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Glutamine synthetase GS, EC 6.3.1.2

Changes in glutamine synthetase activity and in protein pattern of wheat leaves after Fusarium infection Attila Pécsváradi Zoltán Nagy, Edit Németh Dept . of Plant Biology , University of Szeged. Glutamine synthetase GS, EC 6.3.1.2. In vivo biosynthetic reaction :

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Glutamine synthetase GS, EC 6.3.1.2

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  1. Changesinglutamine synthetase activity and in protein pattern of wheatleavesafterFusariuminfectionAttila PécsváradiZoltán Nagy, Edit NémethDept. of PlantBiology, University of Szeged

  2. Glutamine synthetase GS, EC 6.3.1.2 In vivo biosynthetic reaction: NH4+ + L-glutamate + ATP  L-glutamine + ADP + Pi

  3. Eukaryotic GS

  4. Introduction • Technical aspects: • in vitro activity detection • separation by PAGE • identification of isoforms • Physiological aspect: • senescence: indicator of stress

  5. Detection of GS In vitro synthetase reaction: NH2OH + L-glutamate + ATP  -glutamyl hydroxamate + ADP + Pi

  6. Polyacrylamide gelelectrophoresis

  7. Nondenaturing PAGE Natív PAGE Coomassie staining western blot activity in gel

  8. Root Chloroplast Leaf GS isoforms in wheat

  9. Nondenaturing PAGE Rubisco (CBB staining) GS (western blot) GS1 GS2 474 483 488 501 Controls 1a 2a Controls 1a 2a

  10. Nondenaturing PAGE - Protein content Rubisco (CBB staining) 474 483 488 501 Controls 1a 2a

  11. Nondenaturing PAGE - GS GS (western blot) GS1 GS2 474 483 488 501 Controls 1a 2a

  12. IEF (isoelectric focusing) pH 10.5 pH gradient pH 5.5 pH 3.2

  13. IEF - Density plot Natív PAGE 501 K501 1a

  14. IEF - Density plot Natív PAGE 474 K474 1a

  15. IEF - Density plot Natív PAGE 483 K483 1a

  16. IEF - Density plot 488 K488 1a

  17. 2D Natív PAGE IEF SDS Silver staining

  18. 2D 474 501

  19. 2D Natív PAGE 474 K 474 1a

  20. 2D 483 488 501

  21. Summary Fusarium infection: • decrease of in vitro GS → accelerated senescence • decrease of protein content → accelerated senescence • decrease of Rubisco → breakdown of chloroplast • decrease of GS2 → breakdown of chloroplast • 2D pattern: protein degradation • 2D pattern: new dominant spots • Fusarium strains : virulence is different

  22. Summary Inhibition (%) GS activity / FW GS activity / protein Sensitivity for 1a (F. culmorum): 501 >> 474  488 >> 483 Sensitivity for 2a (F. graminearum): 483  501 >>> 474 = 488

  23. Thank you….

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