Large-Scale Arabidopsis Phosphoproteome Profiling Reveals Novel Chloroplast Kinase Substrates and Phosphorylation Networks  

Abstract

We have characterized the phosphoproteome of Arabidopsis (Arabidopsis thaliana) seedlings using high-accuracy mass spectrometry and report the identification of 1,429 phosphoproteins and 3,029 unique phosphopeptides. Among these, 174 proteins were chloroplast phosphoproteins. Motif-X (motif extractor) analysis of the phosphorylation sites in chloroplast proteins identified four significantly enriched kinase motifs, which include casein kinase II (CKII) and proline-directed kinase motifs, as well as two new motifs at the carboxyl terminus of ribosomal proteins. Using the phosphorylation motifs as a footprint for the activity of a specific kinase class, we connected the phosphoproteins with their putative kinases and constructed a chloroplast CKII phosphorylation network. The network topology suggests that CKII is a central regulator of different chloroplast functions. To provide insights into the dynamic regulation of protein phosphorylation, we analyzed the phosphoproteome at the end of day and end of night. The results revealed only minor changes in chloroplast kinase activities and phosphorylation site utilization. A notable exception was ATP synthase b-subunit, which is found phosphorylated at CKII phosphorylation sites preferentially in the dark. We propose that ATP synthase is regulated in cooperation with 14-3-3 proteins by CKII-mediated phosphorylation of ATP synthase b-subunit in the dark.

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.…”

“…Typically, a low-pH offline strong cation exchange (SCX) LC is used for LC-based phosphopeptide enrichment and fractionation. The principle is that under the acidic pH condition (pH 2.7), the negatively charged phosphopeptides tend to elute earlier and are thus separated from the majority of nonphosphopeptides [9,14,21]. However, there are some clear drawbacks for these offline LC separation methods such as time-consuming procedures and big sample loss caused by repeated desalting and lyophilization steps.…”

“…The most commonly used affinity-based methods are the (Fe 3+ )-based immobilized metal affinity chromatography (Fe 3+ -IMAC) [6,21,[26][27][28] and titanium dioxide (TiO 2 ) affinity chromatography [9,14,29,30]. Recently, on the basis of these methods, some new enrichment strategies have been developed by adopting different metal oxides (ZrO 2 and Nb 2 O 5 ) or IMAC with alternative metal ions (Ga 3+ , Zr 4+ , and Ti 4+ ) [19,20].…”

“…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.…”