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Workpackage 5. POLYAMINES (PAs) and AROMATIC AMINES (AAs). WP5: Partners. Partner 1: Katholieke Universiteit Leuven (KULeuven), Belgium, Partner responsible. Partner 5: Fruit Tree Research Institute (ISF), Italy. Partner 2: University of Abertay Dundee (UAD), UK.
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Workpackage 5 POLYAMINES (PAs) and AROMATIC AMINES (AAs)
WP5: Partners Partner 1: Katholieke Universiteit Leuven (KULeuven), Belgium, Partner responsible Partner 5: Fruit Tree Research Institute (ISF), Italy Partner 2: University of Abertay Dundee (UAD), UK Partner 3: University of Derby (UD), UK
WP5: Period 30 months: Starting date: 01-11-2002 Completion date: 30-04-2005
WP5: Background Polyamines bind to important cellular polyanions as DNA, RNA, phospholipids and acidic protein residues and cell wall components; Polyamines are involved in various cell functions such as cell division, growth, differentiation etc; Polyamine metabolism is also strongly involved in plant response towards environmental challenges such as osmotic stress, salinity, hypoxia, cold stress and environmental pollutants; Polyamine biosynthesis in plants happens through ADC and/or ODC pathway.
WP5: Objectives To refine techniques – polyamine analysis To scale down analysis methods To define changes in polyamine composition – different cryo-protocols; To determine relationship between polyamines and survival after cryopreservation To find treatments for improving the cryo-ability of plant tissues
Analysis parameters in WP5 PAs: - DAP (diaminopropane) - PUT (putrescine) - SPD (spermidine) - SPM (spermine) AAs - PEA (phenethylamine) - TYR (tyramine) - DA (dopamine) - SAL (salsolinol) ADC and ODC
HPLC of PAs and AAs Best Column packing: Spherisorb S5 ODS 2 (5 µm) Columns lab: home-made, 100x3 mm Too short for complete resolution of 4 PAs and 4 AAs in one run However: Alltech ODS2 is not as good as Spherisorb (longer gradient required)
ODC and ADC • At 2 d: ADC in controls enhanced • At 2 d: Very low ODC activity • From 15 d on: huge increase, mainly of ODC (about 17 x more than ADC at 21 days • PAs analysed after 2 weeks: synthesised mainly by ODC pathway
Conclusions • Many of the changes of PAs and AAs are positively or negatively correlated with survival percentages of the cultivars • DAP: higher in bad cvs (more PA breakdown?) • SPM: higher in good survivers before treatment: better radical scavenger capacity? • PUT: Higher in good survivers: protection?
Conclusions (2) • AAs: before treatment: positively correlated with survival • AAs: after treatment: negatively correlated with survival • Anti-oxidative protection before and during sucrose treatment? • Production of radicals in bad survivers?? • Many questions, but few answers yet!
Suggestions for Cryo • Adding PAs (different concentrations) • Adding inhibitors (DFMO, DFMA, to be synthesised again) • Adding AAs • Adding inhibitors of AAs biosynthesis • All combinations of the above • Study of biosynthesis of AAs, function in radical scavenging (and/or production!) • Study of metabolism of AAs
Polyamine levels in shoot apex from Apple cv. Annurca dehydrated with mannitol (results by P5) pmol polyamine / mg protein Mannitol Mannitol Silica gel Liq. N2 Control Mannitol Silica gel
Polyamine levels in shoot apex from Apple cv. Annurca dehydrated with sucrose pmol polyamine / mg protein Control Sucrose Sucrose Silica gel Sucrose Silica gel Liq. N2
Polyamine content of 5 Ribes genotypes. Explants were analysed 6 weeks after sub-culturing, using leaf and stem tissue. Means and standard error of the means are shown (n = 5 individual explants). Different letters indicate genotypic differences according to analysis of variance and least significant difference (P < 0.05).
Main problems resolved / to be resolved Two quantitatively very important unknown peaks in chromatograms have been identified: Dopamine and Salsolinol ADC and ODC should be further studied Studies with DFMA and DFMO, and reversal of inhibition by PUT Idem for AAs
Milestones in WP5 M5.1: Development of methods for polyamine analysis for banana tissues (month 6) M5.2: Development of methods for polyamine analysis for different plant species (month 18) M5.3: Determination of polyamine changes in relation with survival after cryopreservation (month 24)
Deliverables in WP5 D5.1: Detailed description of methods (month 18) D5.2: Correlation between polyamines and cryopreservation (month 24) D5.3: Scientific publication (month 24)
Acknowledgements of Leuven group • We acknowledge the excellent technical assistance of Marta Ledo, Johan De Rue, Tom Struyf, Hilde Verlinden, René De Boer (MS) and F. Compernolle (MS)