Large-Scale Phosphoprotein Analysis inMedicago truncatulaRoots Provides Insight into in Vivo Kinase Activity in Legumes  

Abstract

Nitrogen fixation in legumes requires the development of root organs called nodules and their infection by symbiotic rhizobia. Over the last decade, Medicago truncatula has emerged as a major model plant for the analysis of plant-microbe symbioses and for addressing questions pertaining to legume biology. While the initiation of symbiosis and the development of nitrogenfixing root nodules depend on the activation of a protein phosphorylation-mediated signal transduction cascade in response to symbiotic signals produced by the rhizobia, few sites of in vivo phosphorylation have previously been identified in M. truncatula. We have characterized sites of phosphorylation on proteins from M. truncatula roots, from both whole cell lysates and membrane-enriched fractions, using immobilized metal affinity chromatography and tandem mass spectrometry. Here, we report 3,457 unique phosphopeptides spanning 3,404 nonredundant sites of in vivo phosphorylation on 829 proteins in M. truncatula Jemalong A17 roots, identified using the complementary tandem mass spectrometry fragmentation methods electron transfer dissociation and collision-activated dissociation. With this being, to our knowledge, the first large-scale plant phosphoproteomic study to utilize electron transfer dissociation, analysis of the identified phosphorylation sites revealed phosphorylation motifs not previously observed in plants. Furthermore, several of the phosphorylation motifs, including LxKxxs and RxxSxxxs, have yet to be reported as kinase specificities for in vivo substrates in any species, to our knowledge. Multiple sites of phosphorylation were identified on several key proteins involved in initiating rhizobial symbiosis, including SICKLE, NUCLEOPORIN133, and INTERACTING PROTEIN OF DMI3. Finally, we used these data to create an open-access online database for M. truncatula phosphoproteomic data.Medicago truncatula has become a model for studying the biology of leguminous plants such as soybean (Glycine max), alfalfa (Medicago sativa), and clover (Trifolium spp.; Singh et al., 2007). Most members of this vast family have the ability to fix atmospheric nitrogen by virtue of an endosymbiotic association with rhizobial bacteria, through which legumes undergo nodulation, the process of forming root nodules (Jones et al., 2007). Legumes are central to modern agriculture and civilization because of their ability to grow in nitrogen-depleted soils and replenish nitrogen through crop rotation. Consequently, there is great interest in understanding the molecular events that allow legumes to recognize their symbionts, develop root nodules, and fix nitrogen. Nod factors are lipochitooligosaccharidic signals secreted by the rhizobia and are required, in most legumes, for intracellular infection and nodule development. In recent decades, an elegant combination of genetics, biochemistry, and cell biology has shown that Nod factors activate intricate signaling events within cells of legume roots, including protein phosphorylation cascades and intracellular ion flu...

LYR3, a high‐affinity LCO‐binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor

“…However, in MtLYR3, a Ser immediately precedes the region which was cloned into the vector, suggesting that this modification could also occur in MtLYR3. Three more phosphosites are localized in highly variable regions in the juxtamembrane domain (Ser314, Thr315) and the C-terminal domain (Thr628), but at different positions to those previously identified in MtLYR4, extracted from M. truncatula roots [35,36]. An additional phosphosite (Ser399) occurs in a nonconserved residue inside the core kinase-like domain (Fig.…”

“…The fourth phosphosite of MtLYR3 (Ser399) is located just after the predicted aC-helix, which is a key structural element in the N-terminal lobe of protein kinases [42]. The fifth phosphosite (Thr628) is located in the C-terminal domain of the protein, a region commonly subjected to phosphorylation [43], including in vivo for MtLYR4 and the Arabidopsis LRR-RLK, AtBRI1 [35,44]. Taken together, the phosphorylation of MtLYR3 by MtLYK3 revealed the existence of phosphosites that could be of relevance in a biological context.…”

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.…”

“…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].…”

Phosphoproteomic Analysis of Seed Maturation in Arabidopsis, Rapeseed, and Soybean

“…The coupling of phosphopeptide enrichment strategies to high-resolution mass spectrometry has led to a renaissance in phosphorylation site mapping, and several recent large-scale studies have expanded the knowledge of phosphorylation in plants (Sugiyama et al, 2008;Reiland et al, 2009;Grimsrud et al, 2010;Nakagami et al, 2010). These studies discovered phosphorylation sites for hundreds of phosphorylated proteins in roots, shoots, and cell culture and suggested roles for phosphorylation in chloroplasts and root nodulation (Reiland et al, 2009;Grimsrud et al, 2010).…”