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.

Fig 1. Phosphorylation of SNF1-related kinase (Medtr6g01290) upon treatment with NF.

A) Representative chromatograms for SNF1-related kinase in the mock-treated samples. Blue trace corresponds to the heavy standard, and red corresponds to the light endogenous peptide. B) SNF1 showed changes in phosphorylation for site s364 after 15 min of treatment with NF in wild-type seedlings. Two closely related phosphopeptides (s359 and s364) were measured in all samples. C) Protein domain structure of the SNF1-related kinase. Only site s364, which showed significant phosphorylation in wild-type seedlings, is represented here. D) Amino acid sequence alignment of various plant SNF1-related kinases. Red box indicates the two phosphorylation sites s359 and s364. Site s359 is specific to legumes except for Glycine max, whereas site s364 is conserved in legumes and non-legumes, with the exception of Hordeum vulgare. E) One-hour time course to monitor SNF1-related kinase phosphorylation in wild-type seedlings, along with the 15-min time point for nfp, dmi2 and dmi3 mutants. In addition, for the dmi3 mutants, the phosphorylation changes were also monitored at 60 min. A 2-fold increase in phosphorylation was observed in NF-treated wild-type (A17) seedlings within the first 15 min; by 30 min, there was no difference between the treated and control samples. Additionally, at 15 min, all three mutants clustered together near a 1.3-fold increase in phosphorylation in treated seedlings. Error bar represents standard error of mean. F) Phylogenetic analysis of the amino acid sequences of SNF1-related kinases using Mr. Bayes method. The analysis indicates that monocots clustered together, and within the dicots, the non-symbiotic Arabidopsis plant formed a separate clade. M. truncatula clustered together with the other legumes.

Fig 2. Phosphorylation of other peptides that exhibited significant differences upon treatment with NF.

A) Zinc finger peptide displays a similar response profile as that of SNF1-related kinase to NF treatment in wild-type seedlings in the first 30 min; by 60 min, the treated seedlings begin to exhibit a decrease in phosphorylation. In the mutant seedlings at 15 min the peptide displays a differential phosphorylation decrease with NF treatment compared to the wild-type treated with Nod factors. B) Phosphorylation of the s8 residue of MtAHA4 increases significantly at the 15-minute sampling point and then decreases significantly at 30 min in wild-type seedlings. C) This peptide was derived from the hypothetical protein Medtr8g104290 of unknown function. NF treatment results in significant increase in phosphorylation at this site in wild-type seedlings at 15 min. D) Similar to Medtr8g104290, the peptide that derived from Medtr7g068220 (Unknown function) also exhibited a significant change in phosphorylation level at the 15-min time point for the wild-type in comparison to the mutants. Error bar represents standard error of mean for all these figures.