Amal A.A. Mohamed, Jan M.C. Geuns, Wim Van den Ende & Marc De Ley

2. Molecular aspects of early steps in steviol biosynthesis Amal A.A. Mohamed, Jan M.C. Geuns, Wim Van den Ende & Marc De Ley Proceedings of the 3rd Stevia symposium, organised by EUSTAS July 1st and 2nd 2009

3. History • - In 1999, Richman et al. cloned the first two genes- copalyl diphosphate synthase (CPS) and kuerene synthase (KS) –in the committed steps leading to the synthesis of gibberellins and steviol. • - They declared presence of one copy of CPS and two copies of KS • - They profiled their expression patterns in both old and young leaves using northern blotting and claimed that their expressions are high in old leaves and owed that to the contribution of these two genes in steviol glycosides biosynthesis which present in quantatities of 107 in comparison to gibbrillins. • - They couldn’t interpret the mechanism for regulation between synthesis of gibberellins and steviol gylcosides in the light of their outputs.

4. Aims • - Profiling the expression patterns of these two genes -using real time quantitative polymerase chain reaction (RT-q-PCR) technique- in Stevia rebaudiana plants grown at different light conditions. • - Comparing their expressions between old and young leaves. • - Estimation of the accumulation patterns of the steviol glycosides in these plants.

5. 1.1. Methods for profiling gene expression • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives Nuclease protection assays Northern blotting RT-q-PCR -is the most sensitive technique -to quantify mRNA levels from much smaller samples. -sensitive enough to enable quantitation of RNA from a single cell.

6. 1.2. Biosynthetic pathway to steviol and steviol glycosides Bank accession numbers: CPS: AF034545; KS1-1 & KS22-1: AF097310 & AF097311,respectively • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives

7. 1.3. Designing the primers -For studying the expression by RT-q- PCR, specific characters for primers should be achieved: • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives length 18-25bp No more than 2G/C in 3’ Meting temperature > 50°C No 4 similar bases continously Amplicon about 150bp

8. 1.4. Primers for KS1.1 and KS22-1 in an untranslated region • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives

9. 1.5. Choosing the house keeping gene The most internal controls used in RT-q-PCR • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives are rRNAs B-actin GAPDH cyclophilin 18S rRNA is being recommended as the best internal control because it shows less variance in expression than B-actin and GAPDH.

10. 1.6. Choosing the detector - SYBR green provides the simplest and most economical format for detecting and quantitating PCR. • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives

11. Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives But - For single PCR product reactions with well designed primers, SYBR Green can work extremely well, with spurious nonspecifiec background only showing up in very late cycles.

12. 1.7. Three outputs for succesful Q-PCR reaction: 1- Dissociation curve should has one peak at the same melting temperature • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives 3- The strandard curve for the relation between log of cencentration of different dilution folds and Ct value should has a value of the slope not less than -3.2 and the r2>0.99 2- Amplification plot the different dilutions cross the threshold line at equal distance of cycle number

13. 12/12 light/dark 16 light/8 dark 2.1. Methodology RT • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives RNA extraction RNA cDNA House keeping gene Target gene 1 CPS Target gene 2 (KS1) RT-q-PCR instrumentation Data analysis

14. 3 1 2 5 6 4 1 2 Four weeks young leaves 3 Three weeks young leaves Two weeks young leaves 4 Three weeks old leaves 5 Four weeks old leaves 6 Two weeks young leaves 3.1. Comparison of transcript levels of SrCPSent in both young and old leaves • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives

15. It shows one melting temperature for the standard series 3.2. Dissociation curve showing melting temperature for CPS • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives But! For some samples ( specifically in old leaves), a different meting temperature was noticed.This may suggest a presence of another copy of CPS according to Totté et al. (2003)

16. Young Leaves 3.3. The expression patterns of ent- CPS: • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives The expression declined after the first week in both old and young leaves

17. old leaves young leaves 3.4. The expression patterns of ent-KS: • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives There is no big difference in the KS expression beween young and old leaves d

18. 3.5. Accumulation of different steviol glycosides in comparison to CPS expression patterns in young leaves: (a)16/8 full light, (b) 16/8 shady, ( c )12/12 full light • Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives

19. Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives The expression of SrCPS ent was very small in old leaves in comparison to young leaves. The presence of one copy of the gene to trigger the biosynthesis of steviol and gibberellic acid at the same time with an unknown mechanism of regulation (Richman et al., 1999) is ambiguous. The experiments showed that there was no big variance in SrKS ent expression between old leaves and young leaves and this confirmed the tight control of CPS activity over KS in the pathway (West et al., 1982; Yamaguchi et al., 1996; Hedden, 1999).

20. Introduction • 2. Materials and Methods • 3. Results and Discussion • 4. Conclusion • 5- Future Perspectives To design the primers for RT-q-PCR profiling of KO, KAO & KAH

21. References Hedden, P. (1999) Recent advances in gibberellin biosynthesis. Journal of Experimental Biology 50(334), 553-563. Richman, A. S.; Gijzen, M.; Starrat, A. N.; Yang, Z. and Brandle, J. E.(1999). Plant J. 19, 411-421. Totté, N.; Van den Ende, W.; Van Damme, E. J. M.; Compernolle, F.; Baboeuf, I. and Geuns, J. M. C. (2003) Can. J. Bot. 81, 517-522. West, C.A.; Shen-Miller, J. and Railton, I. D. (1982) Regulation of Kaurene synthase. In: Wareing PF, ed. Plant growth substances 1982. London: Acadmic Press,81-90. Yamaguchi, S.; Saito, T.; Abe, H.; Yamane, H.; Murofushi, N. and Kamiya,Y. (1996).The Plant Journal 10, 203-213.

22. Mr.Tom Struyf Mrs. Hilde Verlinden Mrs. Sofie Van Soest Mr. Stijn Ceunen Mrs. Pegah Maghdooni Bagheri Acknowledgements

23. Thanks for your attention!