Cryopreservation Stress-Related Changes in Cytoskeletal Proteins Report

1. Partner 3 Report Susan M. Trigwell, Ayesha Siddika, Graham Souch, Mohannad Al-Majathoub and Paul T. Lynch. University of Derby, U.K. Alan J. Hargreaves and Philip L.R. Bonner. Nottingham Trent University, U.K.

2. P3 mandate crops: Garlic stem-discs (encapsulation/dehydration) Olive somatic embryos (controlled-rate freezing) WP3 Sugars WP4 Membrane Components * WP6 * Cytoskeletal Proteins WP7 Oxidative Stress WP8 Protocol development

3. WP3: Sugars (with P4) • Previously: Garlic stem-discs Olive somatic embryos Successful analysis; problems with data analysis (weight loss after freezing) Confirmed uptake of sucrose from preculture medium

4. WP4: Membrane Components (with P1) • Previously: • This year: Analysis of cryopreserved olive somatic embryos Garlic stem-discs Olive somatic embryos Successful trials (FA and PA) Successful trials (FA and PA) Analysis of cryopreserved tissue

5. WP7: Oxidative Stress(with P2) • Previously: • This year: Extended the desferrioxamine study to olive & strawberry. GR assay Olive somatic embryo Headspace analysis Schiff’s base determination CAT assay SOD assay Proline determination

6. WP8 (oxidative stress) for olive Determine effect desferrioxamine/ cation-free post-thaw recovery medium on olive post-thaw recovery frequency. WP7 Oxidative Stress Desferrioxamine: Limited beneficial effect in terms of fresh weight gain. C- medium: better embryogenesis/quality of growth

7. WP8 (oxidative stress) for strawberry Determine effect desferrioxamine/ cation-free post-thaw recovery medium on strawberry post-thaw recovery frequency. WP7 Oxidative Stress Desferrioxamine: Limited beneficial effect

8. *WP6*: Cytoskeletal proteins

9. Partners in WP6 • University of Derby, UK (P3) • Paul Lynch (WP leader), Susan Trigwell, Ayesha Siddika, Mohannad Al-Majathoub and Graham Souch • The University of Nottingham Trent, UK • (Subcontractor to P3) Philip Bonner and Alan Hargreaves • Fruit Tree Research Institute, Italy (P5) • Carmine Damiano • University of Tor Vergata • (Subcontractor to P5) Simone Beninati, Cinzia Forni and Alessandro Lentini

10. WP6 Key Objective • To relate the stages of cryopreservation protocols, particularly preculture treatments, to stress-related changes in the cytoskeleton and in the enzymes and signalling pathways associated with its regulation.

11. Two main areas of research in final year of CRYMCEPT • Characterise apparent modification/ degradation of tubulin observed after freezing in garlic stem-discs • Characterise potential ‘ERK-like’ proteins detected in both garlic stem-discs and olive somatic embryo tissue

12. Modification/degradation of tubulin after freezing

13. 50 kD Garlic stem-disc extracts– tubulin modification/degradation Preliminary result with B512 (anti-total a tubulin): Protein modification/ degradation apparent after freezing – varies with desiccation time [Equal protein loading] 8h des 4h des 6h des untreated encaps only 6h des/freeze * pre-treat only 8h des/freeze * 4h des/freeze

14. ‘Modification’ analysis

15. 250 250 160 160 105 105 75 75 50 50 35 35 ‘Modification’ analysis – post-thaw garlic UT 4h 6h* 8h* M UT 4h 6h* 8h* M TAP 9311 (a tubulin N-terminal epitope) B512 (a tubulin C-terminal epitope) • Different epitopes, but similar trend in staining intensity

16. Modification or degradation/loss? • No signal observed with modification-specific antibodies (poly-E and poly-G) • B512 antibody (C-term a tubulin) and TAP 9311 antibody (N-term a tubulin) signal profile implies degradation/loss of tubulin, not B512 epitope modification

17. 250 250 160 160 105 105 75 75 50 50 35 35 Tubulin-specific degradation/loss? UT 4h 6h* 8h* M UT 4h 6h* 8h* M B512 anti-total a tubulin anti-GAPDH • Similar staining trend for tubulin (50kD) and GAPDH (37kD)

18. Tubulin-specific degradation/loss? • The housekeeping protein GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and a-tubulin show a similar trend in amount present in lysates • This implies general protein degradation/loss

19. Potential mechanisms ofprotein ‘loss’ • In vivo protein degradation • Diffusion from damaged cell membranes into the bead during rehydration • Reduced efficacy of protein extraction after freezing (polyphenol binding) • Crosslinking of protein (localised to cell debris fraction after protein extraction procedure)

20. Effect of different rehydration t/T on protein loss in cryo-treated garlic 1 2 3 4 5 6 7 8 M After 4 hour desiccation (sub-optimum): 50 Rehydration t /T 1. 2h /25oC 2. 4h /25oC 3. 7h /25oC 4. overnight /25oC 5. 2h /4oC 6. 4h /4oC 7. 7h /4oC 8. overnight /4oC B512 anti-total a tubulin 50 TAP 9311 (anti-a tubulin [98-138aa]) 35 FL-335 anti-GAPDH

21. Evidence for/against in vivo protein degradation • For: • Effect is temperature and time dependent (ie, enzymatic) • Protein amount (and total dry weight) per stem-disc quarter is dramatically reduced after freezing • Against: • There are no protein degradation ‘ladders’ observed on the blots (or is it ‘total’ degradation?)

22. Evidence for/against protein diffusion from damaged cells during rehydration • For: • Effect is temperature and time dependent (ie, physical diffusion) • Protein amount (and total dry weight) per stem-disc quarter is dramatically reduced after freezing • Against: • There was no evidence of sucrose diffusion after freezing (small molecule!)

23. Further analysis of protein loss • Observed higher proportion of protein in the cell debris fraction after freezing • Currently analysing the amount of TG-mediated protein crosslinking (iso di-peptide quantification) in: CELL DEBRIS SUPERNATANT vs.

24. 250 160 105 75 50 35 Cryotreated olive somatic embyro tissue extracts with B512 post-thaw T1 T2 T3 T4 T5 T6 T7 T8 M

25. WP8 (protein loss) • Potential membrane damage is currently being assessed by MDA assay of garlic (lipid peroxidation determination) • Membrane damage will be reduced by alteration of the cryoprotocol

26. ERK-like protein characterisation

27. Why look at the ERK signalling pathway? • Evidence of ERK-like proteins in plants that are activated by cold and drought (Jonak et al., 1996) • ERK homologue AtMPK4 is rapidly activated by cold and osmotic stress (Ichimura et al., 2000)

28. Antibodies used to probe ERK-like proteins in garlic and olive • NP2 - immunogen HTGFLTEYVAT - recognises non p-ERK 1/2 • PT115 - immunogen HTGFLpTEYVAT - recognises mono p-ERK and di p-ERK (not non p-ERK)

29. desicc post-thaw desicc post-thaw 250 160 250 160 105 105 75 75 Garlic PT-115 Garlic NP2 post-thaw post-thaw 250 250 160 160 105 105 75 75 Olive PT-115 Olive NP2 Cryo-treated olive and garlic extracts probed with NP2 and PT-115

30. Olive ERK-like protein characterisation • Potential dimerisation (90-180kD) after freezing • Phosphorylated derivative is 180kD • Confirmed that observed dimerisation is not artefact of extraction procedure (NEM and 283 added to extraction buffer)

31. MS procedure • Covalently link antibody (NP2 and PT-115) to resin • Selectively bind protein from tissue extract • TCA precipitate • Load concentrated protein on SDS PAGE • Coomassie stain (Pierce high sensitivity) • Excise band of correct molecular weight • MS analysis for identification

32. WP8 (cell signalling) • Awaiting identity results of the ERK-like proteins in garlic and olive by MS

33. Publications and Outputs • Publications: S.M. Trigwell, P.T. Lynch, M. Griffin, A.J. Hargreaves, and P.L.R. Bonner (2004) An improved colorimetric assay for the measurement of transglutaminase (type II) e-(g-glutamyl) lysine cross-linking activity. Analytical Biochemistry330 164-166 W. Harris, P.T. Lynch, A.J. Hargreaves, and P.L.R. Bonner (2004) Cryopreservation of Helianthus tuberosus cell suspension cultures: The effect of preculture treatments on cytoskeletal proteins and transglutaminase activity. CryoLetters25 213-217

34. Publications and Outputs • Presentations: Susan M. Trigwell, Satyagar Govindaraju, Paul T. Lynch, Martin Griffin, Alan J. Hargreaves and Philip L.R. Bonner (2005) The use of biotinylated substrates to assay for transglutaminases. 8th International Conference on Protein Crosslinking and Transglutaminases, Lübeck, September 2005. Paul T. Lynch. Life in the Freezer - Use of Cryopreservation to Support Biodiversity. National Academy of Science, Malaysia, Kuala Lumpur, April 2005 (Sponsored by the British Council). Paul T. Lynch. Life in the Freezer- Biotechnology for Plant Conservation. Malaysian Agriculture Development Institution, Kuala Lumpur, April 2005.

35. Deliverable 6.1 • Description of analysis method to assess cytoskeleton and associated enzyme activities • Protocol descriptions have been prepared and posted on the CRYMCEPT website.

36. Milestones 6.1 & 6.2 and Deliverables 6.2, 6.3 & 6.4 • Association between cytoskeletal stability, TG activity and post-thaw regrowth. • Protein crosslinking assay - Analytical Biochemistry (2004). • Identification of potential post-thaw tubulin degradation/ modification marker. • Further characterisation has attributed this to protein loss/degradation. • The first draft of a publication describing these findings is currently in progress.

37. Milestone 6.3 and Deliverables 6.2, 6.3 & 6.4 • Association of stress related cell signalling pathways and post-thaw regrowth. • Anti-ERK antibodies detected proteins related to post-thaw recovery in garlic and olive somatic embryo tissue. • MS analysis of this potential marker is currently in progress. • The first draft of a publication describing these findings is currently in progress.

40. Controversial thought • Are the effects we are studying actually those occurring in the numerous non-viable cells in the post-thaw tissue (ie, background)? • Maybe there are so few remaining viable cells in the post-thaw tissue that the effects occurring in these cells (the ‘signal’) are not measurable over the background?