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![Page 1: Changes in glutamine synthetase activity and in protein pattern of wheat leaves after Fusarium infection Attila Pécsváradi Zoltán Nagy, Edit Németh Dept.](https://reader035.fdocuments.us/reader035/viewer/2022081516/5697c00e1a28abf838cc9f10/html5/thumbnails/1.jpg)
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
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Glutamine synthetase GS, EC 6.3.1.2
In vivo biosynthetic reaction:
NH4+ + L-glutamate + ATP
L-glutamine + ADP + Pi
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Eukaryotic GS
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Introduction
• Technical aspects:• in vitro activity detection• separation by PAGE• identification of isoforms
• Physiological aspect:• senescence: indicator of stress
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Detection of GS
In vitro synthetase reaction:
NH2OH + L-glutamate + ATP -glutamyl hydroxamate + ADP + Pi
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Polyacrylamide gelelectrophoresis
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Natív PAGE
GS1
GS2
western blot
activity in gel
Coomassie
staining
Nondenaturing PAGE
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GS isoforms in wheat
Root Chloroplast Leaf
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Nondenaturing PAGE
GS (western blot)Rubisco (CBB staining)
Controls 1a 2aControls 1a 2a
474
483
488
501
GS1
GS2
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Nondenaturing PAGE - Protein content
Rubisco (CBB staining)
Controls 1a 2a
474
483
488
501
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Nondenaturing PAGE - GS
GS (western blot)
Controls 1a 2a
474
483
488
501
GS1
GS2
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IEF (isoelectric focusing)
pH gradient
pH 10.5
pH 3.2
pH 5.5
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Natív PAGEIEF - Density plot
501 K 501 1a
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Natív PAGEIEF - Density plot
474 K 474 1a
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Natív PAGEIEF - Density plot
483 K 483 1a
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IEF - Density plot
488 K 488 1a
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Natív PAGE2D
IEF
SDS
Silver staining
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2D
474 501
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Natív PAGE2D
474 K
474 1a
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2D
501488483
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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
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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 (%)
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Thank you….