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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
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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
Partners
University Nijmegen Plant Research International Companies:
ENZA Zaden De Ruiter Seeds Rijkzwaan Zaadteelt en Zaadhandel BV SVS Holland BV H.J. Heinz BV
Fruit development
Ratio
The focus is on the early events during fruit development
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
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
Fw2.2 is involved in timing of division phase
Cell expansion in fruit is correlated with endoreduplication
Endoreduplication:
DNA synthesis not followed by mitosis
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
Following cell size increase during tomato development
Focussing on pericarp
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
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
Ovule
Mesocarp
Epidermis/exocarp
Vascular
bundle
Vascular
bundle
Some observations: stage 0; periclinal and anticlinal divisionsSome observations: stage 0;
Stage 3: cell division continues, some expansion occurs
Stage 5: exocarp/mesocarp become distinguishable
Stage 8: very large mesocarp cells
Image processing steps
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
Identification of cell division or –expansion specific gene expression
Cornell fruit development experiments
Gene expression analysis using a 12,000 element (representing app. 8,000 genes) cDNA microarray
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
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
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
Processing tomato M82
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
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
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
Fruit-specific overexpression or knock-down of cell cycle genes
Fruit specific promoter TPRP-F1 (TFM7)
Northern blot (Santino et al.)
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
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
788 51111.1
793 51111PL.1
791 521111.2
706 51111PL.2
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
Principal component analysis (PCA)
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
229
692
Reason: overall more cell expansion and slightly more cell layers in the bigger fruit
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
Development of tools for monitoring cell division and expansion during early fruit development
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)
