Sam White M.S. Defense.

1. A SMALL MITOCHONDRIAL HEAT SHOCK PROTEIN CAUSES GENE SUPER-INDUCTION DURING HEAT SHOCK Presented by Samuel J. White Committee Members: David M. Rhoads, Chair Scott E. Bingham Michael E. Salvucci

9. Research 1) Heat-shock induction kinetics of HSPs. 2) What happens to a less thermotolerant plant species (Arabidopsis) when a mt sHSP from a more thermotolerant plant species (maize) is expressed constitutively at a high level? 3) Mitochondrial retrograde regulation of expression of HSPs.

10. HSP23.6 HSP17.4(CI) HSP21/25.3 HSP70-1 HSP70-6 HSP70-9 HSP17.6C HSP23.5 Continuous HS @ 42 o C (hrs) 0 0.5 1 2 4 6 8 12 24 Mito. Cp Cyto. Wild-type HSP Gene Expression at 42 o C

11. 1. Mitochondrial sHSP genes upregulated quickly & strongly 2. Transcript kinetics vary Wild-type HSP Gene Expression at 42 o C

12. Research 1) Heat-shock induction kinetics of HSPs. 2) What happens to a less thermotolerant plant species (Arabidopsis) when a mt sHSP from a more thermotolerant plant species (maize) is expressed constitutively at a high level? 3) Mitochondrial retrograde regulation of expression of HSPs.

13. ZmHSP22 rRNA 2-8 12-2 C24 Time (hrs) exposed to heat shock 42 o C 0 1 4 0 1 4 0 1 4 Characterization of Arabidopsis thaliana Transgenic Lines 2-8 & 12-2

14. WT 1 2 3 4 5 6 7 8 12-2 12-2 12-2 WT WT HS Zm C Zm mt cp pellet Image courtesy of David Rhoads Characterization of Arabidopsis thaliana Transgenic Lines 2-8 & 12-2

15. Characterization of Arabidopsis thaliana Transgenic Lines 2-8 & 12-2 2-8 12-2 B B K K E E

16. Characterization of Arabidopsis thaliana Transgenic Lines 2-8 & 12-2 Plant Line Leaf Wet Weight (% Change) Wild-type (C24) Experiment -22 % 12-2 -7.8 % 2-8 Wild-type (C24) -17.1 % -3.9 % 1 2 42C heat shock for 30 mins. over 10 day period.

17. Characterization of Arabidopsis thaliana Transgenic Lines 2-8 & 12-2 1. ZmHSP22 transgene is expressed constitutively at a high level. 2. ZmHSP22 localizes to mitochondria. 3. Single insertion of ZmHSP22. 4. Transgene present different loci. 5. Increase in thermotolerance caused by high level of ZmHSP22 expression and mitochondrial localization.

18. Research 1) Heat-shock induction kinetics of HSPs. 2) What happens to a less thermotolerant plant species (Arabidopsis) when a mt sHSP from a more thermotolerant plant species (maize) is expressed constitutively at a high level? 3) Mitochondrial retrograde regulation of expression of HSPs.

19. Signal from mitochondria in response to stress Environmental stimulus - heat stress Retrograde signaling Interaction of heat stress & MRR to affect gene expression Mitochondria Nucleus HSP23.6

20. Retrograde regulation of nuclear gene expression during heat stress 2-8 12-2 C24 AtHSP23.6 rRNA Time (hrs) exposed to heat shock 42 o C 0 1 4 0 1 4 0 1 4

21. AtHSP25.3 AtHSP17.4(CI) AtHSP70-1 AtHSP70-6 AtHSP70-15 rRNA rRNA rRNA rRNA rRNA Time (hrs) exposed to heat shock 42 o C 0 1 4 0 1 4 0 1 4 2-8 12-2 C24 Retrograde regulation of nuclear gene expression during heat stress

22. HSP Expression: Wt vs. At-ZmHSP22-2-8 C24 2-8 HSP23.6 HSP17.4(CI) HSP17.6C HSP23.5 HSP17.6(CII) HSP17.7 HSP18.1 HSP18.5 HSP70-8 HSP70-14 HSP70-17 0 0.5 1 2 4 6 8 12 0 0.5 1 2 4 6 8 12 Continuous HS @ 42 o C (hrs) Continuous HS @ 42 o C (hrs) HSP21/25.3

23. Model for mitochondrial retrograde regulation in mt sHSP CE lines during heat stress Nucleus

24. Model for mitochondrial retrograde regulation in mt sHSP CE lines during heat stress Nucleus Heat stress MRR ↑ sHSPs ↑ HSP70s what else? Heat stress

25. HSP Expression Wt vs. At-ZmHSP22-2-8 3. Some, but not all HSP70 genes subject to superinduction. 2. All sHSP genes examined subject to superinduction. 1. At-ZmHSP22-2-8 line exhibits superinduction of endogenous HSP genes during heat shock.

26. Constitutive Expression of HSP23.6 in A. thaliana PCR AtmtHSP23.6 Subclone into pRTL2 downstream of 35s promoter Subclone 35s:HSP23.6 into plant transformation vector pPZP221 Transform A. tumefaciens Transform A. thaliana ecotype Col-0 via floral dip Select for transformants with 35s:HSP23.6 to point of homozygosity Image: David Rhoads

27. Constitutive Expression of HSP23.6 in A. thaliana HSP23.6 +C 7-8-1 7-8-2 7-8-3 17-4-1 17-4-2 103-4-3 17-4-3 17-4-4 103-4-2 103-4-4 105-4-1 105-4-2 105-4-3 105-4-4 123-8-4 Untreated homozygous lines screened for HSP23.6 constitutive expression

28. Constitutive Expression of HSP23.6 in A. thaliana HSP23.6 +C 7-8-1 7-8-2 7-8-3 17-4-1 17-4-2 103-4-3 17-4-3 17-4-4 103-4-2 103-4-4 105-4-1 105-4-2 105-4-3 105-4-4 123-8-4 Untreated homozygous lines screened for HSP23.6 constitutive expression Check for superinduction of HSP17.4(CI)

29. Constitutive Expression of HSP23.6 in A. thaliana HSP23.6 +C Wt 7-8-1 7-8-2 103-4-2 103-4-3 103-4-4 +C HSP17.4(CI) 0 0 0 0 0 0 1 4 1 1 1 1 1 4 4 4 4 4 Heat shock @ 42 o C (hrs)

30. Constitutive Expression of HSP23.6 in A. thaliana HSP23.6 +C Wt 7-8-1 7-8-2 103-4-2 103-4-3 103-4-4 +C HSP17.4(CI) 0 0 0 0 0 0 1 4 1 1 1 1 1 4 4 4 4 4 Heat shock @ 42 o C (hrs) Use for additional experiments

31. Constitutive Expression of HSP23.6 in A. thaliana Superinduction effect in HSP23.6CE lines & At-ZmHSP22 lines

32. Antibody Production Peptide region selected by David Rhoads Sequence synthesized by ProSci & used for polyclonal Ab production *Alignment from Scharf et al., Cell Stress & Chaperones 6 (2001) * CVRSYDDDGENGDGVDLYRRSVPR

33. 116 94 Duration (hrs) of Heat Shock at 42 o C kDa 201 50 37 29 19 7 103-4-3 Col-0 0 1 2 4 6 0 1 HSP23.6 Protein Levels

34. HSP23.6 Protein Levels 0 0 1 2 1 2 α - HSP23.6 α - Porin C24 At-ZmHSP22-2-8 Duration (hrs) of Heat Shock at 42 o C

35. HSP23.6 Antibody 1. Antibody recognizes ~21 kDa protein in mitochondria. 2. Protein accumulation follows increase in RNA levels. 2. Superinduction occurs at protein level in At-ZmHSP22-2-8

36. HSP23.6 KO Lines Obtain T-DNA insertion knock-out seeds from ABRC Screen initial plants for lack of HSP23.6 expression Outcross KO lines with Wt (Col-0) Heat shock experiments Backcross KO lines to develop homozygous lines

37. HSP23.6 KO Lines HSP23.6 1hr heat shock @ 42 o C A B C D E F G H I J

38. HSP23.6 KO Lines HSP23.6 1hr heat shock @ 42 o C A B C D E F G H I J Outcross with Wt & use for experiments

39. P1S2S1S8 KO-H KO-D HSP23.6 KO Lines: Continued Screening HSP23.6 P1S1S1S4 P1S2S1S2 P1S2S1S3 P1S2S1S4 P1S2S1S5 P1S2S1S6 P1S2S1S7 P1S2S1S6 P1S2S1S4 +C KO-C +C P1S2S1S1 P1S2S1S2 P1S2S1S3 P1S2S1S4 P1S2S1S5 P1S2S1S6 P1S2S1S7 +C KO-D HSP23.6 2 hrs heat shock @ 42 o C 2 hrs heat shock @ 42 o C

40. HSP17.4 (CI) HSP23.6 KO Lines 0 0.5 1.0 2.0 4.0 6.0 8.0 Wt HSP23.6KO H HSP17.4 (CI) Time (hrs) of heat shock @ 42 o C 12.0

41. HSP23.6 KO Lines At mtHSP23.6 required for "standard" sHSP HS induction.

43. Summary These data provide strong, novel evidence of the involvement of sHSPs in mitochondrial retrograde regulation during heat stress.

44. Acknowledgements Committee: David Rhoads Scott Bingham Mike Salvucci (USDA) Colleagues: Jelena Zarkovic Rajani George Danijel Dojcinivic Elizabeth Vierling (U of A) Jan Miernyk (USDA) ABRC Therese McCartney Anne Nelson (Pre-wife) Funding: NSF Additional Support: