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

Jinjuan Tan,Yujie Niu,Zhongjing Zhou,Zhiping Deng,Hanqian Feng,Jiayun Xing

doi:10.3390/ijms222312856

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.

Highlights

Cold stress, which is generally categorized into chilling stress (0–15 ◦C) and freezing stress (
Among the 24,277 unique high-confidence p-sites [29], 86.9% sites of phosphorylation were localized at serine residues, 12.8% were at threonine residues, and 0.2% were at tyrosine residues, which showed a similar residues phosphorylation proportion to the recent report of Arabidopsis phosphoproteome (86.0% at serine, 13.64% at threonine and 0.36% at tyrosine residues) [30]
Comparison of the p-sites identified in this study with a recent large-scale phosphoproteomics profiling in Arabidopsis [30] showed that only 61% of the high-confidence p-sites identified in this study were in the class I p-sites [31], identified by Mergner et al (Figure 2B and Table S2), supporting that Ga3+-based immobilized metal affinity chromatography (Ga-IMAC) and Fe-IMAC are complementary in providing a more comprehensive mapping of the phosphoproteome [32]