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MTR Report WP6: Cytoskeletal Proteins

This report explores the manipulation of cytoskeletal proteins to enhance cryopreservation success. The study aims to identify freeze and desiccation tolerance markers and develop novel treatments for improved stability. Detailed protocols and experimental results on garlic stem-disc cryopreservation are presented.

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MTR Report WP6: Cytoskeletal Proteins

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  1. MTR ReportWP6: Cytoskeletal Proteins Paul Lynch

  2. Partners • University of Derby, UK (P3) • Paul Lynch (WP leader) Susan Trigwell, Ayesha Siddika 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

  3. Why look at cytoskeletal proteins? Helianthus tuberosus cell suspension cultures: cytoskeletal protein modification cryo pretreatment modulation of transglutaminase activity (Harris et al. 2004)

  4. Why look at cytoskeletal proteins? microtubule stability post-thaw recovery (Spinacia oleracea L.) Bartolo and Carter (1991) actin filament stability post-thaw recovery (Daucus carota L.) Morisset et al. (1993)

  5. WP6 Objectives • To relate the stages of cryopreservation protocols, particularly preculture treatments, to stress related changes in the cytoskeleton and enzymes associated with its regulation. • Identification of molecular markers of freeze and desiccation tolerance. • Development of novel cryopreservation treatments to manipulate cytoskeleton stability and the activity of associated enzymes.

  6. Garlic stem-disc cryopreservation protocol Excise Encapsulate Pre-treat (0.5M sucrose 3 days) Desiccate 6 hours Freeze/thaw 24-hr post-thaw recovery on GSD

  7. Immunofluorescent staining of longitudinal garlic stem-disc sections, non-frozen and frozen

  8. Immunofluorescent staining of garlic stem-disc longitudinal sections with anti-crosslink antibody Non-frozen 1d post-thaw

  9. Immunoprobing of protein blots of cryo-treated garlic stem-disc extracts – tubulin modification

  10. 50 kD 50 kD Immunoprobing of protein blots of cryo-treated garlic stem-disc extracts Anti-a tubulin (tyrosinated) Anti-a tubulin (total) 8h des 4h des 6h des 6h des 4h des 8h des untreated untreated encaps only encaps only 8h des/freeze pre-treat only 6h des/freeze pre-treat only 4h des/freeze 8h des/freeze 4h des/freeze 6h des/freeze

  11. Antibody Result Anti-ERK ~200kD band Anti-pERK ~200kD band Anti-ppERK No bands detected Anti-pTyrosine No bands detected Anti-pSerine No bands detected Anti-pThreonine Novel 50kD pThreonine protein detected Immunoprobing of protein blots of cryo-treated garlic stem-disc extracts – cell signalling

  12. Immunoprobing of protein blots of cryo-treated garlic stem-disc extracts 250 160 Anti- ERK 105 75 50 35 30 4h des 6h des 8h des untreated encaps only pre-treat only 4h des/freeze 6h des/freeze 8h des/freeze

  13. ~200kD band identification Immunogen sequence H T G F L T E Y V A T H T G F L 1 hit (33kD) T G F L T 11 hits, all <160kD G F L T E 8 hits, all <160kD F L T E Y 4 hits, all <160kD L T E Y V 10 hits – 215kD glucan synthase T E Y V A 5 hits, all <160kD E Y V A T 8 hits, all <160kD

  14. Immunoprobing of protein blots of cryo-treated garlic stem-disc extracts 250 Anti- pThreonine 160 105 75 50 35 30 6h des 8h des untreated 4h des encaps only pre-treat only 4h des/freeze 6h des/freeze 8h des/freeze

  15. In vitro transglutaminase assays biotin cadaverine- incorporation protein- crosslinking compromised by other polyamine-incorporating enzymes present in cell extracts formation of the e (g -glutamyl)lysine isodipeptide bond is TG - specific

  16. -TVQQEL COLOUR REACTION TG Progress with protein crosslinking assay • Not suitable for crude garlic stem-disc extracts (inhibitory) • Not suitable for crude olive somatic embryo extracts (false positive signal) extravidin- HRPO detection casein substrate

  17. In vivo TG activity in apple shoot apices – P5 cv.Gala mannitol dehydration cv.Annurca mannitol dehydration Contl Mann Mann Mann Si Gel Si Gel Liq.N2 Contl Mann Mann Mann Si Gel Si Gel Liq.N2 pmol 3H-put / mg protein / 20h pmol 3H-put / mg protein / 20h cv.Gala sucrose dehydration cv.Annurca sucrose dehydration Contl Sucr Sucr Sucr Si Gel Si Gel Liq.N2 Contl Sucr Sucr Sucr Si Gel Si Gel Liq.N2

  18. COLOUR REACTION -cadaverine TG Progress with biotin-cadaverine incorporation assay • TG activity in the apple shoot tips was undetectable • Olive somatic embryos and shoot tips have detectable levels of TG activity extravidin- HRPO detection N, N-dimethyl casein substrate

  19. Technical Problems • Availability of quality garlic cloves • Contamination of olive somatic embryo cultures • Almond shoot cultures have not been established at UD • Transfer of techniques from mammalian to plant systems • TG protein crosslinking assay • immunoprobing antibodies

  20. Milestones 6.1 & 6.2 • Association between cytoskeletal stability, TG activity and post-thaw regrowth. • Identification of post-thaw tubulin degradation/ modification marker in garlic which correlates with optimum desiccation times. • Potential marker requires further characterisation. • Changes in TG activity in apple are genotype dependent and appear to be reduced by desiccation. • Milestones in progress

  21. Milestone 6.3 • Association of stress related cell signalling pathways and post-thaw regrowth. • Anti-ERK, Anti-pThreonine antibodies shown to be related to post-thaw recovery in garlic. • Potential markers require further characterisation. • Assessment to be extended to other plant species and tissue types. • Milestone in progress and on schedule.

  22. Deliverable 6.1 • Description of analysis method to assess cytoskeleton and associated enzyme activities • Reliable protocols now being established • Immunofluorescent staining - (tubulin modification) • Immunoprobing - (tubulin modification & cell signalling) • TG assay • Protocol descriptions to be prepared for CRYMCEPT website. • Deliverable in progress and on schedule

  23. Deliverable 6.2 • Changes in cytoskeleton, associated enzyme activities and stress related cell signalling • Garlic cryopreservation protocol analysed • Immunofluorescent staining - (protein cross-linking) • Immunoprobing - (tubulin modification & phosphorylation [cell signalling]) • TG assay • Preparation for analysis of olive SE cryopreservation protocol • Deliverable in progress and on schedule

  24. Deliverable 6.3 • Publications • Protein crosslinking assay - Analytical Biochemistry (2004) • Data currently being collected towards identifying changes in cytoskeleton, associated enzyme activities and cell signalling pathways • Deliverable in progress and on schedule

  25. Deliverable 6.4 • Molecular markers for cryopreservation • Initial identification of potential markers underway (Anti-a tubulin, Anti-ERK, Anti-pThreonine). • Potential markers to be further characterised and assessed using different plant species and cryopreservation protocols. • Deliverable in progress and on schedule.

  26. WP6: Cytoskeletal Proteins

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