Résumé des résultats (Abstract)
(Anglais)
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Contribution of Partner 04: Universität Bern
One of the aims of OPTIMISTICK is to increase the sulfur amino acid content in the storage proteins of maize, pea and potato. To achieve this goal, it is necessary to firstly increase the flux through the pathway of assimilatory sulfate reduction, thus opening the possibility for increased formation of cysteine, the end product of assimilatory sulfate reduction, and of glutathione (GSH), which is one of the transport molecules for reduced sulfur of plants. Our efforts in this project have focused on the increase of the flux through the assimilatory sulfate reduction pathway.
During the three years of the project, we have studied enzymes involved in or related to the cysteine biosynthesis, namely, adenosine 5'-phosphosulfate reductase (APR), adenosine 3'-phosphate 5'-phosphosulfate reductase (PAPR) from E. coli, acetyl CoA synthase (ACS) from yeast, serine acetyltransferase (SAT) from E. coli and g-glutamyl-cysteine synthetase (gECS), respectively. In order to investigate the effect of these proteins on the sulfur assimilation pathway we have overexpressed most of them in different plant species, mainly maize, potato and Arabidopsis thaliana in collaboration with P02 and P09. Several transgenic lines of each of the key enzymes mentioned above have been analysed on the molecular, biochemical and physiological level. In all cases it was possible to increase the corresponding enzyme activity. E.g. transgenic Arabidopsis thaliana plants over-expressing the 5'-phosphosulfate reductase showed a ten- to fifteen-fold increase in enzyme activity. Furthermore, the increase in enzyme activity of APR and SAT, respectively, resulted also in a two- to threee-fold elevated levels of cysteine and glutathione in leaves. However, in summary these results indicate that the flux coefficients of APR and SAT are low. Interestingly, there seems to be a threshold level for glutathione since it was not possible to overcome the three-fold increase in all approaches tried so far. Taken together, the results obtained show that production of cysteine and glutathione can be achieved by metabolic pathway engineering.
Further results of importance can be summarised as follows:
§ Substantial reduction of sulfate occurs in the dark both in roots and shoots.
§ Nitrogen availability is essential for a high flux through sulfate assimilation.
§ The flux through the pathway of assimilatory sulfate reduction is high during the light period both in leaves and roots. During the dark period, the flux of sulfur into proteins is 50 and 80% lower in roots and shoots, respectively.
§ Finally, preliminary results indicate that the element distribution in transgenic APR lines is different compared to wild type plants.
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