Large-Scale Comparative Phosphoproteomics Identifies Conserved Phosphorylation Sites in Plants    

Abstract

Knowledge of phosphorylation events and their regulation is crucial to understand the functional biology of plants. Here, we report a large-scale phosphoproteome analysis in the model monocot rice (Oryza sativa japonica 'Nipponbare'), an economically important crop. Using unfractionated whole-cell lysates of rice cells, we identified 6,919 phosphopeptides from 3,393 proteins. To investigate the conservation of phosphoproteomes between plant species, we developed a novel phosphorylation-site evaluation method and performed a comparative analysis of rice and Arabidopsis (Arabidopsis thaliana). The ratio of tyrosine phosphorylation in the phosphoresidues of rice was equivalent to those in Arabidopsis and human. Furthermore, despite the phylogenetic distance and the use of different cell types, more than 50% of the phosphoproteins identified in rice and Arabidopsis, which possessed ortholog(s), had an orthologous phosphoprotein in the other species. Moreover, nearly half of the phosphorylated orthologous pairs were phosphorylated at equivalent sites. Further comparative analyses against the Medicago phosphoproteome also showed similar results. These data provide direct evidence for conserved regulatory mechanisms based on phosphorylation in plants. We also assessed the phosphorylation sites on nucleotide-binding leucinerich repeat proteins and identified novel conserved phosphorylation sites that may regulate this class of proteins.

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

“…By genome annotation, protein kinases were found to make up about 5.5% of the Arabidopsis genome [4], which is nearly twice of that in human [5], indicating the high specificity and a complex network of phosphorylation events in plants [4]. In recent years, benefitting from the new advances in proteomics technologies including phosphopeptide enrichment, high-accuracy mass spectrometry (MS), and associated bioinformatics, large-scale analyses of protein phosphorylation have been carried out in variety of plant species including Medicago Truncatula [6], Oryza sativa [7], Glycine max [8], Brassica napus [8], and Arabidopsis thaliana [8][9][10][11][12][13][14][15][16][17][18]. However, our understanding of plant phosphoproteomes remains very limited with respect to their complexity and functions.…”

“…In Arabidopsis phosphoproteome, nearly 500,000 potential phosphorylation sites have been predicted with high confidence [4]; however, currently only about 14,000 were identified by MS-based experiments [16]. In addition, the current identification capacity of phosphorylation sites from a single MS run in plants is only about 100-200 [7,9,13]. This low coverage and throughput reflect that there is a high demand in developing more efficient phosphopeptide enrichment strategies to facilitate the large-scale profiling of phosphorylation events in plants.…”

“…First, the Ti 4+ -IMAC was used for phosphopeptide enrichment, and then the online MudPIT equipped with a RP-SCX biphasic column was employed for phosphopeptide fractionation. Through two duplicated MS runs, a total of 5348 unique phosphopeptides were identified, which represent 2552 unique phosphoproteins and are among the largest phosphorylation data sets reported in plants by far [7][8][9][10][11][12][13][14][15][16][17]. Further phosphorylation motif and pathway analysis revealed known and novel kinase recognition motifs and provided new insights into the phosphorylation network regulating both metabolic and signaling pathways.…”

Structure, function, and evolution of plant NIMA-related kinases: implication for phosphorylation-dependent microtubule regulation

“…These modifications affect the microtubule dynamics, MAP binding affinity to microtubules, and microtubule motor activity. Sirajuddin et al (2014) analyzed the motility of kinesins on microtubules, which were composed of homogeneous Plant tubulin phosphorylation has been detected via the phosphoproteomics and immunological studies (Sugiyama et al 2008; Blume et al 2008;Nakagami et al 2010).…”

“…These modifications affect the microtubule dynamics, MAP binding affinity to microtubules, and microtubule motor activity. Sirajuddin et al (2014) analyzed the motility of kinesins on microtubules, which were composed of homogeneous Plant tubulin phosphorylation has been detected via the phosphoproteomics and immunological studies (Sugiyama et al 2008; Blume et al 2008;Nakagami et al 2010).Although the biological roles of tubulin phosphorylation are not clear, identification of several tubulin kinases, as well as stress-inducible phosphorylation of α-tubulin, shed new light on (Ben-Nissan et al 2008; Motose et al 2011; Ban et al 2013; Fujita et al 2013). In this review, We aim to provide an overview of phosphorylation-dependent microtubule regulation with special emphasis on Never in mitosis A (NIMA)-related kinases (NEKs).…”