Glutamine synthetase GS, EC 6.3.1.2
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Transcript of Glutamine synthetase GS, EC 6.3.1.2
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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 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 (%)
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Thank you….