Arik Honig STSM at University of Bonn
COST-STSM-Reference number: FA0605-5155
Grantee : Arik Honig, Depertment of Plant Sciences,
Hosts: Prof. Dr. D. Bartels and Dr. H. Rohrig. Institute of Molecular Physiology and Biotechnology of Plants (IMBIO),
.Duration: 07.09.09- 12.09.09
Purpose of the Visit : Study the methods for phosphoprotein enrichment and Two dimensional (2D) gel analysis of proteins that were developed in Prof. Bartels lab by Dr. Rohrig and perform an initial estimation of the phosphoylation state of CWLP in plants grown under normal and drought stress conditions.
Objectives:
1) Perform total-protein extraction and Phosphoprotein-enrichment from leaves of CWLP::GFP-OE plants subjected to two treatments: plants grown in normal conditions and dehydrated plants.
2) Use Isoelectric focusing (ISF) and SDS-PAG (2D analysis) to compare the total Phospho-proteins pattern of the drought stressed and non-stressed plants using extracts from CWLP::GFP-OE plants.
3) Perform a series of 1D gels with Total-proteins, Phospho-proteins and Immunoprecipitated proteins to reveal possible phosphorylation of the CWLP, and compare its phosphorylation state in plants grown under normal and drought stress conditions.
Main Results
· Total protein extraction and Phospho-protein enrichment were very successful for both treatments. Coomassie staining revealed equal loading of samples (TP, PP, IP) and demonstrated the efficiency of the method of enriching Phosphoproteins. However, considering the lack of staining of the Immunoprecipitated proteins, and the detection of the CWLP::GFP band by the coomassie staining, suggest that CWLP::GFP is not phosphorylated in plants grown under both conditions.
· Western blot analysis detected the CWLP::GFP in the IP lanes (an expected 90KDa band). There was no difference between the band intensity in the two treatments, suggesting equal levels of the protein. The GFP-AB identified several bands that correspond to CWLP::GFP. This phenomenon may result from a destructive effect caused by the organic extraction steps during the TP preparation. If this is the case, it can be concluded that a different method for TP extraction should be applied in similar experiments in the future in order to maintain the integrity of the CWLP::GFP protein.
· 2D gels were stained successfully by Pro-Q. There were significant differences in the Phosphoprotein pattern between the gels (e.g. treatments). The coomassie staining confirmed that equal amounts of PP were loaded on both gels. The portion of PP in the dehydrated sample is higher than in the control sample. In many cases there was no correlation between the coomassie staining and the PP dots on the gel. The direct identification of CWLP::GFP in the 2D gels was impossible and required further probing of the SDS-PAG with anti-GFP AB.
Future collaborations with the host: The set of experiments described above was a preliminary effort to learn certain strategies in phosphoproteomics analysis, using the CWLP during normal and stressful conditions as the model. The results of those experiments have shown the feasibility of the methods and pointed the conditions for the experiments that will be carried out in the future.
References:
Röhrig H, Schmidt J, Colby T, Bräutigam A, Hufnagel P, Bartels D (2006) Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation. Plant Cell Environ. 29, 1606-1617.
Röhrig H, Colby T, Schmidt J, Harzen A, Facchinelli F, Bartels D (2008) Analysis of desiccation-induced candidate phosphoproteins from Craterostigma plantagineum isolated with a modified metal oxide affinity chromatography procedure. Proteomics. 3548-3560.
Wang W, Scali M, Vignani R, Spadafora A, Sensi E, Mazzuca S, Cresti M (2003) Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds. Electrophoresis 24, 2369-2375.
