Glutamine synthetase GS, EC 6.3.1.2
30
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
description
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 : - PowerPoint PPT Presentation
Transcript of Glutamine synthetase GS, EC 6.3.1.2
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 strainsGS 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….
