Anna Czerednik Early fruit development and its role in tomato fruit production and quality Lab discussion meeting

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.

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

Fruit development Endoreduplication Fruit set

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?

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.

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.

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.

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

Cell cycle and expression pattern of candidate genes

Cdk

E2F DP Rb E2F DP Rb P Cdk

DEL endoreduplication KRP Cdk

KRP P Cdk endoreduplication

Candidate genes CDK-Cyc Rb E2F-DP Del KRP

The choice of unigenes sequences

Gene name GenbankUnigenes Cornell Gene name GenbankUnigenes Cornell CDK-A1 gi| CycB2gi| CDK-A2 gi| CycD1gi| CDK-B1 gi| CycD2gi| CDK-B2 gi| CycD3;1gi| CDK-C1 gi| CycD3;3gi| CDK D gi| E2Fgi| CycA-1 gi| E2Fgi| gi| CycA-2 CycA3 CycB1;1 gi| gi| gi| Rb KRP DEL gi| gi| gi| gi| gi|

Design of primers

Processing tomato M82

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

C Sample collection

Results

Diameter of tomato fruits at the early stages of development

H2O Cyc B2 Uni Examples PCR expression profiles H2O CDK B2 Uni H2O Cyc D4 Uni

Examples qRT-PCR expression profiles

Summary qRT-PCR expression profiles

Clustering of expression profiles

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

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)

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)