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1

A proteomic approach analysing the Arabidopsis thaliana response to virulent and avirulent Pseudomonas syringae strains

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Sghaier-Hammami B., Redondo-Lopez I., Maldonado-Alconada A. M., Echevarria-Zomeno S., Jorrin-Novo J. V.
Acta Physiologiae Plantarum
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2012
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tom 34
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nr 3
The present work is directed at studying changes at the proteome level in Arabidopsis thaliana leaves in response to Pseudomonas syringae virulent (Pst) and avirulent (Pst avrRpt2) strains. Arabidopsis leaves were sampled from challenged plants at 4, 8 and 24 h post inoculation. Proteins were TCA–acetone–phenol extracted and subjected to 2-DE (5–8 pH range) and MS/MS (MALDI–TOF–TOF) analysis. Out of 800 matched spots on each of the 36 gels analysed, 147 spots were either absent in at least one of the conditions studied (time or treatments; qualitative variable spots) or differentially accumulated between time and treatments (quantitative variable spots). Out of the 24 proteins successfully identified over TAIR10 database, 23 have not been reported previously in similar proteomics studies of the Arabidopsis thaliana–Pseudomonas syringae interaction. The exhaustive statistical analysis performed, including principal component and heat map, showed that 24 h post inoculation can clearly discriminate the challenged plants from the control. The protein change occurred early (4 h post inoculation) following the virulent pathogen infection, whereas the change occurred later (24 h post inoculation) following the avirulent pathogen inoculation. Concerning the variable proteins, three behavioural groups can be observed: group 1 (common protein changes in response to virulent and avirulent pathogen infection), group 2 (protein changes in response to virulent pathogen infection) and group 3 (protein changes in response to avirulent pathogen infection). Differential identified proteins following the pathogen infection belonged to different groups including those of oxidative stress defence, enzymes of metabolic pathways and molecular chaperones.
2

Simple, rapid and reliable methods to obtain high quality RNA and genomic DNA from Quercus ilex L. leaves suitable for molecular biology studies

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Echevarria-Zomeno S., Abril N., Ruiz-Laguna J., Jorrin-Novo J., Maldonado-Alconada A. M.
Acta Physiologiae Plantarum
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2012
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tom 34
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nr 2
Isolation of high-quality RNA and genomic DNA (gDNA) from many samples is a necessary step before accomplishing molecular biology studies. The particular composition of Quercus ilex leaves, specially hard and rich in cell wall material, polyphenolics and secondary metabolites, usually results in preparations contaminated with non-nucleic acid compounds. Although many methods have been developed, each case of study demands a protocol adapted to the specific plant sample and the pursued research objectives. We have evaluated several protocols to establish the methodology that best suited to our current genetic and molecular studies on Q. ilex. Our priority was to select the simplest methods reducing the plant starting material and the time employed, without compromising yield, quality and integrity of the isolated nucleic acids. Our results point to two protocols based on silicamembrane purification, as the most convenient for Q. Ilex leaf tissue, and both procedures are greatly improved by adding insoluble polyvinyl polypyrrolidone during the isolation process. The protocols optimized here can be completed at the microfuge scale and allow a researcher to process 48 samples in 1 h, producing high quality preparations suitable for the routinely molecular biology applications with higher efficiency than other more labour and time-consuming protocols.
3

Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with Pseudomonas syringae

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Maldonado-Alconada A. M., Echevarria-Zomeno S., Lindermayr C., Redondo-Lopez I., Durner J., Jorrin-Novo J. V.
Acta Physiologiae Plantarum
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2011
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tom 33
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nr 4
Nitric oxide (NO) is a key signaling molecule in plants, being its biological effects mainly mediated through S-nitrosylation of cysteine thiols. Using the biotin switch method combined with mass spectrometry analysis we have identified 127 targets of S-nitrosylation in Arabidopsis cell suspension cultures and leaves challenged with virulent and avirulent isolates of Pseudomonas syringae pv. tomato. The NO targets are proteins associated with carbon, nitrogen, and sulpfur metabolism, photosynthesis, the cytoskeleton, stress-, pathogen- and redox-related and signaling proteins. Some proteins were previously identified in plants and mammals, while others (63%) represent novel targets of S-nitrosylation. Our data suggest that NO might be orchestrating the whole plant physiology, presumably through covalent modification of proteins.
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