Legumes play an essential role in agriculture due to their ability to ‘fix’ nitrogen in a symbiotic relationship with the soil bacteria known as rhizobia. This relationship is dependent on the development, and subsequent rhizobial infection, of specialized organs called nodules, which are located in the roots of the plant. Medicago truncatula is a well-established model for studying both symbiotic nitrogen fixation (SNF) and legume biology.
Modern advancements in mass spectrometer acquisition rate have enabled vast improvements in the depth and speed of large-scale proteomic analyses using liquid chromatography tandem mass spectrometry (LC-MS/MS). We have exploited these high-throughput methods to carry out a large-scale, quantitative proteomic, phospho-proteomic, and acetyl-proteomic study of Medicago truncatula to establish a global picture of protein expression in the major organs of the Medicago plant. Included in this comparison are nodule tissues harvested at 10, 14, and 28 days past rhizobial inoculation, permitting the assessment of the dynamic protein and PTM expression changes that accompany the progression of SNF. The discovery-based LC-MS/MS methods employed permit the simultaneous analysis of both nodule and rhizobial proteins over this time-course.
Utilizing isobaric tag-based technologies and deep-sequencing label-free methods, our study not only enabled the identification of over 18,000 proteins in Medicago and over 3,000 proteins in rhizobia, but also permitted the collective detection of over 21,000 sites of phosphorylation and over 500 sites of acetylation within the two species. Together, these data comprise the most holistic proteomic analysis of Medicago truncatula to date, enabling the elucidation of tissue-specific protein functionality and providing unique insight into the proteome and PTM changes that accompany the progression of SNF.