Comparative Qualitative Phosphoproteomics Analysis Identifies Shared Phosphorylation Motifs and Associated Biological Processes in Flowering Plants

Phosphorylation is regarded as one of the most prevalent post-translational modifications and plays a key role in regulating cellular processes. In this work we carried out a comparative bioinformatics analysis of phosphoproteomics data, to profile two model species representing the largest subclasses in flowering plants the dicot Arabidopsis thaliana and the monocot Oryza sativa, to understand the extent to which phosphorylation signaling and function is conserved across evolutionary divergent plants. Using pre-existing mass spectrometry phosphoproteomics datasets and bioinformatic tools and resources, we identified 6,537 phosphopeptides from 3,189 phosphoproteins in Arabidopsis and 2,307 phosphopeptides from 1,613 phosphoproteins in rice. The relative abundance ratio of serine, threonine, and tyrosine phosphorylation sites in rice and Arabidopsis were highly similar: 88.3: 11.4: 0.4 and 86.7: 12.8: 0.5, respectively. Tyrosine phosphorylation shows features different from serine and threonine phosphorylation and was found to be more frequent in doubly-phosphorylated peptides in Arabidopsis. We identified phosphorylation sequence motifs in the two species to explore the similarities, finding nineteen pS motifs and two pT motifs that are shared in rice and Arabidopsis; among them are five novel motifs that have not previously been described in both species. The majority of shared motif-containing proteins were mapped to the same biological processes with similar patterns of fold enrichment, indicating high functional conservation. We also identified shared patterns of crosstalk between phosphoserines with motifs pSXpS, pSXXpS and pSXXXpS, where X is any amino acid, in both species indicating this is an evolutionary conserved signaling mechanism in flowering plants. However, our results are suggestive that there is greater co-occurrence of crosstalk between phosphorylation sites in Arabidopsis, and we were able to identify several pairs of motifs that are statistically significantly enriched to co-occur in Arabidopsis proteins, but not in rice.