1. Anna Czerednik Early fruit development and its role in tomato fruit production and quality Lab discussion meeting 25-01-06

2. Prof. dr. C. Mariani Prof. dr. G. Angenent Dr. W. Vriezen Dr. R. de Maagd Ing. M. Busscher Enza Zaden, De Ruiter Seeds, H.J. Heinz B.V., SVS B.V., Rijk Zwaan Zaadteelt en Zaadhandel B.V.

3. Utilization Horticultural products grown 3,5 billion EU annually Third biggest tomato exporter in the world

4. Fruit development Endoreduplication Fruit set

5. Approaches/questions The overall objective of this project is to establish which molecular and cell biological parameters in early fruit development co-determine eventual fruit quality, size, and shape. Which genes are involved in fruit growth?

6. Is there a relation between cellular context and fruit quality characteristics? The working hypothesis More cell division at the cost of cell expansion gives better fruit.

7. Can we modify cell division and/or expansion? Increasing the ratio cell division/cell expansion during early fruit development will positively influence taste through its influence on dry matter content and fruit texture.

8. Can we follow/quantify cell division and expansion in a non-destructive manner? The methods to do this allow to follow individual fruits throughout development. Using this plants as a tools it would be possible to determine the effects of genetic variation and various cultivation conditions and practices on division and expansion activities.

9. Goals Identification of genes involved in early fruit development of tomato, and controlling their regulation –cell division –cell expansion –endoreduplication

10. Cell cycle and expression pattern of candidate genes

11. Cdk

12. E2F DP Rb E2F DP Rb P Cdk

13. DEL endoreduplication KRP Cdk

14. KRP P Cdk endoreduplication

15. Candidate genes CDK-Cyc Rb E2F-DP Del KRP

16. The choice of unigenes sequences www.sgn.cornell.edu

17. Gene name GenbankUnigenes Cornell Gene name GenbankUnigenes Cornell CDK-A1 gi|3123614215179 227206 CycB2gi|5420282 238171 241637 CDK-A2 gi|3123616215180 CycD1gi|25989347 222190 CDK-B1 gi|11125683222599 231845 CycD2gi|4160298 238097 CDK-B2 gi|11125685237651 225483 CycD3;1gi|5679622 223956 237649 CDK-C1 gi|15215944226229 229134 CycD3;3gi|6434201 214054 224976 CDK D gi|15419985218632 E2Fgi|6328415 238159 CycA-1 gi|5420274231612 239734 E2Fgi|18399985 gi|6328415 216275 235025 CycA-2 CycA3 CycB1;1 gi|540276 gi|5420278 gi|5420280 242338 235688 223396 229622 239271 Rb KRP DEL gi|6681366 gi|26190464 gi|23899381 gi|23899378 gi|22330789 223873 217044 221733 224188 221655

18. Design of primers

19. Processing tomato M82

20. Day 0 Day 2 Day 5 Day 8 Day 11Day 15 Day 20 Day 25

21. -160 0 C Sample collection

22. Results

23. Diameter of tomato fruits at the early stages of development

24. 0 2 5 8 11 15 20 25 H2O Cyc B2 Uni 238171 Examples PCR expression profiles 0 2 5 8 11 15 20 25 H2O CDK B2 Uni 237651 0 2 5 8 11 15 20 25 H2O Cyc D4 Uni 216435

25. Examples qRT-PCR expression profiles

27. Summary qRT-PCR expression profiles

28. Clustering of expression profiles

29. Conclusions Several candidate genes revealed a clear pattern, either strongly down-regulated or up-regulated during tomato fruit development.

30. Next steps Repeat experiments with the new set of samples (fruits harvested based on size) Histological analysis of pericarp Selections of genes for functional analysis (RNAi and overexpression)

31. Transformation of M82 with binary vectors containing candidate genes Fruit specific promoter:TFM5, expressed in early stages Construction of marker lines to monitor cell division and expansion Cell cycle::YFP and Expansion::GFP transformation to tomato (M82, Moneymaker)