anna czerednik early fruit development and its role in tomato fruit production and quality lab...
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Anna Czerednik
Early fruit development and its role in tomato fruit production and
qualityLab discussion meeting 25-01-06
• 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/cellexpansion during early fruit development willpositively influence taste through itsinfluence 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
E2FDP
Rb
P
Cdk
DEL endoreduplicationKRP
Cdk
KRPP
KRP
Cdk
endoreduplication
Candidate genes
• CDK-Cyc
• Rb
• E2F-DP
• Del
• KRP
Gene name
Genbank Unigenes
Cornell
Gene name
Genbank Unigenes
Cornell
CDK-A1 gi|3123614 215179
227206CycB2 gi|5420282 238171
241637
CDK-A2 gi|3123616 215180 CycD1 gi|25989347 222190
CDK-B1 gi|11125683 222599
231845CycD2 gi|4160298 238097
CDK-B2 gi|11125685 237651
225483CycD3;1 gi|5679622 223956
237649
CDK-C1 gi|15215944 226229
229134CycD3;3 gi|6434201 214054
224976
CDK D gi|15419985 218632 E2F gi|6328415 238159
CycA-1 gi|5420274 231612
239734E2F gi|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|6681366gi|26190464
gi|23899381
gi|23899378
gi|22330789
223873
217044
221733
224188
221655
Design of primers
Processing tomato M82
Day 0 Day 2 Day 5 Day 8
Day 11 Day 15 Day 20 Day 25
-1600C
Sample collection
Results
0
5
10
15
20
25
30
35
40
45
day0 day2 day5 day8 day11 day20 day25
dia
mete
r (m
m)
Diameter of tomato fruits at the early stages of development
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
0
10
20
30
40
50
60
70
80
0 2 5 8 11 15 20 25 BR
day
tran
scri
pt
abu
nd
ancy
CDKA1 Uni227206
CDKA1 Uni215179
CDKA2 Uni 215180
0
10
20
30
40
50
60
70
80
90
100
0 2 5 8 11 15 20 25 BR
day
tran
scri
pt
abu
nd
ancy
CDKB1Uni 231845
CDK B1 Uni222599
0
200
400
600
800
1000
1200
0 2 5 8 11 15 20 25 BR
day
tran
scri
pt a
bund
ancy
CDKB2 Uni237651
0
2
4
6
8
10
12
0 2 5 8 11 15 20 25 BR
day
tran
scrip
t abu
ndan
cy
Cyc A1 Uni 216180
CycA3 Uni223396
0
50
100
150
200
250
300
350
400
450
0 2 5 8 11 15 20 25 BR
day
tran
scrip
t abu
ndan
cy
CycA1 Uni231612
CycA2 Uni 242338
0
20
40
60
80
100
120
140
160
180
0 2 5 8 11 15 20 25 BR
day
trans
crip
t abu
ndan
cy
Cyc B2 Uni238171
CycB2 Uni 241637
Examples qRT-PCR expression profiles
Examples qRT-PCR expression profiles
0
5
10
15
20
25
30
35
40
45
0 2 5 8 11 15 20 25 BR
day
tran
scri
pti
on
ab
un
dac
y
Cyc D1 Uni222190
Cyc D2 Uni 238097
Cyc D3 Uni 228249
Cyc D3 Uni224976
0
1
2
3
4
5
6
0 2 5 8 11 15 20 25 BR
day
tra
ns
cri
pt
ab
un
da
nc
y CDK C1Uni 226229
CDK D Uni 218632
0
2
4
6
8
10
12
14
16
18
0 2 5 8 11 15 20 25 BR
day
tran
scri
pt
abu
nd
ancy
E2F Uni238159
E2F Uni216275
E2F Uni235025
E2F Uni235586
0
5
10
15
20
25
30
35
40
0 2 5 8 11 15 20 25 BR
day
tran
scri
pt
abu
nd
ancy
KRP Uni 221733
KRP Uni 224188
KRP Uni 21925
Summary qRT-PCR expression profiles
da
y 0
da
y 2
da
y 5
da
y 8
da
y 1
1
da
y 1
5
da
y 2
0
da
y 2
5
sta
ge
of b
rea
kin
g (
BR
)
CDK A1CDK A1CDK A2CDK B1CDK B1CDK B2CDK CCDK DCycA1CycA2CycA3CycB2CycB2CycD1CycD2CycD2CycD3CycD3CycD3E2FE2FE2FE2FKRPKRPKRP
227206215179215180231845222599237651226229218632231612242338223396238171241637222190238097223956228249224976214054238159216275235025235586221733224188219425
Clustering of expression profiles
10
0
80
60
40
20
0-20
da
y 0
da
y 2
da
y 5
da
y 8
da
y 1
1
da
y 1
5
da
y 2
0
da
y 2
5
sta
ge
of b
rea
kin
g (
BR
)
CDK A2E2FKRPCycD3CycA3CDK CCDK A1CDK A1CDK DCycD1E2FCycD2KRPE2FCycD2CycB2CycA1CDK B2CycB2CycD3CycD3CDK B1KRPCDK B1CycA2E2F
215180216275221733228249223396226229227206215179218632222190238159223956219425235025238097241637231612237651238171214054224976222599224188231845242338235586
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)
