ros and Del together are sufficient to up-regulate all the genes required for the production of delphinidin-type anthocyanins. The key gene required for the accumulation of anthocyanins in Ros/Del plants is F3’5’H, which is required for the formation dihydromyricitin, the precursor of delphinidin-type anthocyanins. In addition to phenylpropanoid and flavonoid genes we surprisingly observed a strong upregulation of the gene encoding the antimicrobial protein gamma-thionin in Ros/Del tomatoes. Althoug we can not exclude the possibility that the upregulated gamma-thionin gene reveals a secundary effect of the transgenic plants, it is also possible that these trancription factors may activate other metabolic pathways involved in the production of defence-related metabolites.

- Gene expression in XPT/FLS fruits. Comparison of gene expression profiles of XPT/FLS fruits with WT microtom fruits did not reveal any significant changes in gene expression in the transgenic plants analysed.

- Gene expression in F3Hi and CHI fruits. F3Hi fruits are transgenic plants in which the F3H gene is downregulated by an RNAi approach. This resulted in plants whith strongly reduced levels of the flavonols quercetin and kaempferol (A. Bovy, personal communication). Microarray analyses of F3Hi fruit peel revealed that, except for the down-regulated F3H gene, no other significant effects on gene expression were observed in these plants. Similar results were obtained with CHI overexpressing tomato plants, which accumulate high levels of quercetin glucosides in the fruit peel.

- Gene expression in CHSi fruits. In CHSi plants, the complete flavonoid pathway is blocked by RNAi-mediated suppression of both tomato CHS genes. This resulted in flavonoid-lacking tomato fruits with an altered fruit morphology. Microarray analysis revealed that blocking the flavonoid pathway leads to up-regulation of both upstream phenylpropanoid and downstream flavonoid genes, suggesting the existence of metabolic control mechanisms, which regulate the flux through the flavonoid pathway. In addition to phenylpropanoid and flavonoid genes, a number of NAM-family transcription factor genes were upregulated in CHSi plants as well.

The microarray analyses of these transgenic tomato lines revealed a very good insight into the regulation of the flavonoid pathway and the possibilities and limitations of engineering metabolic pathways in general and the flavonoid pathway in particular. The results can be used to design novel strategies for engineering tomato plants with an optimal content of flavonoids, and with minimal pleiotrpoic side effects.
In addition, identified key genes involved in fruit ripening and/or flavonoid production can be used as targets for marker development in breeding programs.

Informations connexes

Contact

Arnaud BOVY, (Senior scientist)
Tél.: +31-317476864
E-mail