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.
Highlights
Most plants belonging to the legume family utilize symbiotic associations with nitrogen—fixing bacteria called rhizobia, to meet their nitrogen requirement [1, 2]
The nfp and dmi2 mutants were Nod factors (NF)-treated for 15 min and the dmi3 mutant was NF-treated for 15 min and 60 min
In our prior work [26], an isobaric tagging strategy was used to probe the effects on phosphorylation of Nod factor treatment in wild type and mutant plants grown by different conditions and across several time points, This earlier work used a combination of isobaric tagging reagents and fragmentation mechanisms, including ETD, CID, and HCD
Summary
Most plants belonging to the legume family utilize symbiotic associations with nitrogen—fixing bacteria called rhizobia, to meet their nitrogen requirement [1, 2] In this mutualistic association, rhizobia reside in specialized organs called nodules that are formed in the plant roots, where they fix atmospheric dinitrogen into plant-available ammonia, in exchange for a carbon source [1, 3]. Rhizobia reside in specialized organs called nodules that are formed in the plant roots, where they fix atmospheric dinitrogen into plant-available ammonia, in exchange for a carbon source [1, 3] This interaction is highly specific and is facilitated by the exchange of chemical signals between plant and microbes. Root hair swelling followed by root hair deformation occurs within one hour of NF application [9, 10]
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