Tomato Seed Development
and Germination
Theresa Nguyen
Department of Biology
Mentor: Dr. Hiro Nonogaki
Why Seeds?

Agriculture

Food source rich in
antioxidants

Carries genetic
information
http://union.osu.edu/mgarden/images/tomato.jpg
http://birdsandbeesonline.com/tomato.gif
Tomato Seed Morphology
Embryo
Testa
Endosperm
Seed Germination
Embryo
Mechanical
Growth
Endosperm
ResistanceCap Potential
Seed Germination
Radicle
Endosperm
Cap
Gal
Gal
—
—
- - Man — Man — Man — Man — Man — Man — Man
Endosperm Cap Degradation
α-Galactosidase
Gal
Gal
—
—
- - Man — Man — Man — Man — Man — Man — Man
β - Mannanase
β - Mannosidase
β – Mannanase enzyme is encoded by LeMAN2 gene.
LeMAN2 Gene
LeMAN2 is highly
expressed in the
endosperm cap
TISSUE PRINTING
0.5
Asymmetric Leaves 2
0.4
0.3
0.2
0.1
0
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
12h
24h
36h
Imbibition Time (h)
Multi-protein Bridging
Factor
24h
36h
Imbibition Time (h)
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
48h
MBF
12h
Relative mRNA levels
AS2
0.6
48h
Relative mRNA levels
Relative mRNA levels
Relative mRNA levels
Stage Specific Expression
0.05
0.045
0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
C2C2-GATA1
12h
24h
36h
Imbibition Time (h)
48h
C2C2-GATA2
12h
24h
36h
Imbibition Time (h)
48h
0.05
0.05
0.04
0.04
0.03
0.03
Relative mRNA levels
levels
Relative mRNA
levels
mRNA
Relative
Stage Specific Expression
C2C2-GATA2
0.02
0.02
0.01
0.01
0
0
12
24
36
Imbibition Time
Time (h)
Imbibition
(h)
0.12
0.1
LeMAN2
0.08
0.06
0.04
0.02
0
12
24
36
Imbibition Time (h)
Tissue Specificity
Endosperm cap (EC)
Cotyledon (C)
Lateral endosperm (LE)
Root (R)
0.3
0.2
0.1
0
EC
LE
C
Seed Tissues
R
Multi-protein Bridging Factor
0.03
MBF
0.02
0.01
0
EC
LE
C
Seed Tissues
R
0.25
C2C2-GATA1
0.2
0.15
0.1
0.05
Relative mRNA levels
AS2
0.4
Relative mRNA levels
Relative mRNA levels
Asymmetric Leaves 2
Relative mRNA levels
Tissue Specific Expression
0
EC
0.04
LE
C
Seed Tissues
R
C2C2-GATA2
0.03
0.02
0.01
0
EC
LE
C
Seed Tissues
R
Tissue Specific Expression
0.04
LeMAN2
0.03
0.02
0.01
0
EC
LE
C
Seed Tissues
R
Relative mRNA levels
Relative mRNA levels
LeMAN2
0.04
C2C2-GATA2
0.03
0.02
Notice the localization of gene
expression in the
0.01
endosperm cap.
0
EC
LE
C
Seed Tissues
R
Hypothetical Regulation

GATA2 gene is upstream of LeMAN2
gene in tomato
Amino Acid Sequence Alignment
GATA2
(tomato)
AGP1
(tobacco)
http://www.plantcare.com/oldSite/httpdocs/images/namedImages/Tomatoes.jpg
http://upload.wikimedia.org/wikipedia/commons/a/ae/Nicotiana_Tobacco_Plants_1909px.jpg
AGP1 Pathway in Tobacco
AGP1
AGACCAA
Myb
Prediction

GATA2 is the regulatory protein that binds indirectly
to promoter region of LeMAN2
GATA2
AGACCAA
Promoter
Myb
Myb
LeMAN2
Protein Expression


GATA2 = 35.993 kD
MBP = 50.8 kD
86.793kD
pMal
vector
Results

Fusion protein MBP-GATA2 is clearly induced with
Isopropyl β-D-1-thiogalactopyranoside (IPTG)
Molecular
Marker
209 kD
124
80
49.1
34.8
28.9
20.6
7.1
–
+
MBP-GATA2
Microarray Analysis
Gene Chip®
 Massive approach versus gene-by-gene


Tomato chip contains ≈10,000 genes
http://www.colorado.edu/news/reports/renewable
-energy/images/genechip.jpg
Gene Functional Classification

There was a
total of 166
endosperm
cap specific
genes
DNA binding
(Transcription
Factors) 4%
Kinase 3%
Cell-wall related
12%
Metabolism 10%
Phosphatase
1%
Unclassified
68%
Peroxidase 2%
Acknowledgments




Dr. Kevin Ahern
Howard Hughes Medical Institute
Dr. Hiro Nonogaki
Seed Biology Lab








Wioletta Pluskota, Ph.D
Masa Asahina, Ph.D
George W. Bassel II, Ph.D
Arun Kumar
Tammy Chan
Jing Sun
Jen Coppersmith
Natalya Goloviznina
Thank you!