1. Delineating the relationship between the chloroplast and the vacuole during natural leaf senescence in Arabidopsis. Ian Evans 4/28/08 Biol 466H

2. Defining Senescence in Plants Foliar senescence is the final stage of leaf development in which nutrients are remobilized to younger tissues

3. Lim et al. (2007) Annual Review of Plant Biology 58:122. Senescence is tightly regulated

4. Chlorophyll degradation is well characterized, but degradation of the LHC proteins that bind Chl is not well understood. Hörtensteiner, S (2006) Annual Review of Plant Biology 57:55. Chloroplast (Cp) Senescence

5. Spatial Relationships in Senescence

6. Evidence for Vacuolar Degradation Endopetidases are present in vacuole and their activity is upregulated in senescence TEMs show Cps in the center of the cell during senescence but not during normal growth

7. Wittenbach et al. Plant Physiol. 1982 69:98 Wheat Mesophyll Cell

8. Minamikawa et al. Protoplasma. 2001 218:144 Bean Mesophyll Cell

9. Evidence for Internal Degradation Upregulation of Cp-localized proteases Initial Chl catabolism occurs in the stroma Early literature shows internal changes in Cps

10. Barton et al. Planta. 1966 71:314 Bean Mesophyll Cell senescing Cps

11. Freeman et al. 1978 Protoplasma 94:221 Citrus Mesophyl Cell senescing Cp

12. Aims of my research Aim 1: Characterize the ultrastructure of the Cp in senescence Aim 2: Define the relationship between the Cp and the vacuole in senescence

13. Rationale for Approach to Aim 1 TEM will allow subcellular structure to be analyzed within senescent cells Morphological changes in Cp will be evident

14. Zoning an Arabidopsis leaf Green Zone 3 Zone 2 Zone 1

15. Chlorophyll levels Chl levels decrease as leaf senescence progresses

16. Rubisco levels Through zoning system Rubisco is serially degraded Zone 1 shows absence of intact Rubisco with increased non- specific Ab binding Marker Green Zone 3 Zone 2 Zone 1 55kD

17. Ultrastructure of Leaves Use Transmission Electron Microscope (TEM) to visualize subcellular structure within each zone

18. Healthy Green Cp Green 15,000X

19. Plastoglobuli formation Zone 3 15,000X

20. Separation of Thylakoids Zone 2 15,000X

21. Dismantling of Thylakoid System Zone 1 20,000X

22. Circularization and loss of distinguishable grana Zone 1 40,000X

23. Late Stage Separation from Plasma Membrane Zone 1 40,000X

24. Aim 1 Conclusions While Chl and Rubisco are degraded, Cps remain intact along the plasma membrane Ultrastructural changes include separation of thylakoids, formation of plastoglobuli, and circularization of Cps.

25. Rationale for Approach to Aim 2 TEM is impractical even with serial sectioning or immunogold staining Newer techniques are looked upon favorably Confocal has less artifacts and preparation problems

26. Structure of the Vacuole The tonoplast surrounds the central vacuole in plant cells

27. Confocal Microscopy To visualize the tonoplast - use a 35S-GFP::  -TIP line. To visualize the chloroplast - use autofluorescence.

28. Avila et al. 2003. Plant Phys. 133:1674 GFP lines treated with EMS to induce single nt mutations Above, tvs mutant’s vacuoles transected by transvacuolar strands To the right, bub mutants have many small vacuoles

29. Eggink, et al. 2004. BMC Plant Biology, 4:5 http://icecube.berkeley.edu/~bramall/work/astrobiology/images/chlorophyllspectra.jpg

30. What would results look like? During normal growth: During senescence:

31. Conclusions and Significance Degradation of chloroplast will be characterized in Arabidopsis Relevance of vacuole in senescence of the chloroplast will be assessed Areas of high impact: - plant molecular biology - agriculture - CSULB community

32. THANK YOU Questions???