Data-Independent Acquisition-Based Proteome and Phosphoproteome Profiling Reveals Early Protein Phosphorylation and Dephosphorylation Events in Arabidopsis Seedlings upon Cold Exposure

Abstract

Protein phosphorylation plays an important role in mediating signal transduction in cold response in plants. To better understand how plants sense and respond to the early temperature drop, we performed data-independent acquisition (DIA) method-based mass spectrometry analysis to profile the proteome and phosphoproteome of Arabidopsis seedlings upon cold stress in a time-course manner (10, 30 and 120 min of cold treatments). Our results showed the rapid and extensive changes at the phosphopeptide levels, but not at the protein abundance levels, indicating cold-mediated protein phosphorylation and dephosphorylation events. Alteration of over 1200 proteins at phosphopeptide levels were observed within 2 h of cold treatment, including over 140 kinases, over 40 transcriptional factors and over 40 E3 ligases, revealing the complexity of regulation of cold adaption. We summarized cold responsive phosphoproteins involved in phospholipid signaling, cytoskeleton reorganization, calcium signaling, and MAPK cascades. Cold-altered levels of 73 phosphopeptides (mostly novel cold-responsive) representing 62 proteins were validated by parallel reaction monitoring (PRM). In summary, this study furthers our understanding of the molecular mechanisms of cold adaption in plants and strongly supports that DIA coupled with PRM are valuable tools in uncovering early signaling events in plants.

Integrative Proteome and Phosphoproteome Profiling of Early Cold Response in Maize Seedlings

“…Interestingly, in the global proteome analysis, the control samples and the 30 min samples were mostly separated by the second component of the PCA plot, while in the phosphoproteome analysis, the control samples and the 30 min samples were separated by both the first and the second components of the PCA plots. These results suggest that the phosphoproteome responds more quickly than the global proteome, in agreement with similar studies in Arabidopsis [ 31 , 32 ].…”

“…Interestingly, there were more down-regulated proteins than up-regulated proteins observed after 30 min of cold treatment, but more up-regulated proteins after 2 h of cold treatment. The same pattern held true for phosphopeptides; there were more down-regulated phosphopeptides at 30 min while slightly more up-regulated ones in 2 h. These observations were consistent with those observed in phosphopeptides in the early cold response of Arabidopsis seedlings [ 32 ]; however, only 7 out of 6733 Arabidopsis proteins (about 1%) were observed with altered protein accumulation, even after 2 h of cold treatment, indicating that the response in the global proteome in maize is much more rapid and pronounced than that in Arabidopsis .…”

“…In the global (or total) proteome, 8885 proteins were identified at the FDR cutoff of 1%, and 8567 proteins were quantified across the nine samples (each three biological repeats from the three time points, and proteins with missing values were discarded) ( Table S1 ). In this workflow, phosphopeptides were enriched using Ga 3+ -based immobilized metal ion affinity chromatography [ 32 ]. In the phosphoproteome analysis, a total of 21,641 phosphopeptides (defined as phosphopeptide isoforms in Proteome Discoverer 2.4) ascribed to 5632 proteins were identified ( Table S1 ), and the majority of phosphopeptides (18,342 or 84.8%) featured a single p-site ( Table S1 ).…”

“…A rapid response at the phosphoproteome levels were observed in both maize and Arabidopsis in as early as 30 min. Additionally, after 2 h of cold shock, a total of 2024 phosphopeptides representing 1055 proteins were cold responsive in maize, while alterations of over 2038 phosphopeptides representing 1208 proteins at phosphopeptide levels were observed in Arabidopsis [ 32 ]. KEGG and GO enrichment analysis suggest that photosynthesis, spliceosome, endocytosis, and response to biotic and abiotic stresses were the major pathways associated with rapid cold response in maize, in agreement with similar studies in Arabidopsis [ 31 , 32 ].…”

“…Cold-induced subcellular translocation of DPE2 is involved in the rapid accumulation of maltose that acts as compatible solute to protect cells from cold damage [ 30 ], while the activation of different members of MAPKs is critical for the induction or attenuation of the expression of CBF genes [ 28 ]. Recently, two large-scale phosphoproteome profiling studies on early cold response in Arabidopsis (within 2 h of cold shock) have provided significant resources to understand early molecular events during a sudden temperature drop in plants [ 31 , 32 ]. In this study, we have performed a multiplex isobaric tandem mass tags (TMT)-based quantitative proteomics approach to uncover the early cold signaling events in maize, and we have identified similarities and differences in early cold response in maize, when compared to previous similar studies in Arabidopsis .…”