1. 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

2. Glutamine synthetase GS, EC 6.3.1.2 In vivo biosynthetic reaction: NH 4 + + L-glutamate + ATP  L-glutamine + ADP + P i

5. Eukaryotic GS

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

7. Detection of GS In vitro synthetase reaction : NH 2 OH + L-glutamate + ATP   -glutamyl hydroxamate + ADP + P i

8. Polyacrylamide gelelectrophoresis

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

10. GS isoforms in wheat RootChloroplastLeaf

11. Nondenaturing PAGE GS (western blot)Rubisco (CBB staining) Controls1a 2aControls1a 2a 474 483 488 501 GS1 GS2

12. Nondenaturing PAGE - Protein content Rubisco (CBB staining) Controls1a 2a 474 483 488 501

13. Nondenaturing PAGE - GS GS (western blot) Controls1a 2a 474 483 488 501 GS1 GS2

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

15. Natív PAGE IEF - Density plot 501 K 501 1a

16. Natív PAGE IEF - Density plot 474 K 474 1a

17. Natív PAGE IEF - Density plot 483 K 483 1a

18. IEF - Density plot 488 K 488 1a

19. Natív PAGE 2D IEF SDS Silver staining

20. 2D 474501

21. Natív PAGE 2D 474 K 474 1a

27. 2D 501488483

28. 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

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

30. Thank you….