Large-scale phosphoprotein analysis in Medicago truncatula roots provides insight into in vivo kinase activity in legumes.

Journal Article (Journal Article)

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 nitrogen-fixing 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.

Full Text

Duke Authors

Cited Authors

  • Grimsrud, PA; den Os, D; Wenger, CD; Swaney, DL; Schwartz, D; Sussman, MR; Ané, J-M; Coon, JJ

Published Date

  • January 2010

Published In

Volume / Issue

  • 152 / 1

Start / End Page

  • 19 - 28

PubMed ID

  • 19923235

Pubmed Central ID

  • PMC2799343

Electronic International Standard Serial Number (EISSN)

  • 1532-2548

Digital Object Identifier (DOI)

  • 10.1104/pp.109.149625

Language

  • eng

Conference Location

  • United States