Integrated analysis of zone-specific protein and metabolite profiles within nitrogen-fixing Medicago truncatula-Sinorhizobium medicae nodules

Abstract

Symbiotic nitrogen fixation (SNF) between rhizobia and legumes requires metabolic coordination within specialized root organs called nodules. Nodules formed in the symbiosis between S. medicae and barrel medic (M. truncatula) are indeterminate, cylindrical, and contain spatially distinct developmental zones. Bacteria in the infection zone II (ZII), interzone II-III (IZ), and nitrogen fixation zone III (ZIII) represent different stages in the metabolic progression from free-living bacteria into nitrogen fixing bacteroids. To better understand the coordination of plant and bacterial metabolism within the nodule, we used liquid and gas chromatography coupled to tandem mass spectrometry (MS) to observe protein and metabolite profiles representative of ZII, IZ, ZIII, whole-nodule, and primary root. Our MS-based approach confidently identified 361 S. medicae proteins and 888 M. truncatula proteins, as well as 160 metabolites from each tissue. The data are consistent with several organ- and zone-specific protein and metabolite localization patterns characterized previously. We used our comprehensive dataset to demonstrate how multiple branches of primary metabolism are coordinated between symbionts and zones, including central carbon, fatty acid, and amino acid metabolism. For example, M. truncatula glycolysis enzymes accumulate from zone I to zone III within the nodule, while equivalent S. medicae enzymes decrease in abundance. We also show the localization of S. medicae's transition to dicarboxylic acid-dependent carbon metabolism within the IZ. The spatial abundance patterns of S. medicae fatty acid (FA) biosynthesis enzymes indicate an increased demand for FA production in the IZ and ZIII as compared to ZI. These observations provide a resource for those seeking to understand coordinated physiological changes during the development of SNF.

Characterization of the Sinorhizobium meliloti HslUV and ClpXP Protease Systems in Free-Living and Symbiotic States

“…Interestingly, HslU-and HslV-eGFP also appeared to form punctate loci, although to a lesser extent than ClpXP1-eGFP. The lack of zone specificity for HslUV-eGFP and ClpXP1-eGFP was consistent with previous studies (8,9).…”

“…HslUV and ClpXP protein distributions are not zone specific. Nodules formed in the symbiosis between M. sativa and S. meliloti are indeterminate and form developmental zones along the nodule longitudinal axis, each containing unique protein, metabolite, and transcript profiles (8,9). Therefore, if HslUV and ClpXP proteins accumulate in particular zones within the nodule, this may indicate that they play a specific role in SNF development or that their protein-protein interactions would be limited to gene products in that region.…”

“…Relative to free-living bacteria, bacteroids are larger, are often branched, and have undergone multiple rounds of endoreduplication. Nitrogenfixing bacteroids have been shown to have a drastically altered transcriptome (7,8) and proteome (9) compared to those of their non-nitrogen-fixing counterparts.…”

Multidisciplinary approaches for studying rhizobium–legume symbioses

“…Furthermore, nodule proteomics has been used to examine the proteome during field growth conditions (Delmotte et al 2010), to compare root nodules and stem nodules (Delmotte et al 2014), and to identify NifA-regulated proteins (Salazar et al 2010). Studies have compared the proteomes of nodules of different maturities or ages (Delmotte et al 2014;Nambu et al 2015;Marx et al 2016), and recently, proteomes of three zones from the same nodules were independently isolated and evaluated (Ogden et al 2017). These studies have revealed how the proteome changes during symbiotic development, and they complement and extend conclusions generated using transcriptomics.…”

“…Others have compared the metabolite profile of effective and noneffective nodules to identify metabolic processes specifically active during SNF (Ye et al 2013;Gemperline et al 2015). As with transcriptomics and proteomics, metabolomics of nodules of different age or maturity has been performed to examine developmental progression (Lardi et al 2016), and zone-specific metabolite analyses have been performed (Ogden et al 2017). Metabolomics has further been employed to compare the metabolome of nodules from different plants containing the same rhizobial strain (Lardi et al 2016).…”