Changes in glutamine synthetase activity and in protein pattern of wheat leaves

after Fusarium infection

Attila PécsváradiZoltán Nagy, Edit Németh

Dept. of Plant Biology, University of Szeged

Glutamine synthetase GS, EC 6.3.1.2

In vivo biosynthetic reaction:

NH4+ + L-glutamate + ATP

L-glutamine + ADP + Pi

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:

NH2OH + L-glutamate + ATP -glutamyl hydroxamate + ADP + Pi

Polyacrylamide gelelectrophoresis

Natív PAGE

GS1

GS2

western blot

activity in gel

Coomassie

staining

Nondenaturing PAGE

GS isoforms in wheat

Root Chloroplast Leaf

Nondenaturing PAGE

GS (western blot)Rubisco (CBB staining)

Controls 1a 2aControls 1a 2a

474

483

488

501

GS1

GS2

Nondenaturing PAGE - Protein content

Rubisco (CBB staining)

Controls 1a 2a

474

483

488

501

Nondenaturing PAGE - GS

GS (western blot)

Controls 1a 2a

474

483

488

501

GS1

GS2

IEF (isoelectric focusing)

pH gradient

pH 10.5

pH 3.2

pH 5.5

Natív PAGEIEF - Density plot

501 K 501 1a

Natív PAGEIEF - Density plot

474 K 474 1a

Natív PAGEIEF - Density plot

483 K 483 1a

IEF - Density plot

488 K 488 1a

Natív PAGE2D

IEF

SDS

Silver staining

2D

474 501

Natív PAGE2D

474 K

474 1a

2D

501488483

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

Wheat lines 1a 2a474 37 no483 no 63488 21 no501 71 60

Fusarium strainsWheat lines 1a 2a

474 26 no483 no 61488 26 no501 59 41

Fusarium strains

GS activity / FW GS activity / protein

Sensitivity for 1a (F. culmorum): 501 >> 474 488 >> 483

Sensitivity for 2a (F. graminearum): 483 501 >>> 474 = 488

Inhibition (%)

Thank you….