2. Introduction: stepping into the science What kind of research is being done on the project? What is an Arabidopsis plant? How does the ABE workshop fit in? What kind of results might you expect?

3. What the heck does that mean? National Sciences Foundation (NSF) funds the ABE workshop through a research project entitled: “Functional Genomics of Protein Disulfide Isomerase Gene Family: Unraveling Protein Folding and Redox Regulatory Networks” What kind of research is being done on the project?

4. “Functional Genomics of Protein Disulfide Isomerase Gene Family: Unraveling Protein Folding and Redox Regulatory Networks”

5. Functional Genomics: New field of biological science Genetics-based Genome: all of the genes encoded in DNA in a living organism. Function: Conduct research to figure out what the genes are doing. What proteins do they encode and what jobs in the cell are they responsible for?

6. What jobs do the proteins do in a cell? 1. Structure: hold things up 2. Enzymes: activity make and burn energy. Stimulate growth and biomass production. 1000’s different enzymes -> unique activities Figure out their activities. ENZ A -----------> B Where the enzyme is located in the cell? Do they need other protein partners to do their job?

7. “Functional Genomics of Protein Disulfide Isomerase Gene Family: Unraveling Protein Folding and Redox Regulatory Networks”

8. ENZYME: Protein Disulfide Isomerase Protein = chain of amino acids. Results from de- coding the mRNA sequence transcribed from DNA. Disulfide: “Two sulfurs” The amino acid containing sulfur is cysteine Protein with 2 cysteines | SH | SH

9. ENZYME: Protein Disulfide Isomerase Isomer: Different substances that have the same components. = Different molecules with same chemical formula Alter chemical bonding --> different “shapes” --> activities and functions. Isomerase: an enzyme that can make different molecular shapes out of the same substance.

10. | SH | SH Protein Disulfide Isomerase (PDI) can make different protein shapes based on altered sulfur bonding -S- S- An Isomer with new activity !

11. Proteins do not do their job unless they are folded correctly So, PDIs fold other proteins correctly in cells. A major responsibility for keeping cells normal, development, metabolism and growth.

12. Protein Disulfide Isomerase (PDI) Gene Family Study all the PDIs in the genome of a small plant. All the PDIs in the same related family. but they go off and have different jobs at various locations in the cell.

13. PDI Protein folding- oxidoreductase PDI = cys Inactive state Active state

14. Oxidation of 2 cysteine sulfhydryls --> disulfide bridge –Remove 2 electrons and 2 H+ Reduction --> breaks bridge --> 2 sulfhydryls –Add 2 electrons and 2 H+ All proteins have to fold to proper states Chemical Mechanism

15. But what about plants???? In Yeast and humans - PDIs located in the endoplasmic reticulum (ER)

16. Arabidopsis thaliana Plants

17. First plant to have its genome completely sequenced – Smallest Plant Genome known -> 26,000 genes but makes a plant!! – 40 days from seed to seed. – Easily genetically engineered. – Easy to knock out genes to see what they do. Arabidopsis

18. There are 11 different PDI’s in Arabidopsis plants

19. Research goals of workshop Learn some recombinant DNA methods Mapping genes that have been tagged by a T-DNA Microscopy to locate where PDIs are using PDI-seeking antibodies.

20. What kind of results might you expect?

21. Ooops, Not this….

22. Any kind of result is a success Learn by doing !!! Have fun while you learn ! Nothing has to work perfectly to be a valuable learning experience.

23. M 1-a 1-b 3- Ⅰ 3- Ⅱ 3- Ⅲ 4- Ⅰ 4- Ⅱ 4- Ⅲ W.T T-DNA Mapping

24. PDI antisera binds to chloroplasts of Arabidopsis

25. Localization to chloroplasts and cell wall

26. Localization to chloroplasts: Red chlorophyll fluorescence

27. Alignment of Arabidopsis PDI2 with Chlamydomonas PDI M N R W N L L A L T L G L LD D D V D A L P A V T V E D A D K L K S L E A D A E V V V V T E D V V F V Q T T S A D V A K A A G L D A V D T V S V V K N F A G E D R A T A V L A T D I D T D S L T A F V KG G A TN E S V G Y E F S L D E L G D D A K S E

28. PDI2 is processed by ER microsomes indicating it has a N-terminal signal peptide destined for ER or secrettion 0 20 60 90 min

29. Transmission electron microscopy: Immunolocalization

30. TEM: Immunolocalization to periphery of starch in chloroplasts

31. TEM: Asymmetry starch biogenesis