11/16/2012
Adapting Kernel Metabolism to Enhance
Cereal Yield Under Adverse Conditions
L. Curtis C. Hannah, Alan Myers, William Tracy, Jon
Stewart , Karen Koch, A. Mark Settles, Donald McCarty,
Tracie Hennen-Bierwagon and Susan Boehlein
University of Florida
Iowa State University
University of Wisconsin
Goals of the project:
Goals of the project:
Identify important, rate limiting, heatlabile, biochemical steps in the seed
Engineer cognate gene to enhance
heat stability of the product or over
express the product
Place transgene into maize and
monitor for enhanced yield
Glucose-1-phosphate + ATP
Follow from a success story
Bt2
Sh2
Bt2
Sh2
ADP-glucose
pyrophosphorylase
ADP-glucose + PPi
ADP-Glucose Pyrophosphorylase
•First unique step in starch
synthesis
ADP-Glucose Pyrophosphorylase
•Heat labile
•Allosteric enzyme
- 3-PGA activates
- Pi anti-activates (inhibits)
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11/16/2012
ADP-Glucose Pyrophosphorylase
- Rate limiting step
Sh2-Rev 6
1.68 kb Ds
Sh2-HS 33
sh2-m1
Greene & Hannah, 1998, PNAS
Greene & Hannah, 1998, Plant Cell
Giroux et al, 1994. PNAS
Iodine staining of AGPase variants
Sh2HS33
His
1
Tyr
333
516
SH2 aa sequence
- enhances glycogen in E. coli
- enhances enzyme activity
- enhances heat stability
- enhances subunit interactions
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11/16/2012
HS33/Rev6 Enhanced seed yield
First Generation
Wheat
Rice
Corn
38%
23%
68%
Large Subunit
- Rev6
- HS33
Smidansky et al., 2002, PNAS
Smidansky et al., 2003, Planta
Hannah et al 2012, Plant Cell
Construct by Maureen Clancy
Maize Yield
9000
1% increase/year
Kg/ha
Temperature during early kernel
development is important
Donald Duvick
Pioneer Hi-Bred
3000
1930
1990
Year of hybrid release
2006
2008
1.4
1.2
0.8
0.6
0.4
0.2
30
31
32
33
34
35
Temperature, C
Trans / Endogenous
1.4
Ratio, seed number
Trans / Endogenous
Ratio, seed number
1.6
1
1.2
1
0.8
0.6
0.4
0.2
31
32
33
34
35
36
37
38
Temperature, C
Average high temperature days 1 – 4 post-pollination
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11/16/2012
Correlation, seed number and temperature,
average high first 4 days post-pollination
Year
2006
2008
+trans
0.8441
0.4476
- trans
0.0460
0.0024
Mosaic small subunit
1
199
MP is more heat stable
475
MP
Maize endosperm
(brittle-2, (bt2) gene
The second transgene tested in the field:
Potato tuber
Genotype
T½ 42 C
T½ 55 C
WT
MP
4.5 min
18.0
0.35
1.4
Boehlein et al 2008b Plant Physiol
Boehlein et al 2005 Plant Physiol
Cross et al 2005 Plant J
Cross et al 2004 Plant Physiol
2007
MP is quite active particularly in the absence of 3-PGA
Construct
Genotype
Kcat -3PGA
WT
5.9
98
MP
32.3
137
Kcat is the number of reactions/active site/second
Ratio ear weights
(substitution lines)
Kcat + 3PGA
WT
WT
0.86
0.86
MP
MP
2.32
2.59
MP is not as dependent on 3-PGA for activity
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11/16/2012
Yield Increase
MP
2.32
2007
MP
2.22
2008
Goals of the project:
Identify important, rate limiting, heatlabile, biochemical steps in the seed
Engineer the gene to enhance heat
stability of the product or over
express the product
Place transgene into maize and
monitor for enhanced yield
Goals of the project:
UniformMu maize
Identify important, rate limiting, heatlabile, biochemical steps in the seed
Identify important steps in the seed
controlled by one gene.
Knock out mutants giving seed
phenotypes.
UniformMu maize
-- A reverse genetic resource
8256 F3 W22 inbred families
-- A reverse genetic resource
-- Transposon insertions with
Robertson’s Mutator
-- Indexed by sequence
-- Mapped in the maize genome
-- Stable, sustainable seed stocks
UniformMu maize:
-- Mutants to go
-- On line now
41,523 sequenced insertions
-- Blast them ----- MaizeGDB.org
> 14,000 genes
-- Browse them -- MaizeGDB.org
-- Request them - MaizeGDB.org
Follow links to Maize
Genetics Cooperation
Stock Center
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11/16/2012
UniformMu maize: Key features
1. Uniform background: W22 inbred
facilitates phenotypic analyses:
1. Uniform background: W22 inbred
}
Mu–off
stable
}
2. Genetic stability:
Mu-activity monitored with
bronze color marker
wildtype
}
}
}
}
Mu–on
mutagenic
BZBZ
BZbz
Mu
bzbz
Mu
MuDR
3. Mapped, heritable insertions: 10.3 per line if a visible phenotype is present
5.3 per line if no visible phenotypes are present
Defined ancestral inserts:
4. Sustainable seed resource
5. Now accessible:
Blast-search genes of interest: MaizeGDB, NCBI or UnformMu.UF-Genomics.org
Get seeds: Link from MaizeGDB.org to Maize Genetics Cooperation Stock Center
Figure 1. Forward Genetics: Gene Insertions co-segregating
with Seed Phenotypes.
DNA-binding transcription factor
embryo lethal
1 cm
5 mm
5 mm
Unknown protein
embryo lethal
1 cm
5 mm
5 mm
Pentatricopeptide Repeat Protein
empty pericarp
Forward Genetics: Ear segregating a
novel shrunken mutant tagged with Mu.
1 cm
5 mm
5 mm
1 cm
5 mm
5 mm
RNA-binding protein
small kernel
1.5 kb
Reverse Genetics
Gene Annotation
Seed phenotype
Alcohol dehydrogenase
Plant phenotype
Co-segregating?
small plant
homozygous
Alcohol dehydrogenase
empty pericarp
No
PEP carboxylase
empty pericarp
6-phosphofructokinase
empty pericarp
6-phosphofructokinase
defective kernel
small plant
No
Enolase
empty pericarp
narrow leaves
N.D.
small plant
No
No
Fructose-PP Aldolase
No phenotype
NAD kinase
Homozygous/BC
completed
Putative sugar
translocator
small plant
Goals of the project:
Identify important, rate limiting, heatlabile, biochemical steps in the seed
Identify important, rate limiting,
biochemical steps in the seed
Possibly Yes
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11/16/2012
Rate limiting step
Rate limiting step
Should exhibit a dosage effect
Weight of a seed with the genotype
+/m/m should be less than that of
the genotype +/+/+
Weight of a seed with the genotype
+/m/m should be less than that of
the genotype +/+/+
+/m female X +/+ male
+/m/m and +/+/+ endosperms
Select 20 heaviest and 20 lightest
seed. Test for non-random
distribution of embryo genotypes.
Rate limiting step
Goals of the project:
Seed weight of a seed with the
genotype +/m/m should be less than
that of the genotype +/+/+
Identify important, rate limiting, heatlabile, biochemical steps in the seed
Identify important, rate limiting, heatlabile, biochemical steps in the seed
+/+ female X +/m male
+/+/m and +/+/+ endosperms
Select 20 heaviest and 20 lightest
seed. Test for non-random
distribution of embryo genotypes.
Central carbon metabolism and cereal endosperm starch biosynthesis
ADP-Glucose
Sucrose/
Glucose
Hexose-P
PEP
ADP-Glucose
STARCH
ADP-Glucose
Hexose-P
OPPP
Triose-P
Metabolic labelling studies show the majority of glucose is metabolized prior
to starch synthesis – Much of the glucose passes through the OPPP
Triose-P
Pentose-P
PLASTID
Generally
assumed major
pathway is
shown with thick
arrows.
Sucrose/
Glucose
Hexose-P
PGD3
Hydrolysis
20 %
Triose-P
PEP
Triose-P
Pentose-P
PLASTID
80 %
TCA
TCA
MITOCHONDRION
MITOCHONDRION
CYTOSOL
Spielbauer et al. (2006) Phytochemistry. 67(14):1460-75.
13C-NMR
111111
OPPP
Glycolysis
111111
STARCH
ADP-Glucose
Hexose-P
CYTOSOL
111000
000111
110000
001111
000011
011000
000110
100000
010000
001000
000100
000010
000001
Spielbauer et al. (2006) Phytochemistry. 67(14):1460-75.
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11/16/2012
PGD3 has an endosperm specific function in maize seeds
Heterozygous Ear
normal
mutant
Translocation Uncovering
mutant mutant
normal endosperm embryo
A
PGD3 is a heat labile enzyme in developing endosperm
Non-concordant, B-A
translocation uncovering
kernels show a mutant
endosperm phenotype.
pgd3
Cannot express in E. coli even when codon optimized for E. coli.
Indicates 6PGDH is needed in
the endosperm.
B
Express with transit peptide, heat stable cytosolic PGD in plastid.
The pgd3 mutant has a loss
of starch in the endosperm
and embryo development is
defective.
dek11
C
bt2
Goals of the project:
Three approaches:
1.)Screen in E. coli at high temperatures
Identify important, rate limiting, heatlabile, biochemical steps in the seed
2.) Evolution:
Engineer the gene to enhance heat
stability of the product or over
express the product
a) Amino acids showing tissue-specific
conservation
b) Amino acids under positive selection
(missense/silent ratios)
Place transgene into maize and
monitor for enhanced yield
3.) Structure: motifs that don’t crystalize
Rate of catalysis at 37oC and 55oC
K
Mutant
K
D
P
R
R
D
E
R
K
L
wt
Sh2-1
Sh2-2
Sh2-3
Sh2-4
Sh2-5
Sh2-6
Sh2-7
Sh2-8
Sh2-9
Sh2-10
Sh2-11
Sh2-12
Sh2-13
Sh2-14
Sh2-15
Sh2-16
Sh2-17
Sh2-18
Sh2-2:25
Sh2-19
Sh2-20
Sh2-21
Sh2-22
Sh2-23
Sh2-24
ⱡ
Normalizedⱡ rate at
37oC
20000
26108
4819
2509
6192
6237
10938
7459
12545
16611
21850
9070
4313
11672
15123
10514
12413
29870
10952
14493
14375
21905
26286
37442
24605
44160
37oC rate as % of
wt
100
131
24
13
31
31
55
37
63
83
109
45
22
58
76
53
62
149
55
72
72
110
131
187
123
221
Normalizedⱡ rate at
55oC
*
*
1075
*
855
*
2214
1906
3655
1533
5980
*
*
732
3108
1095
535
*
*
1512
*
*
*
2282
*
*
% of rate remaining at
55oC
0
0
22
0
14
0
20
26
29
9
27
0
0
6
21
10
4
0
0
10
0
0
0
6
0
0
Rates were normalized for varying enzyme purity using coomassie stained gels and ImageJ
at 55oC was determined from 2.5-7.5 minutes and was linear with time.
◊ Activity
*Activity was <200 nmol/min/mg
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11/16/2012
Km’s are not altered in the mutants.
Evolutionary mutants selected for pyramiding
Mutant
42°C colony
spot staining
T½
0-7.5
min
Activity
at
37°C*
Activity
at 55°C*
3PGA Ka
Activity
-3PGA
Sh2-2
very lt.
brown
12.7
4819
1075
0.59
0
Sh2-10
very lt.
brown
3.0
21850
5980
0.05
2860
Sh2-22
lt. brown
1.8
37442
2282
0.088
0
Sh2-4
very lt.
brown
8.9
6192
855
0.12
0
Sh2--8
lt. brown
9.7
12545
3655
0.14
0
Sh2-14
very lt.
brown
3.1
15123
3108
0.27
0
Sh2-17
dark brown
2.5
29870
<200
0.31
0
WT
brown
3.1
20000
<200
0.28
0
Determination of activity at 370C and
550C
Enzyme
Activity
at 550C
Activity at
370C
Maize
-
25000
-
MP-TI
20,340
12,270
1.65
Sh2-10
6,000
16,500
0.36
Synthetic
24,290
22,400
1.08
550C/370C
•Activity of purified enzyme is measured at 15 min
•All assays were linear up to 15 min
Thanks!
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