Phosphoproteomic Analyses Reveal Early Signaling Events in the Osmotic Stress Response    

Abstract

Elucidating how plants sense and respond to water loss is important for identifying genetic and chemical interventions that may help sustain crop yields in water-limiting environments. Currently, the molecular mechanisms involved in the initial perception and response to dehydration are not well understood. Modern mass spectrometric methods for quantifying changes in the phosphoproteome provide an opportunity to identify key phosphorylation events involved in this process. Here, we have used both untargeted and targeted isotope-assisted mass spectrometric methods of phosphopeptide quantitation to characterize proteins in Arabidopsis (Arabidopsis thaliana) whose degree of phosphorylation is rapidly altered by hyperosmotic treatment. Thus, protein phosphorylation events responsive to 5 min of 0.3 M mannitol treatment were first identified using 15 N metabolic labeling and untargeted mass spectrometry with a high-resolution ion-trap instrument. The results from these discovery experiments were then validated using targeted Selected Reaction Monitoring mass spectrometry with a triple quadrupole. Targeted Selected Reaction Monitoring experiments were conducted with plants treated under nine different environmental perturbations to determine whether the phosphorylation changes were specific for osmosignaling or involved cross talk with other signaling pathways. The results indicate that regulatory proteins such as members of the mitogen-activated protein kinase family are specifically phosphorylated in response to osmotic stress. Proteins involved in 59 messenger RNA decapping and phosphatidylinositol 3,5-bisphosphate synthesis were also identified as targets of dehydration-induced phosphoregulation. The results of these experiments demonstrate the utility of targeted phosphoproteomic analysis in understanding protein regulation networks and provide new insight into cellular processes involved in the osmotic stress response.

Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants*

“…The median represents this variability, which may occur at any step following protein extraction or be biological variation inherent to an in vivo system. This result is consistent with the previously published study, which indicated that statistical significance decreases drastically when monitored phosphorylation fold changes are Յ1.5 (29). DISCUSSION We used deep phosphoproteomic analyses to determine what ABA-regulated phosphorylation occurs in WT A. thaliana within 5 min of ABA treatment, a time point considerably earlier than has been previously examined in depth.…”

“…This CV value may explain why smaller magnitude fold changes are not reproduced; variation associated with experimental processing and biological variability is 20%, convoluting reproducible determination of smaller magnitude fold changes when performing targeted analyses using the described pipeline. This result is in line with a recent publication from this laboratory, which showed that for fold changes determined by SRM to be Յ1.5, statistical significance greatly decreases (29). Interestingly, when examining the targeted dataset, higher biological CVs were obtained when comparing the mutant data to the WT data (Table S3), suggesting a higher degree of variability in general in the mutant background, although the cause of this remains unclear.…”

“…1). The largest ABA-responsive phosphorylation increases were on the three SnRK2 kinases responsible for ABA signaling, corroborating data from a recently published targeted study from this laboratory focusing on crosstalk between signaling pathways (29). We also identified a phosphorylation event ambiguous between SnRK2.1, 4, 5, and 10 that increased in response to ABA treatment.…”

“…This panel included 20 of the SRM peptides previously analyzed and 35 from a recently published targeted study from this laboratory (29). A median coefficient of variation of 20% was observed after data were processed identically to the ABA experiment (Fig.…”

Rapid Oligo-Galacturonide Induced Changes in Protein Phosphorylation in Arabidopsis

“…The experiment was performed in triplicate. To control for any possible effects of the isotope, the N 14 and N 15 were switched between the treatment type and the experiment was performed in triplicate again, for a total of six biological replicates (18,(47)(48)(49). From the peptides identified (Supplemental Table 1), phosphopeptides were selected that appeared in at least one of either experimental set's replicates and whose fold change had an average value between experimental sets of Ն1.2.…”