Oxidative Stress and Plant Proteomics Group

 

Sabine Lüthje
 Dr.rer.nat.habil.
 Private Lecturer


 

Structure and Function of Plasma Membrane Redox Systems

The plasma membrane builds the outer permeability barrier of the cell that achieves important functions in cell signaling, transport processes, plant development and stress response. About 40 years ago, a constitutive transmembrane electron transport system - the so-called standard system - was demonstrated for plasma membranes of eucaryotic cells. Besides the standard system, a second transmembrane electron transport system was demonstrated in root plasma membranes of non-grass monocots and dicots that is induced by iron deficiency (so-called turbo system or ferric-chelate reductase, FRO).

Several redox proteins could be identified in highly enriched plasma membrane fractions prepared by aqueous polymer two-phase partitioning (APTPP): b-type cytochromes and NAD(P)H oxidoreductases. The structure, electron transfer mechanism and function of plasma membrane-bound redox systems are one of our major topics.

An increasing evidence suggests association of plasma membrane redox components with microdomains (so-called lipid rafts) and protein assemblies. Beside physicochemical studies and lipid analysis of plasma membranes, proteomic approches were established in our laboratory to investigate the participation of redox proteins in putative protein assemblies. Both, blue native and clear native electrophoresis (hrCNE) confirmed an interaction of redox proteins with high molecular mass protein assemblies. Composition of those complexes depends on the state of development, physiological conditions and plant material investigated.

Class III peroxidases (secretory pathway)

Our team was the first that identified membrane bound heme peroxidases in plant plasma membranes. Class III peroxidases are a multigene family with at least 143 isoenzymes in maize. It appears that these enzymes build a cellular network of ROS scavenging and producing enzymes (PeroxiNET) that has to be tightly regulated. We established protocols for cell fractionation and gel-based proteomics to investigate the regulation and function of peroxidases within this network in plant development and oxidative stress. Beside studies on the structure and localization of these proteins by in silico analysis and GFP-fusions, heterologous expression and gene silencing (RNAi) are used for functional analysis.

Oxidative stress and plant development

Plants are sessil organisms that have to cope with multiple abiotic and biotic stress factors during their life cycle. We are interested in plant nutrition, toxic elements, weather extremes and multiple stresses that occurred during plant development.

Phenotyping

To study stress response and adaptation of plants setups for phenotyping rosette-like structures were established. Pictures are taken from Arabidopsis, Medicago or Mesembryanthemum with digital cameras from the top every 10-20 minutes during growth and stress treatment. For data evaluation pictures were analyzed using Image-J 1.49t Fiji software and a self-made plugin (S.Fraas, University of Hamburg). By defining HSV settings, identification of the leaves allows automatic analysis of all pictures. Different parameters (e.g. perimeter, major axis) can be quantified and assessed. Experimental setup for non-rosette-like structures is also available. Beside morphological monitoring, phenotyping on cellular level (physiological, histological and biochemical data) is routinely done.

Heavy metals

Studies of our team showed alterations in the plasma membrane proteome of iron uptake strategy I and strategy II plants (pea and maize) by both iron deficiency and iron toxicity for the first time. Peroxidases, redox systems and several other proteins are regulated by these stressors. The combination of iron deficiency with elicitor treatment support important functions of heme containing enzymes, but also of other plasma membrane proteins, in pathogen response.

Emissions of industrial activities cause accumulation of cadmium and other toxic elements in the environment. Ions of these elements are taken up by unspecific transporters. Inside the cell, they cause oxidative stress and thereby changes in the cellular redox state. The molecular mechanisms of these reactions are not very well understood. So far, total peroxidase activity of samples is used as a general stress marker for many factors. My team established protocols to study the regulation of different peroxidase isoenzymes side by side in the same sample. We found that abundance and activity of different soluble, cell wall and membrane bound class III peroxidases changed under cadmium exposure.

Weather extremes

Due to changing climate heat, drought and flooding events increases in northern hemisphere. Additionally, the use of heavy machinery in agriculture cause soil compaction, which supports waterlogging. Flooding provokes alterations in pH and oxygen levels (hypoxia and anoxia) in the soil and thereby iron toxicity. A recent study of our team showed alterations in abundance and activity of soluble class III peroxidases in maize by waterlogging.

 
tb-root
Redox staining
APTPP
APTPP
PM
Plasma membranes
hrCNE
hrCNE
peroxidase
In silico analysis
Micro assay
Micro assay
phenotyping
Phenotyping

iron deficiency
Iron deficiency
Turbo system
Induction of FRO
2D-PAGE
2D-PAGE
flooding
native IEF-PAGE


DFG   DAAD   Xunta da Galicia   
COST   Erasmus   

 
 

 
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