1. Tomato Seed Development and Germination Theresa Nguyen Department of Biology Mentor: Dr. Hiro Nonogaki

2. Why Seeds? Agriculture Food source rich in antioxidants Carries genetic information http://union.osu.edu/mgarden/images/tomato.jpg http://birdsandbeesonline.com/tomato.gif

3. Tomato Seed Morphology Embryo Testa Endosperm

4. Seed Germination Endosperm Cap Mechanical Resistance Embryo Growth Potential

5. Seed Germination Radicle Endosperm Cap - - Man — Man — Man — Man — Man — Man — Man Gal Gal — —

6. Endosperm Cap Degradation - - Man — Man — Man — Man — Man — Man — Man Gal Gal —— α-Galactosidase β - Mannanaseβ - Mannosidase β – Mannanase enzyme is encoded by LeMAN2 gene.

7. LeMAN2 Gene TISSUE PRINTING LeMAN2 is highly expressed in the endosperm cap

8. Stage Specific Expression Imbibition Time (h) 12h C 2 C 2 -GATA1 24h36h48h Relative mRNA levels 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 0.15 0.2 0.3 0.35 0.4 0.45 0.5 Imbibition Time (h) MBF 12h24h36h48h Relative mRNA levels 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 Imbibition Time (h) 12h24h 36h 48h Relative mRNA levels 0.005 0.015 0.025 0.035 0.045 0 0.01 0.02 0.03 0.04 0.05 Imbibition Time (h) C 2 C 2 -GATA2 0 0.1 0.2 0.3 0.4 0.5 0.6 12h24h36h48h 0 0.1 0.2 0.3 0.4 0.5 0.6 Relative mRNA levels AS2 Asymmetric Leaves 2 Multi-protein Bridging Factor

9. Stage Specific Expression 0 0.02 0.04 0.06 0.08 0.1 0.12 LeMAN2 Relative mRNA levels 12 24 36 ImbibitionTime (h) 0 0.01 0.02 0.03 0.04 0.05 Relative mRNA levels ImbibitionTime (h) 0 0.01 0.02 0.03 0.04 0.05 Relative mRNA levels 12 24 36 ImbibitionTime (h) C 2 C 2 -GATA2

10. Tissue Specificity Lateral endosperm (LE)Cotyledon (C)Root (R) Endosperm cap (EC)

11. Tissue Specific Expression AS2 0 0.1 0.2 0.3 0.4 Relative mRNA levels EC LE C R Seed Tissues C 2 C 2 -GATA1 0 0.05 0.1 0.15 0.2 0.25 Relative mRNA levels EC LE C R Seed Tissues MBF 0 0.01 0.02 0.03 Relative mRNA levels EC LE C R Seed Tissues C 2 C 2 -GATA2 0 0.01 0.02 0.03 0.04 Relative mRNA levels EC LE C R Seed Tissues Asymmetric Leaves 2 Multi-protein Bridging Factor

12. Tissue Specific Expression LeMAN2 Relative mRNA levels 0 0.01 0.02 0.03 0.04 EC LE C R Seed Tissues LeMAN2 C 2 C 2 -GATA2 0 0.01 0.02 0.03 0.04 Relative mRNA levels EC LE C R Seed Tissues Notice the localization of gene expression in the endosperm cap.

13. Hypothetical Regulation GATA2 gene is upstream of LeMAN2 gene in tomato

14. Amino Acid Sequence Alignment 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 GATA2 (tomato)

15. AGP1 Pathway in Tobacco AGACCAAMyb AGP1

16. Prediction GATA2 is the regulatory protein that binds indirectly to promoter region of LeMAN2 PromoterLeMAN2AGACCAAMyb GATA2 Myb

17. Protein Expression MBP GATA2 GATA2 = 35.993 kD MBP = 50.8 kD 86.793kD pMal vector

18. 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

19. 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

20. Gene Functional Classification There was a total of 166 endosperm cap specific genes

21. 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!