![Page 1: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/1.jpg)
Early fruit development
and its role in tomato fruit production and quality
STW Project NPB.6705 BU Meeting 13 June 2006
![Page 2: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/2.jpg)
Partners
University Nijmegen Plant Research International Companies:
ENZA Zaden De Ruiter Seeds Rijkzwaan Zaadteelt en Zaadhandel BV SVS Holland BV H.J. Heinz BV
![Page 3: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/3.jpg)
Fruit development
Ratio
The focus is on the early events during fruit development
![Page 4: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/4.jpg)
Goals
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
![Page 5: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/5.jpg)
Starting hypothesis
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
![Page 6: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/6.jpg)
Fw2.2 is involved in timing of division phase
![Page 7: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/7.jpg)
Cell expansion in fruit is correlated with endoreduplication
Endoreduplication:
DNA synthesis not followed by mitosis
![Page 8: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/8.jpg)
Early in fruit development, there is a switch from mitosis to endoreduplication
Endoreduplication
E2F
DP
RbP
P
P
E2FDP
Rb
Progression of the cell cycle is regulated by cyclin’s and cdk’s
![Page 9: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/9.jpg)
Following cell size increase during tomato development
Focussing on pericarp
![Page 10: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/10.jpg)
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 2.5-3.0 mm. 3: diameter (equator) fruit is 3.2-4.2 mm. 4: diameter (equator) fruit is 4.5-6.0 mm. 5: diameter (equator) fruit is 7.0-9.0 mm 6: diameter (equator) fruit is 20.0-23.0 mm 7: diameter (equator) fruit is 32.0-40.0 mm 8: diameter (equator) fruit is 54.0-60.0 mm
![Page 11: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/11.jpg)
Equatorial diameter of fruits (mm). Average of 4 fruits.
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
0 1 2 3 4 5 6 7 8
Stage
![Page 12: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/12.jpg)
Ovule
Mesocarp
Epidermis/exocarp
Vascular
bundle
Vascular
bundle
Some observations: stage 0; periclinal and anticlinal divisionsSome observations: stage 0;
![Page 13: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/13.jpg)
Stage 3: cell division continues, some expansion occurs
![Page 14: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/14.jpg)
Stage 5: exocarp/mesocarp become distinguishable
![Page 15: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/15.jpg)
Stage 8: very large mesocarp cells
![Page 16: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/16.jpg)
Image processing steps
![Page 17: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/17.jpg)
Distribution of cell size classes
12
34
56
78
9
10
11
Mean 0
Mean 1
Mean 2
Mean 3
Mean 4
Mean 5
Mean 6Mean 7
Mean 80%
5%
10%
15%
20%
25%
30%
35%
40%
Size class, 2-fold increments,
0.0002-0.2 mm2
Stage
![Page 18: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/18.jpg)
Identification of cell division or –expansion specific gene expression
![Page 19: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/19.jpg)
Cornell fruit development experiments
Gene expression analysis using a 12,000 element (representing app. 8,000 genes) cDNA microarray
![Page 20: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/20.jpg)
PCA of all Cornell ratio’s, relative to 7 DAP
0 20 40 60 80
-40
-20
0
20
40
7DAP
17DAP27DAP
39DAP
41DAP
42DAP
43DAP
47DAP52DAP57DAP
![Page 21: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/21.jpg)
Selection of major changes DAP 7-39 (MG)
100
50
0-50 7D
AP
17D
AP
27D
AP
39D
AP
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.l.......................................................................................................................
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2log ratios of -4 to +4
`7 17 27 39
![Page 22: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/22.jpg)
Cell cycle genes are under-represented on the Cornell array
Gene name Alt. name Genbank Unigene #ESTs On array Protein motif FunctionCyclinscycA1;1 AJ243251 U231612/239734 2/1 0/0cycA2; 1 AJ243252 U235688 1 0cycA3;1 AJ243253 U229622/223396 2/4 2/1cycB1;1 AJ243254 U239271 1 0cycB2;1 AJ243255 U238171/241637 1/1 0 Strongly up in early dev.;cycB3 -cycD1 CAC15503 U222190 5 2cycD2.1 - U238097 1 0cycD3.1 CAB51788 U223956 4 0 Strongly up in early development and later in gelcycD3.2 CAB60837 U223044 3 0cycD3.3 CAB60838 U214054 38 0cycD4cycD5cycD6cycD7cycH
CDK'scdkA1 (p34-)cdc2 Y17225 ? PSTAIRE M-phase associated histone H1 kinasecdkA2 cdc2 Y17226 U215180/215179 22/3 12/0 PSTAIREcdkB1;1 cdc2 AJ297916 U222599 5 0 PPTALRE Unique for plants; induces mitosis; dominant neg. mutations availablecdkB2;1 AJ297917 U225483/237651 3/1 0 PPTTLRE idem; both expressed early in dev and later in gelcdkC;1 AJ294903 U229134/226229 2/3 0 PITAIREcdkD - U218632 10 1cdkE -cdkF
CDK subunit proteinscks1 docking factors for cdk substrates and regulatorscks2
E2FE2Fb - U238159 1 0 Transcription factor; induces G1-S transition? - U235025 1 0
DELDEL3 - U221655 6 0 E2F-like repressor; does not dimerize with DP
DP- U223495 4 0 Dimerization partner for E2F
RbU223873 4 0 Binds and inhibits E2F/DP in phosphorylated state
OtherWEE1 LeWEE1 AJ715533 U224768 4 1 Cell cycle inhibiting phoshorylation of CDK's
Krp1 - U217044 14 4 cyclin/cdk-inhibitorsKrp2Krp3 LeKRP2 CAD29649 U221733 6 0
LeKRP1 CAD29648 U224188 4 0- - U219425 9 0
Krp4Krp5Krp6Krp7
![Page 23: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/23.jpg)
Processing tomato M82
![Page 24: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/24.jpg)
Developmental stages of fruit
1 : diameter 1.8-2 mm – pollination 2 : diameter 2.1-2.2 mm – 1 DAP 3: diameter 2.3-2.5 mm – 2 DAP 4: diameter 2.6-3 mm – 3 DAP 5: diameter 4- 4.9 mm – 4-5 DAP 6: diameter 5.5-6 mm – 5 DAP 7: diameter 6.5-7.3 mm – 6 DAP 8: diameter 7.6-9.4 mm – 7-9 DAP 9: diameter 11-11.7 mm – 10-11 DAP 10: diameter 16.4-16.8 mm – 11 DAP 11: diameter 18.8-20.8 mm – 12 DAP 12: diameter 22.1-25 mm – 13-15 DAP 13: diameter 25.4-28 mm – 15-19 DAP 14: diameter 37-38.8 mm – 25 DAP 15: diameter 40-48 mm – mature green
0
3
5
10
11
![Page 25: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/25.jpg)
0
1
2
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
4
8
12
16
20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 150
5
10
15
20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CDKA2CDKA1 CDKB1
0
50
100
150
200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CDKB2
0
5
10
15
20
25
1 3 5 7 9 11 13 15
CycA1
0
5
10
15
20
25
30
35
40
45
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CycA2
0
200
400
600
800
1000
1200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CycB1
0
2
4
6
8
10
12
14
16
18
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
CycB2
0
2
4
6
8
10
12
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
CDKD
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CDKC
0
2
4
6
8
10
12
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CycD1
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cyc D2
0
100
200
300
400
500
600
700
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CycD3
RT-PCR analysis of cell cycle gene expression
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Conclusions
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
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Fruit-specific overexpression or knock-down of cell cycle genes
![Page 28: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/28.jpg)
Fruit specific promoter TPRP-F1 (TFM7)
Northern blot (Santino et al.)
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Constructs for fruit-specific over-expression or for suppression by RNAi
X X
TPRP-F1 promoter T NOSatt R1 att R2Gateway cassette
att L1 att L2ORF of interest
NPT II
X X
TPRP-F1 promoter T OCSatt R1 att R2Gw cassette NPT IIatt R2 att R1Gw cassette
X X
intron
att L1 att L2partial mRNA att L2 att L1partial mRNA
![Page 30: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/30.jpg)
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
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788 51111.1
793 51111PL.1
791 521111.2
706 51111PL.2
![Page 32: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/32.jpg)
Cell sizes, DRS
1
3
5
7
9
11
112 22
9 434 43
5 556 56
0 597 69
2 824
BP
28
0%
5%
10%
15%
20%
25%
30%
Size class
Variety
Pericarp cell sizes; DRS
![Page 33: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/33.jpg)
Principal component analysis (PCA)
![Page 34: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/34.jpg)
DRS; in order of increasing cell size
pericarp thickness
0
1
2
3
4
5
6
7
8
9
10
692.169
2.2
aver
age
824.182
4.2
aver
age
112.111
2.2
aver
age
560.156
0.2
aver
age
597.159
7.2
aver
age
556.155
6.2
aver
age
435.143
5.2
aver
age
434.143
4.2
aver
age
229.122
9.2
aver
age
BP28.1
BP28.2
aver
age
weight
0
50
100
150
200
250
692.169
2.2
aver
age
824.182
4.2
aver
age
112.111
2.2
aver
age
560.156
0.2
aver
age
597.159
7.2
aver
age
556.155
6.2
aver
age
435.143
5.2
aver
age
434.143
4.2
aver
age
229.122
9.2
aver
age
BP28.1
BP28.2
aver
age
Smaller cells, thinner pericarp, smaller fruit
![Page 35: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/35.jpg)
229
692
Reason: overall more cell expansion and slightly more cell layers in the bigger fruit
![Page 36: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/36.jpg)
DRS; in order of increasing cell size
pericarp thickness
0
1
2
3
4
5
6
7
8
9
10
692.169
2.2
aver
age
824.182
4.2
aver
age
112.111
2.2
aver
age
560.156
0.2
aver
age
597.159
7.2
aver
age
556.155
6.2
aver
age
435.143
5.2
aver
age
434.143
4.2
aver
age
229.122
9.2
aver
age
BP28.1
BP28.2
aver
age
weight
0
50
100
150
200
250
692.169
2.2
aver
age
824.182
4.2
aver
age
112.111
2.2
aver
age
560.156
0.2
aver
age
597.159
7.2
aver
age
556.155
6.2
aver
age
435.143
5.2
aver
age
434.143
4.2
aver
age
229.122
9.2
aver
age
BP28.1
BP28.2
aver
age
Smaller cells, same pericarp thickness, yet bigger fruit
![Page 37: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/37.jpg)
Development of tools for monitoring cell division and expansion during early fruit development
![Page 38: Early fruit development and its role in tomato fruit production and quality STW Project NPB.6705 BU Meeting 13 June 2006](https://reader034.vdocument.in/reader034/viewer/2022050704/5697bfde1a28abf838cb2434/html5/thumbnails/38.jpg)
In vivo monitoring of mitotic and expansion activity with phase-specific promoter-reporter gene fusions
as tools for following the effects of genes and environment on both processes and on the final
outcome (fruit cell size and number)
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