1. STW Project NPB.6705 BU Meeting 13 June 2006
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Early fruit development and its role in tomato fruit production and quality
STW Project NPB.6705 BU Meeting 13 June 2006

2. Partners University Nijmegen Plant Research International Companies:
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University Nijmegen
Plant Research International
Companies:
ENZA Zaden
De Ruiter Seeds
Rijkzwaan Zaadteelt en Zaadhandel BV
SVS Holland BV
H.J. Heinz BV

3. Fruit development Ratio
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Ratio
The focus is on the early events during fruit development

4. Goals
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Identification of genes involved in early fruit development of tomato, and controlling their regulation
cell division
cell expansion
differentiation
endoreduplication
Modelling fruit development
The relation between division/expansion-ratio and fruit quality
Ratio

5. Starting hypothesis
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More cell division at the cost of cell expansion gives better fruit
We propose that the ratio cell division/expansion may vary in different genetic backgrounds or mutants without having an obvious effect on ripe fruit size but with potentially substantial effects on quality traits

6. Fw2.2 is involved in timing of division phase
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7. Cell expansion in fruit is correlated with endoreduplication
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Endoreduplication:
DNA synthesis not followed by mitosis

8. Progression of the cell cycle is regulated by cyclin’s and cdk’s
Early in fruit development, there is a switch from mitosis to endoreduplication
Progression of the cell cycle is regulated by cyclin’s and cdk’s
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Endoreduplication
E2F DP Rb P
E2F DP Rb P P

9. Following cell size increase during tomato development
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Following cell size increase during tomato development
Focussing on pericarp

10. 25/04/2017
Developmental series of round fruit from CBSG green house experiment (in duplicate)
0: fruit from open flowers, diameter and weight not measured.
1: fruit from pollinated flowers, diameter and weight not measured.
2: diameter (equator) fruit is mm.
3: diameter (equator) fruit is mm.
4: diameter (equator) fruit is mm.
5: diameter (equator) fruit is mm
6: diameter (equator) fruit is mm
7: diameter (equator) fruit is mm
8: diameter (equator) fruit is mm

11. Equatorial diameter of fruits (mm). Average of 4 fruits.
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Equatorial diameter of fruits (mm). Average of 4 fruits.

12. Some observations: stage 0; periclinal and anticlinal divisions
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Some observations: stage 0;
Some observations: stage 0; periclinal and anticlinal divisions
Epidermis/exocarp
Vascular
bundle
Mesocarp
Vascular
bundle
Endocarp
Ovule

13. Stage 3: cell division continues, some expansion occurs
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Stage 3: cell division continues, some expansion occurs

14. Stage 5: exocarp/mesocarp become distinguishable
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Stage 5: exocarp/mesocarp become distinguishable

15. Stage 8: very large mesocarp cells
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16. Image processing steps
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17. Distribution of cell size classes
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18. Identification of cell division or –expansion specific gene expression
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Identification of cell division or –expansion specific gene expression

19. Cornell fruit development experiments
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Gene expression analysis using a 12,000 element (representing app. 8,000 genes) cDNA microarray

20. PCA of all Cornell ratio’s, relative to 7 DAP
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-40
-20
7DAP
17DAP
27DAP
39DAP
41DAP
42DAP
43DAP
47DAP
52DAP
57DAP

21. Selection of major changes DAP 7-39 (MG)
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2log ratios of -4 to +4

22. Cell cycle genes are under-represented on the Cornell array
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Cell cycle genes are under-represented on the Cornell array

23. Processing tomato M82
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24. Developmental stages of fruit
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1 : diameter mm – pollination
2 : diameter mm – 1 DAP
3: diameter mm – 2 DAP
4: diameter mm – 3 DAP
5: diameter mm – 4-5 DAP
6: diameter mm – 5 DAP
7: diameter mm – 6 DAP
8: diameter mm – 7-9 DAP
9: diameter mm – DAP
10: diameter mm – 11 DAP
11: diameter mm – 12 DAP
12: diameter mm – DAP
13: diameter mm – DAP
14: diameter mm – 25 DAP
15: diameter mm – mature green 3 5 10 11

25. RT-PCR analysis of cell cycle gene expression
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RT-PCR analysis of cell cycle gene expression
CDKA1
CDKA2
CDKB1
CDKB2
CDKC
CDKD
CycA1
CycA2
CycB1
CycB2
CycD1
Cyc D2
CycD3

26. Conclusions
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Several non-phase specific (at the transcriptional level) cell cycle genes have been identified
Several putative mitosis-specific cell cycle genes have been identified
No expansion-specific cell cycle genes have been identified yet

27. Fruit-specific overexpression or knock-down of cell cycle genes
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Fruit-specific overexpression or knock-down of cell cycle genes

28. Fruit specific promoter TPRP-F1 (TFM7)
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Northern blot (Santino et al.)

29. Constructs for fruit-specific over-expression or for suppression by RNAi
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TPRP-F1 promoter
att R1
Gateway cassette
att R2
T NOS
NPT II X X
att L1
ORF of interest
att L2
TPRP-F1 promoter
att R1
Gw cassette
att R2
intron
att R2
Gw cassette
att R1
T OCS
NPT II X X X X
att L1
partial mRNA
att L2
att L2
partial mRNA
att L1

30. Relation fruit quality & cell size and number
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Relation fruit quality & cell size and number
Mature green fruits from 10 varieties of each of 3 companies were collected and embedded
Sectioning, staining, photography, and data acquisition
Analysis ongoing

31.
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PL.1
PL.2

32. Cell sizes, DRS
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33. Principal component analysis (PCA)
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34. DRS; in order of increasing cell size
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Smaller cells, thinner pericarp, smaller fruit

35. Reason: overall more cell expansion and slightly more cell layers in the bigger fruit
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692
229

36. DRS; in order of increasing cell size
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Smaller cells, same pericarp thickness, yet bigger fruit

37. 25/04/2017
Development of tools for monitoring cell division and expansion during early fruit development

38. In vivo monitoring of mitotic and expansion activity with phase-specific promoter-reporter gene fusions
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as tools for following the effects of genes and environment on both processes and on the final outcome (fruit cell size and number)

39. 25/04/2017