A novel strategy to uncover specific GO terms/phosphorylation pathways in phosphoproteomic data in Arabidopsis thaliana

Abstract

Background Proteins are the workforce of the cell and their phosphorylation status tailors specific responses efficiently. One of the main challenges of phosphoproteomic approaches is to deconvolute biological processes that specifically respond to an experimental query from a list of phosphoproteins. Comparison of the frequency distribution of GO (Gene Ontology) terms in a given phosphoproteome set with that observed in the genome reference set (GenRS) is the most widely used tool to infer biological significance. Yet, this comparison assumes that GO term distribution between the phosphoproteome and the genome are identical. However, this hypothesis has not been tested due to the lack of a comprehensive phosphoproteome database. Results In this study, we test this hypothesis by constructing three phosphoproteome databases in Arabidopsis thaliana: one based in experimental data (ExpRS), another based in in silico phosphorylation protein prediction (PredRS) and a third that is the union of both (UnRS). Our results show that the three phosphoproteome reference sets show default enrichment of several GO terms compared to GenRS, indicating that GO term distribution in the phosphoproteomes does not match that of the genome. Moreover, these differences overshadow the identification of GO terms that are specifically enriched in a particular condition. To overcome this limitation, we present an additional comparison of the sample of interest with UnRS to uncover GO terms specifically enriched in a particular phosphoproteome experiment. Using this strategy, we found that mRNA splicing and cytoplasmic microtubule compounds are important processes specifically enriched in the phosphoproteome of dark-grown Arabidopsis seedlings. Conclusions This study provides a novel strategy to uncover GO specific terms in phosphoproteome data of Arabidopsis that could be applied to any other organism. We also highlight the importance of specific phosphorylation pathways that take place during dark-grown Arabidopsis development.

Radip Light-Induced Phosphorylation Changes in Microtubule Related Proteins in Arabidopsis

“…Many of the phosphosites identified in this study have already been reported as early light-induced ones: CRY2 is phosphorylated by light in Yp 604 (p-value = 0.017) (Table 1) (Liu et al, 2017), while PHOT1 is phosphorylated in Sp 13 (p-value = 10 0.014), Sp 23 (p-valor = 0.008), Sp 382 (p-valor = 0.051) and Sp 409 (p-valor = 0.022) (Table 1) (Inoue et al, 2008;Sullivan et al, 2008;Deng et al, 2014). In a previous study we found that the GO (Gene Ontology) terms "regulation of mRNA splicing" and "cytoplasmic MTs compounds" were specifically enriched in the phosphoproteome of dark-grown Arabidopsis seedlings (Arico et al, 2021). None of the 40 unique phosphoproteins related to "regulation of mRNA splicing" identified in darkness (Arico et al, 2021) changes their phosphorylation status after 20 min of light.…”

“…In a previous study we found that the GO (Gene Ontology) terms "regulation of mRNA splicing" and "cytoplasmic MTs compounds" were specifically enriched in the phosphoproteome of dark-grown Arabidopsis seedlings (Arico et al, 2021). None of the 40 unique phosphoproteins related to "regulation of mRNA splicing" identified in darkness (Arico et al, 2021) changes their phosphorylation status after 20 min of light.…”

“…Total protein extraction, trypsin digestion of proteins, phosphopeptides enrichment and LC-MS/MS (Supplemental figure 1) is fully described in Arico et al, 2021. Three biological replicates per condition were analyzed.…”

Arabidopsis hypocotyl growth in darkness requires the phosphorylation of a microtubule‐associated protein

“…Total protein extraction, trypsin digestion of proteins, phosphopeptides enrichment and LC–MS/MS (Figure S1) is fully described in Arico et al. (2021). Three biological replicates per condition were analyzed.…”

“…In a previous study we found that the Gene Ontology terms "regulation of mRNA splicing" and "cytoplasmic microtubule compounds" were specifically enriched in the phosphoproteome of dark-grown Arabidopsis seedlings (Arico et al, 2021). None of the 40 unique phosphoproteins related to "regulation of mRNA splicing" identified in darkness changes their phosphorylation status after 20 min of light (Arico et al, 2021). The bioinformatic tool MAPanalyzer allows the analysis of known or predicted ones (Zhou et al, 2015).…”