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 In vivo biosynthetic reaction: NH L-glutamate + ATP  L-glutamine + ADP + P i

Eukaryotic GS

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

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

Polyacrylamide gelelectrophoresis

Natív PAGE western blot activity in gel Coomassie staining Nondenaturing PAGE

GS isoforms in wheat RootChloroplastLeaf

Nondenaturing PAGE GS (western blot)Rubisco (CBB staining) Controls1a 2aControls1a 2a GS1 GS2

Nondenaturing PAGE - Protein content Rubisco (CBB staining) Controls1a 2a

Nondenaturing PAGE - GS GS (western blot) Controls1a 2a GS1 GS2

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

Natív PAGE IEF - Density plot 501 K 501 1a

Natív PAGE IEF - Density plot 474 K 474 1a

Natív PAGE IEF - Density plot 483 K 483 1a

IEF - Density plot 488 K 488 1a

Natív PAGE 2D IEF SDS Silver staining

2D

Natív PAGE 2D 474 K 474 1a

2D

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

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 (%)

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