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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. Embryo.

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tomato seed development and germination

Tomato Seed Development and Germination

Theresa Nguyen

Department of Biology

Mentor: Dr. Hiro Nonogaki

why seeds
Why Seeds?
  • Agriculture
  • Food source rich in antioxidants
  • Carries genetic information

http://union.osu.edu/mgarden/images/tomato.jpg

http://birdsandbeesonline.com/tomato.gif

seed germination
Seed Germination

Embryo Growth Potential

Mechanical Resistance

Endosperm Cap

seed germination1

Gal Gal

- - Man — Man — Man — Man — Man — Man — Man

Seed Germination

Radicle

Endosperm Cap

endosperm cap degradation

Gal Gal

- - Man — Man — Man — Man — Man — Man — Man

Endosperm Cap Degradation

α-Galactosidase

β - Mannanase

β - Mannosidase

β – Mannanase enzyme is encoded by LeMAN2 gene.

leman2 gene

TISSUE PRINTING

LeMAN2 Gene

LeMAN2 is highly expressed in the endosperm cap

stage specific expression

AS2

C2C2-GATA1

0.5

0.5

0.6

0.6

0.45

0.45

0.4

0.4

0.5

0.5

0.35

0.35

0.4

0.4

0.3

0.3

Relative mRNA levels

Relative mRNA levels

0.25

0.3

0.3

0.2

0.2

0.2

0.2

0.15

0.15

0.1

0.1

0.1

0.05

0

0

0

0

12h

24h

36h

48h

12h

24h

36h

48h

C2C2-GATA2

MBF

0.08

0.05

0.07

0.045

0.04

0.06

0.035

0.05

Relative mRNA levels

0.03

0.04

0.025

Relative mRNA levels

0.02

0.03

0.015

0.02

0.01

0.01

0.005

0

0

12h

24h

48h

36h

12h

24h

36h

48h

Imbibition Time (h)

Imbibition Time (h)

Stage Specific Expression

Asymmetric Leaves 2

Imbibition Time (h)

Imbibition Time (h)

Multi-protein Bridging Factor

stage specific expression1

0.05

0.05

0.04

0.04

0.03

0.03

Relative mRNA levels

Relative mRNA levels

0.02

0.02

0.01

0.01

0

0

12 24 36

Imbibition

Time (h)

Imbibition

Time (h)

Stage Specific Expression

0.12

LeMAN2

C2C2-GATA2

0.1

0.08

Relative mRNA levels

0.06

0.04

0.02

0

1224 36

Imbibition

Time (h)

tissue specificity

Endosperm cap (EC)

Lateral endosperm (LE)

Cotyledon (C)

Root (R)

Tissue Specificity
tissue specific expression

AS2

0.4

0.25

C2C2-GATA1

0.3

0.2

0.15

Relative mRNA levels

0.2

Relative mRNA levels

0.1

0.1

0.03

MBF

0.05

0

EC LE C R

0.04

0

C2C2-GATA2

Seed Tissues

EC LE C R

0.02

Relative mRNA levels

Seed Tissues

0.03

Relative mRNA levels

0.01

0.02

0.01

0

EC LE C R

Seed Tissues

0

EC LE C R

Seed Tissues

Tissue Specific Expression

Asymmetric Leaves 2

Multi-protein Bridging Factor

tissue specific expression1

0.04

LeMAN2

0.03

Relative mRNA levels

0.02

0.01

0

EC LE C R

0.04

C2C2-GATA2

Seed Tissues

0.03

Relative mRNA levels

0.02

0.01

0

EC LE C R

Seed Tissues

Tissue Specific Expression

LeMAN2

Notice the localization of gene expression in the endosperm cap.

hypothetical regulation
Hypothetical Regulation
  • GATA2 gene is upstream of LeMAN2 genein tomato
amino acid sequence alignment
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

prediction

Myb

AGACCAA

Myb

GATA2

Promoter

LeMAN2

Prediction
  • GATA2 is the regulatory protein that binds indirectly to promoter region of LeMAN2
protein expression

86.793kD

MBP

GATA2

Protein Expression
  • GATA2 = 35.993 kD
  • MBP = 50.8 kD

pMal vector

results

+

Molecular Marker

209 kD

124

80

49.1

34.8

28.9

20.6

7.1

Results
  • Fusion protein MBP-GATA2 is clearly induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG)

MBP-GATA2

microarray analysis
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
Gene Functional Classification
  • There was a total of 166 endosperm cap specific genes
acknowledgments
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!