WP 2 – Proteins

1. WP 2 – Proteins Objective: The major objective of this work package is to analyse protein patterns of plants and cell cultures in relation to cryoprotection and to investigate underlying physiological mechanisms inducing or enhancing the formation of proteins which can provide cryoprotection.

2. WP 2 – Proteins Specific objectives are: • To establish techniques for the measurement of protein patterns • of plants and cell cultures • To scale down detection methods for the application to • meristems and/or meristematic tissues • To establish protein patterns of plants and cell cultures in • relation to cryopreservation • To define changes of the protein patterns during the application • of different cryopreservation protocols • To define specific protein spots that are related to cryo- • protection • 6. To define the relation of specific protein spots, that provide cryoprotection, to stress reactions.

3. WP 2 – Proteins Scientific Background Cryopreservation of plant cells is difficult: * because of their high water content * because of their subcellular structure Manipulations during application of cryopreservation techniques expose plant cells to a certain stress Metabolic compounds and physiological mechanisms may help the plant cells to overcome these stresses

4. WP 2 – Proteins Scientific Background Proteins related to cryopreservation • Can be structural proteins providing cryoprotection (anti freeze proteins, dehydrins) Can be enzymes or structural proteins providing cryoprotective traits (aquaporines, SOD) Can be enzymes involved in the formation of cryoprotective compounds (enzymes from lipid or sugar metabolism) Can be regulatory enzymes inducing protective mechanisms Can be proteins linked to cryoprotection for unknown reason (COR, heat shock proteins)

5. WP 2 – Proteins Scientific Background Proteins related to cryopreservation: Can be constitutively present in a certain plant tissue or genotype Comparative analysis of different tissues or genotypes Can be induced by a certain step in a cryopreservation procedure Comparative analysis of the same material in different stages of a cryopreservation procedure

6. WP 2 – Proteins Scientific Background Proteins correlated with cryopreservation success: Constitutively present in a certain plant tissue or genotype Can be used as markers to select suitable tissues or genotypes for cryopreservation Induced by a certain step in a cryopreservation procedure Can be used as markers for the optimization of a certain cryopreservation procedure

7. WP 2 – Proteins Steps of a cryopreservation process - In-vitro culture physiol.state, hormone treatment - Pre-culture treatment osmotic stress, chilling stress - Isolation or excision physical damage - Cryo-protective treatment • chemical treatment toxic effects • dehydration drought stress • desiccation salt stress - Freezing chilling stress - Thawing physical damage - Recovery/Regeneration hormone treatment

8. WP 2 – Proteins Specific objectives are: • To establish techniques for the measurement of protein patterns • of plants and cell cultures • To scale down detection methods for the application to • meristems and/or meristematic tissues • To establish protein patterns of plants and cell cultures in • relation to cryopreservation • To define changes of the protein patterns during the application • of different cryopreservation protocols • To define specific protein spots that are related to cryo- • protection • 6. To define the relation of specific protein spots, that provide cryoprotection, to stress reactions.

9. WP 2 – Proteins Partners: P1 Laboratory of Tropical Plant Improvement, University of Leuven, Belgium (Bart Panis, Sebastien Carpentier) P5 Fruit Tree Research Institute, Ciampino, Italy (Carmine Damiano) Subcontractor: Dept of Biology, University of Tor Vergata, Rome (Cinzia Forni) P6 DSMZ- German Collection of Microorganisms and Cell Cultures GmbH – Plant Cell Culture Department (H.M. Schumacher, Y. Merhoffer/E. Müller)

10. WP 2 – Proteins Time Scale for WP2: Year 1 Year 2 Year 3 M 2.1 M2.2 M2.3 M2.4 Input for WP 8 Feedback from WP 8

11. WP 2 – Proteins Situation at Start of Work Partners provide different model plants and different cryopreservation procedures No partner could provide established methods for 2D protein analysis All partners could provide basic equipment for 2D protein analysis

12. WP 2 – Proteins Input at Start Partner 1 Plant material Musa ssp. meristematic clumps Cryo-method Vitrification - in-vitro cultured meristematic clumps - isolation of meristems - vitrification treatment - direct freezing Electroph. Equipment Multiphor, small medium size vertical gel chambers

13. WP 2 – Proteins Input at Start Partner 5 Plant material Apple cv. Annurca, cv. Gala, almond Cryo-method Encapsulation/Dehydration - in-vitro plantlets - excision of apices - pre-culture in sucrose medium - desiccation - direct freezing Electroph. Equipment Multiphor, small medium size vertical gel chambers

14. WP 2 – Proteins Input at Start Partner 6 Plant material Potato (different cultivars) Cryo-method Ultra-rapid droplet freezing - in-vitro plantlets - excision of apices - incubation over night - DMSO treatment - direct freezing Electroph. Equipment Multiphor, small medium size vertical gel chambers

15. WP 2 – Proteins Information and Training Visit at CRPGL For Introduction and training on eletrophoretical techniques a visit was organized for P1 Bart Panis, Sebastien Carpentier 17th – 21st of March 2003 P6 Heinz Martin Schumacher 20th – 21st of March 2003 CRPGL (Public Research Centre-Gabriel Lippmann), Luxemburg Information and Training was provided by and is kindly acknowledged Jean Francois Hausman Jenny Renaut

16. WP 2 – Proteins Work performed in Year 1 – P1 Setup of 2D electrophoretical techniques Aquisition and setup of new equipment for electro- phoresis and spot pattern analysis ! Establishment and comparative studies with four different protein extraction protocols !

17. WP 2 – Proteins Work performed in Year 1 – Partner 5 Refinement of cryopreservation techniques for apple and almond Development of extraction methods for total protein extraction as well as polyamine isolation Setup of SDS electrophoretical techniques to test extraction methods

18. WP 2 – Proteins Work performed in Year 1 – Partner 6 Setup of electrophoretical techniques (using small strips with vertical and horizontal second dimension gels) Comparison of different extraction techniques based of phenol extraction and TCA precipitation First measurements of protein patterns with different cultivars using stem and leaf material and suspension cultures

19. WP 2 – Proteins Meeting in Leuven 2nd – 6th Feb. 2004 Objectives: - Comparative analysis of the best extraction method for different materials - Standardization of extraction methods - Training of all participants in the most advanced lab Results - extraction methods and electrophoresis was refined for apple - extraction methods and electrophoresis was refined for potato - standardization of techniques was achieved

20. WP 2 – Proteins Results obtained in Year 2 – Partner 6 Basic improvement of analytical methods Adaptation of extraction method from Partner 1 Scale down of extraction method to the use of isolated apices (30 apices minimun, 100 apices used) Improvement of spot resolution using 18 cm strips and vertical gels Recently: Setup of Ettan Dalt 6 system for second dimension runs, new documentation system and new software

21. WP 2 – Proteins Results obtained in Year 2 – Partner 6 Protein patterns have been measured of apices of cultivars: Unicopa, Desiree, Ijsselster - directly after excision - after incubation over night - after incubation over night and DMSO treatment Still to be measured: after freezing and two days recovery In all cases differences of the spot patterns occur Results have to be statistically validated Relation of differences to cryopreservation has to be established

22. WP 2 – Proteins Results obtained in Year 2 – Partner 5 Methods for 2D analysis of protein pattern were established Extraction methods for apple cv. Annurca and cv. Gala have been refined

23. WP 2 – Proteins Results obtained in Year 2 – Partner 5 Experiments were started comparing pattern of control and mannitol treated meristems Fig. 2 Apple cv. Annurca: cryopre-served Sample treated with mannitol Fig. 1 Apple cv. Annurca: control sample Tested cultivars: cv. Annurca cv. Gala

24. WP 2 – Proteins Results obtained in Year 2 – Partner 1 Further improvement of extraction procedure Analysis of spot patterns after sucrose treatment Optimization of resolution of differentially expressed spots 250 kD 150 kD 100 kD 75 kD 50 kD 37 kD 25 kD 20 kD IPG strip pH 3 - 10 IPG strip pH 4 - 7

25. WP 2 – Proteins Results obtained in Year 2 – Partner 1 Application of CBB staining technique for future peak identification Silver stained CBB stained

26. WP 2 – Proteins Results obtained in Year 2 – Partner 1 First identification of differentially expressed proteins 8 of 41 proteins identified Control Sucrose treated Identification through MS/MS: Phosphoglycerate kinase Enzyme involved in glycolysis.

27. WP 2 – Proteins Main problems solved Analytical methods set up by all partners Scale down of analytical techniques achieved to practical application Standardization of extraction procedures achieved for all partners Measurements of protein patterns in relation to cryopreservation started by all partners Further standardization of equipment achieved

28. WP 2 – Proteins Problems to be solved in the future: Further detection of proteins related to cryopreservation Statistical validation of relation between spots and cryopreservation in most cases Further standardization of methods by exchange of material between laboratories Further identification of proteins by Western blot or MS Further definition of input for WP 8 from results obtained

29. WP 2 – Proteins Poster Presentations 2003 Sample preparation for 2D PAGE analysis of plant tissues: a balance between gel quality and sample completeness Sebastien C. Carpentier; Bart Panis and Rony Swennen Proteomic Forum Munich 2003, International Meeting on Proteome Analysis, Munich 14th – 17th Sept. 2003

30. WP 2 – Proteins Poster Presentation 2004 Cryopreservation of fruit tree species: effects on proteins, polyamines and Tgase activity Forni, C., Beninati S., Lentini A., Valle G., Frattarelli A., DeBonfis A., Giovinazzi J., Arias M., Caboni E., Montierell S., La Starza S., Gaboni E., Carpentier S., Mathia D., Chiacino T and Damiano C. „Riunione annuale gruppi, Biotechnologie-Differenziamento e Biologia Cellulare-Moleculare“, Belgrad 23rd – 25th June 2004 Preparation of extracts from recalcitrant plant tissue: an evaluation of different methods for 2D page analysis S.C. Carpentier, B. Panis and R. Swennen 6th Siena Meeting from Genome to Proteome: Biomarker Discovery & Proteome Imaging, Siena 30th Aug. – 2nd Sept. 2004

31. WP 2 – Proteins Deliverables: D2.1: Reliable method for protein analysis is described for large amount of tissues (month 6): D2.2: Reliable method for protein analysis is described for small amount of tissues (month 24):. D2.3: Description of change in protein patterns in relation to cryopreservation for the plant and tissue material under investigation (month 27): D2.4: Scientific publication on the fine-tuned protein analysis method and the correlation between the presence of certain proteins and cryoprotection (month 27).

32. WP 2 – Proteins Milestones: M2.1: Development of methods for the measurement of protein patterns in relation to cryopreservation for potato suspension cultures (month 6): M2.2: Scaling down of protein analysis techniques in order to apply them to meristem and meristematic tissue of the plant species included in the Work Package (month 17): M2.3: Measurement of changes of protein patterns for different plants and tissues during the application of different cryopreservation protocols (month 24): M2.4: Measurement of the induction of changes of the protein pattern by different stresses and the consequences for cryopreservation success (month 27):