Changes in glutamine synthetase activity and in protein pattern of wheat leaves after Fusarium infection Attila P

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Changes in glutamine synthetase activity and in
protein pattern of wheat leaves after Fusarium
infectionAttila PécsváradiZoltán Nagy, Edit
NémethDept. 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
Nondenaturing PAGE
Coomassie staining
western blot
activity in gel
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GS isoforms in wheat
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Nondenaturing PAGE
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Nondenaturing PAGE - Protein content
Rubisco (CBB staining)
Controls
1a 2a
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Nondenaturing PAGE - GS
GS (western blot)
GS1
GS2
Controls
1a 2a
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IEF (isoelectric focusing)
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Natív PAGE
IEF - Density plot
501 K 501 1a
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Natív PAGE
IEF - Density plot
474 K 474 1a
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Natív PAGE
IEF - Density plot
483 K 483 1a
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IEF - Density plot
488 K 488 1a
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Natív PAGE
2D
IEF
SDS
Silver staining
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2D
474
501
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Natív PAGE
2D
474 K
474 1a
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2D
501
488
483
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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
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Thank you.