title: Mass Spectrometric-Based Selected Reaction Monitoring of Protein Phosphorylation during Symbiotic Signaling in the Model Legume, Medicago truncatula.
abstract: Unlike the major cereal crops corn, rice, and wheat, leguminous plants such as soybean and alfalfa can meet their nitrogen requirement via endosymbiotic associations with soil bacteria. The establishment of this symbiosis is a complex process playing out over several weeks and is facilitated by the exchange of chemical signals between these partners from different kingdoms. Several plant components that are involved in this signaling pathway have been identified, but there is still a great deal of uncertainty regarding the early events in symbiotic signaling, i.e., within the first minutes and hours after the rhizobial signals (Nod factors) are perceived at the plant plasma membrane. The presence of several protein kinases in this pathway suggests a mechanism of signal transduction via posttranslational modification of proteins in which phosphate is added to the hydroxyl groups of serine, threonine and tyrosine amino acid side chains. To monitor the phosphorylation dynamics and complement our previous untargeted 'discovery' approach, we report here the results of experiments using a targeted mass spectrometric technique, Selected Reaction Monitoring (SRM) that enables the quantification of phosphorylation targets with great sensitivity and precision. Using this approach, we confirm a rapid change in the level of phosphorylation in 4 phosphosites of at least 4 plant phosphoproteins that have not been previously characterized. This detailed analysis reveals aspects of the symbiotic signaling mechanism in legumes that, in the long term, will inform efforts to engineer this nitrogen-fixing symbiosis in important non-legume crops such as rice, wheat and corn.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, 53706, United States of America.
authors.forename: Lori K
authors.initials: LK
authors.lastname: Van Ness
authors.affiliation: Department of Agronomy, University of Wisconsin-Madison, Madison, WI, 53706, United States of America.
authors.forename: Dhileepkumar
authors.initials: D
authors.lastname: Jayaraman
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, 53706, United States of America.
authors.forename: Junko
authors.initials: J
authors.lastname: Maeda
authors.affiliation: Mass Spectrometry/Proteomics Facility, University of Wisconsin-Madison, Madison, WI, 53706, United States of America.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Barrett-Wilt
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, 53706, United States of America.|Biotechnology Center, University of Wisconsin-Madison, Madison, WI, 53706, United States of America.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sussman
authors.affiliation: Department of Agronomy, University of Wisconsin-Madison, Madison, WI, 53706, United States of America.|Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, 53706, United States of America.
authors.forename: Jean-Michel
authors.identifier: 0000-0002-3128-9439
authors.initials: JM
authors.lastname: Ané
mesh_terms: D018506:Gene Expression Regulation, Plant; D013058:Mass Spectrometry / Q000379:methods*; D046913:Medicago truncatula / Q000378:metabolism*; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism*; D012231:Rhizobium / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D013559:Symbiosis / Q000502:physiology
chemical_list: D010940:Plant Proteins