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 leucine-rich repeat proteins and identified novel conserved phosphorylation sites that may regulate this class of proteins.
Highlights
Knowledge of phosphorylation events and their regulation is crucial to understand the functional biology of plants
In order to perform comparative analyses of rice and Arabidopsis phosphoproteomes, a large-scale data set of rice phosphorylation sites was collected from nonstimulated suspension-cultured rice cells using three different phosphopeptide enrichment methods, lactic acid-modified titania and b-hydroxypropanoic acidmodified zirconia-utilized hydroxy acid-modified metal oxide chromatography (Ti-HAMMOC and ZrHAMMOC) and Fe(III)-immobilized metal-ion affinity chromatography (Fe-IMAC), as detailed in our previous Arabidopsis phosphoproteome study (Sugiyama et al, 2008)
Four replicates of the analysis were performed with each method, and a total of 32 liquid chromatographymass spectrometry (LC-MS) runs were performed for each sample
Summary
Knowledge of phosphorylation events and their regulation is crucial to understand the functional biology of plants. Recent progress in phosphoproteomics technology paved the way for the identification of a few thousand phosphorylation sites from unfractionated plant cells by simple one-step phosphopeptide enrichment methods, and we have found more than 2,000 phosphorylation sites in Arabidopsis (Sugiyama et al, 2008). We identified a large number of conserved phosphorylation sites in rice and Arabidopsis, providing, to our knowledge, the first overview of phosphoproteome conservation between dicots and monocots. Using this information, we identified conserved phosphorylation sites in nucleotide-binding leucine-rich repeat (NB-LRR) proteins, most of which are located to important regions for conferring resistance against pathogens. Comparative analyses against the recently published Medicago phosphoproteome revealed highly conserved phosphorylation sites in three distinct plant species
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