Soybean seed germination LC-MS/MS
This study aims at addressing soybean seeds (variety Absolute RR) germination, at 48h, during optimal and salt stressed condition when treated with bacterial signal compounds lipo-chitooligosaccharide (LCO) and thuricin 17 (Th17). Soybean growth is negatively affected when exposed to 40 mM NaCl and exposure to 80 mM NaCl is often lethal. When treated with the bacterial signal compounds lipo-chitooligosaccharide (LCO) and thuricin 17 (Th17), soybean seeds (variety Absolute RR) responded positively at salt stress of up to 150 mM NaCl. Shotgun proteomics of unstressed and 100 mM NaCl stressed seeds (48 h) in combination with the LCO and Th17 revealed many known, predicted, hypothetical and unknown proteins. In all, carbon, nitrogen and energy metabolic pathways were affected under both unstressed and salt stressed conditions when treated with signals. PEP carboxylase, Rubisco oxygenase large subunit, pyruvate kinase, and isocitrate lyase were some of the noteworthy proteins enhanced by the signals, along with antioxidant glutathione-S-transferase and other stress related proteins. These findings suggest that the germinating seeds alter their proteome based on bacterial signals and on stress, the specificity of this response plays a crucial role in organ maturation and transition from one stage to another in the plants life cycle; understanding this response is of fundamental importance in agriculture and, as a result, global food security.
Sample Processing Protocol
For the proteome analysis, germinated seeds from the unstressed condition, including the water-only control, 10-6 M LCO, 10-9 M Th17, and the salt stressed condition, including 100 mM NaCl as the salt-stressed control, and combinations of 100 mM NaCl with 10-6 M LCO and 10-9 M Th17, were sampled and total proteins extracted using a protein extraction kit (Cat. no. PE-0230, Plant total protein extraction kit, Sigma-Aldrich, Co., St. Louis, MO, USA). The total proteins extracted were then digested with trypsin and subjected to LC-MS/MS using a Velos Orbitrap instrument (Thermo Fisher, MA, USA). Tandem mass spectra were extracted, charge state deconvoluted and deisotoped and all MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.3.02). Mascot was set up to search the Soybean_20120801 database (unknown version, 80416 entries) assuming the digestion enzyme trypsin. Mascot searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 15 PPM. Carbamidomethyl of cysteine was specified in Mascot as a fixed modification. Oxidation of methionine was specified in Mascot as a variable modification.
Data Processing Protocol
Scaffold (version Scaffold 4), Proteome Software Inc., Portland, OR), was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0 % probability, as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99.0 % probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides, and could not be differentiated based on MS/MS analysis alone, were grouped to satisfy the principle of parsimony. Scaffold 4 was used to analyze the proteomics data for fold change and Fisher’s exact test of the identified proteins, after subjecting the quantitative value of the spectra to the embedded normalization. The FASTA file generated was analyzed using Blast2GO-Pro 3.1.3, for the functional annotation and analysis of the protein sequences. Apart from these, Enzyme code (EC), KEGG maps and InterPro motifs were queried directly using the InterProScan web service.
Sowmyalakshmi Subramanian, McGill University
Donald L Smith, James McGill Professor Director and CEO, BioFuelNet Canada Director, McGill Network for Innovation in Biofuels and Bioproducts Director, Eastern Canadian Oilseeds Development Initiative Plant Science Department, McGill University 21111 Lakeshore Road Ste. Anne de Bellevue, Quebec Canada H9X3V9 Phone: +1 514 398 7866 Fax: +1 514 398 7990 Website: www.mcgill.ca/plant/faculty/smith/ ( lab head )
Subramanian S, Ricci E, Souleimanov A, Smith DL. A Proteomic Approach to Lipo-Chitooligosaccharide and Thuricin 17 Effects on Soybean GerminationUnstressed and Salt Stress. PLoS One. 2016 Aug 25;11(8):e0160660. eCollection 2016 PubMed: 27560934