cell number control and plant organ size: implications for
TRANSCRIPT
Mei Guo
Pioneer Hi-Bred Int'l Inc, A DuPont Business
Illinois Corn Breeders’ School
Champaign, Illinois, March 8, 2011
Cell Number Control and Plant Organ
Size: Implications for Crop
Improvement and Heterosis
Crop Improvement
Plant and Organ Size
Yield Potential
Cell Number Regulators
Heterosis
1. The Cell Number Regulator (CNR) super family
as negative regulators across diverse species
2. Functional analyses of maize CNR genes:
• Ectopic expression in transgenic maize
• Natural expression regulation
3. Cell number control and plant organ size
• Crop domestication
• Hybrid vigor and crop productivity
• Transgenic manipulation
Outline
BUSINESS CONFIDENTIALPlant and Organ Size Variation in Natural
Populations
Cell number plays a major role in organ size variation in natural populations (Mizukami ,
2001, Curr. Opin. Plant Biol. 4: 533-539 )
• FW2.2 is a cloned QTL, contributing
to 30% of tomato fruit weight variation
• Larger fruits express a lower level,
small fruits express a higher level, of
FW2.2 transcript
• It controls the fruit size by controlling
cell number
• FW2.2 may be involved in the cell
cycle signaling
Frary, et al. 2000, Science
Tomato Fruit Weight Gene (FW2.2) Controls
Fruit Size by Cell Number
The Cell Number Regulator Genes Exist in Diverse Species
Monocot
Expanded
Cell Number
Ca2+
Influx
Fw2.2
ZmCNR1
Animals
Diverse Plants
Fungi
Animals
Algae
Diverse
Plants
PLAC8
Long N-
terminal
Extensions
AtMCA1
AtPCR1,2
AtPCR9
AtPCR8
AtPCR10
ZmCNR2
Dicot
Expanded
(Not CdR)
CL
CC
N-terminal
Extensions
Seed Plants
Maize:
OsPCR1
ZmCNR12
Diverse Plants
Cadmium
Resistance:
1697
99
27
87
97
73
95
82
34
90
39
99
96
23
25
4497
51
99
67 10
80
99
71
36
9686
36
99
63
96
9999 26
28
99
99
99
99
79
8943 18
16
39
99
66
95
79
62
39
40
24
37
98
18
9799
9574
21
23
64
59
9845
16
40
29
54
2676
9796
62
37
87
64
38
19
20
88
10
17
93
9441
99
80
53
64
19
99
99
97
34
40
43
14
993940
21
9460
99
18
15
99
9937
20
Plants
Only
12
The CNR Genes Encode Trans-membrane
Proteins
Computational analysis of the CNR protein functions:
• Most likely plasma membrane associated or extra cellular
• Metal ion interaction or transport: Cd and Ca
The Maize CNR Gene Family Has 13 Members
The ZmCNR1 and ZmCNR2 are the closest orthologs to the tomato
fw2.2 based on sequence homology
LeORFX
ZmORF01
ZmORF02
ZmORF03
ZmORF07
ZmORF09
ZmORF10
ZmORF12
ZmORF05
ZmORF06
ZmORF08
ZmORF04
ZmORF11
Fw2.2
ZmCNR1
ZmCNR2
ZmCNR3
ZmCNR10
ZmCNR9
ZmCNR7
ZmCNR12
ZmCNR5
ZmCNR6
ZmCNR8ZmCNR4
ZmCNR11
ZmCNR1
ZmCNR2
ZmCNR3
ZmCNR4
ZmCNR5
ZmCNR6
ZmCNR7
ZmCNR9
ZmCNR10
ZmCNR13
ZmCNR8
Root
ColeoptileLeafStalk
Apical Meris
tem
Immatu
re EarEmbryo
EndospermPeric
arpSilk
Tassel Spikelet
Pollen
0
100
200
300
400
500
600
700
800
Tra
nscri
pt
Level (P
PM
)
The ZmCNR Members Tissue Expression (MPSS)
ZmCNR1
ZmCNR2
Transgenic
testing
Natural expression
analysis
Functional Understanding of the CNR Genes
Z
Z
Z
0
50
100
150
200
250
Pla
nt H
eig
ht
(cm
)
Transgenic
Control
1 2 3 4 5 6 7
Estim
ate
d B
iom
ass (
g)
TransgenicControl
0200400
600
80010001200
140016001800
1 2 3 4 5 6 7
Ear
length
(cm
)
1 2 3 4 5 6 7
8.0
11.0
14.0
17.0
20.0
23.0
Transgenic
Control
Plant Height
Ear length
Estimated Biomass
EventUbi:ZmCNR1 T3 plants
TG Non TG
Event 2
Event 1
TG Non TG
Over Expressing ZmCNR1 Suppresses Plant and Organ Growth
The Suppressed Plant and Organ Growth Negatively
Correlates with Transgene (CNR1) Expression Level
R2 = 0.73
Estimated biomass
Transgene expression-200
-100
0
100
200
300
400
500
0 100 200 300 400 500 600 700 800
Bio
ma
ss D
iffe
ren
ce
s (
g)
Ea
r le
ng
th d
iffe
ren
ce
(cm
)
TG
-N
on
TG
Transgene expression-2
0
2
4
6
8
10
12R
2= 0.72
0100 200 300 400 500 600 700 800
Ear length
R2= 0.97
Pla
nt h
eig
ht d
iffe
ren
ce
(cm
)
TG
-n
on
TG
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800
Transgene expression
Plant height70
Ubi:ZmCNR1 T3 plants
Higher Expression = Smaller Plant
1-copy events 2-copy events
Ubi::ZmCNR1 T0 plants
Suppressed Plant and Organ Growth Correlates
with Transgene (CNR1) Copy Number
Increased Ear Size from ZmCNR1 Co-Suppression (Event 7)
4
5
3
1
2
6
TG Non TG
Ubi:ZmCNR1 T3 plants
TG Non TG
7
Kernel number/row
34.0
35.0
36.0
37.0
38.0
39.0
40.0
TG Non TG
Ear length
18.5
19
19.5
20
20.5
21
21.5
TG Non TG
Increased Ear Size from ZmCNR1 Co-Suppression (Event 7)
Co-Suppressed ZmCNR1 Expression in Leaf
Tissue (Event 7)
ZmCNR1
tubulin
NT Transgenic plants
ZmCNR1
tubulin
Transgenic plantsNTEvent 7
Co-suppressed event
Event 4—1 of the 6
over expressing events
Ubi::ZmCNR1
Down Regulation of the ZmCNR1 Expression
Enhances Plant Growth
Tubulin
ZmCNR1
Ubi:ZmCNR1 RNAi
Plant Height Ratio of TG to Non TG Control
0.000.200.400.600.801.001.201.40
E.1 E.2 E.3 E.4 E.5
Control TG Control TG
Event 5
Natural Expression Regulation of ZmCNR2
LeORFX
ZmORF01
ZmORF02
ZmORF03
ZmORF07
ZmORF09
ZmORF10
ZmORF12
ZmORF05
ZmORF06
ZmORF08
ZmORF04
ZmORF11
Fw2.2
ZmCNR1
ZmCNR3
ZmCNR10
ZmCNR9
ZmCNR7
ZmCNR12
ZmCNR5
ZmCNR6
ZmCNR8
ZmCNR4
ZmCNR11
ZmCNR2
Transgenically constitutive expression of ZmCNR2 did not
show obvious phenotypes
0
100
200
300
400
500
600
Leaf
Growth activity
hours after pollination2 6 12 24 720
100
200
300
400
500
600Silk
0
20
40
60
80
100
120
10 15 21 24 30 35 40 45
Embryo
days after pollination
Growth activity
0
50
100
150
200
250
300
350
8 12 21 22 30 35 40 45
Endosperm
days after pollination
ZmCNR2 Expression Negatively Correlates
with Tissue Growth Activity (MPSS)
ZmCNR2 Expression Negatively Correlates
with Tissue Growth Activity (RT-PCR)
In different genotypes
0
1
2
3
4
5
6
7
8
9
Ratio o
f C
NR
2/t
ub
ulin
B73 Mo17
Growth activity Growth activity
ZmCNR2
tubulin
ZmCNR2tubulin
The ZmCNR Genes and Hybrid Vigor
1. Does over expressing
ZmCNR1 affect organ
size by altering cell
number or cell size?
2. Is ZmCNR2 expression
reduced in the hybrids
as compared to inbred
parents?
Birchler et al, The Plant Cell 2003
Courtesy of O. Danilevskaya
B73 B/M M/B Mo17
BUSINESS CONFIDENTIALZmCNR1 Reduces Plant and Organ Size: by
Cell Number vs. Cell Size
TG Non TG
Leaf epidermal cell count
Ce
ll co
un
ts p
er
are
a
30.0
60.0
90.0
120.0
150.0
180.0
0.0Event 7 Event 5
TG
Non TG
Leaf position on the plants
(Ear leaf)
Le
af a
rea
(cm
2)
0100
200300
400500
600700
800
-2 -1 0 +1 +2
TGNon TG
Leaf area (cm2)
ZmCNR2 Expression and Hybrid Seedling Vigor
Sh
oo
t d
ry m
ass (
g)
Days after planting (leaf stage)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
6 (V2) 12 (V3) 18 (V4)
B73/Mo17
B73
Mo17/B73
Mo17
Shoot Dry Mass
B73/M
o17
Parent A
20 (V4)
Parent B
22 (V4)
Hi-II (F1)
22 (V4)
0.0
1.0
2.0
3.0
4.0
5.0
Sh
oo
t d
ry m
ass (
g)
Days after planting (leaf stage)
Hi-II
ZmCNR2
Tubulin
B73 Mo17 B73/Mo17 Mo17/B73
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10 CNR2 Expression
(RT-PCR)
Exp
ressio
n le
ve
l(n
orm
aliz
ed
to
tu
bu
lin)
ZmCNR2
Tubulin
Parent A Parent B Hi-II (F1)
0.00
0.20
0.40
0.60
0.80
Expre
ssio
n le
vel
(no
rma
lize
d to
tu
bu
lin)
The ZmCNR Genes and Hybrid Vigor
Birchler et al, The Plant Cell 2003
Inbred Hybrids Inbred
ZmCNR1 affects plant and organ size by cell number,
resembling heterosis effects
ZmCNR2 expression correlates with growth activity
and hybrid vigor
ZmCNR1 transgenic
The ZmCNR Genes, Heterosis Effect
and Crop Improvement
1. The CNR super family as negative cell number regulators
functions in diverse species, and multiple tissue types
2. ZmCNR1 and heterosis both affect organ size by altering cell
number.
3. ZmCNR2 expression correlates with tissue growth activity and
hybrid seedling vigor.
4. ZmCNR and related genes may contribute to native heterosis,
or could mimic heterosis, but may be used to generate more
vigorous and productive crop plants by increasing cell number.
Guo et al, 2010 The Plant Cell 22: 1057–1073
BUSINESS CONFIDENTIAL
Crop
Domestication
Breeding
Hybridization
Tanksley 2004 The Plant Cell; van
der Knaap & S.Tanksley 2003 TAG;
Doganlar et al 2002 Genetics
Birchler et al, The Plant Cell 2003 Guo et al The Plant Cell 2010
(B) (C)(A)
Cell Number Control in Plant Organ Size and
Crop Improvement
Transgenic Crop
Improvement
Acknowledgement
Support:
Bioinformatics
qRT-PCR
Microscopy
Vector Construction
Transformation
Greenhouse
Field Evaluation