Proteomic analysis dissects the impact of nodulation and biological nitrogen fixation on Vicia faba root nodule physiology

Abstract

Symbiotic nitrogen fixation in root nodules of legumes is a highly important biological process which is only poorly understood. Root nodule metabolism differs from that of roots. Differences in root and nodule metabolism are expressed by altered protein abundances and amenable to quantitative proteome analyses. Differences in the proteomes may either be tissue specific and related to the presence of temporary endosymbionts (the bacteroids) or related to nitrogen fixation activity. An experimental setup including WT bacterial strains and strains not able to conduct symbiotic nitrogen fixation as well as root controls enables identification of tissue and nitrogen fixation specific proteins. Root nodules are specialized plant organs housing and regulating the mutual symbiosis of legumes with nitrogen fixing rhizobia. As such, these organs fulfill unique functions in plant metabolism. Identifying the proteins required for the metabolic reactions of nitrogen fixation and those merely involved in sustaining the rhizobia:plant symbiosis, is a challenging task and requires an experimental setup which allows to differentiate between these two physiological processes. Here, quantitative proteome analyses of nitrogen fixing and non-nitrogen fixing nodules as well as fertilized and non-fertilized roots were performed using Vicia faba and Rhizobium leguminosarum. Pairwise comparisons revealed altered enzyme abundance between active and inactive nodules. Similarly, general differences between nodules and root tissue were observed. Together, these results allow distinguishing the proteins directly involved in nitrogen fixation from those related to nodulation. Further observations relate to the control of nodulation by hormones and provide supportive evidence for the previously reported correlation of nitrogen and sulfur fixation in these plant organs. Additionally, data on altered protein abundance relating to alanine metabolism imply that this amino acid may be exported from the symbiosomes of V. faba root nodules in addition to ammonia. Data are available via ProteomeXchange with identifier PXD008548.

Single organelle function and organization as estimated from Arabidopsis mitochondrial proteomics

“…Solubilized proteins were purified, concentrated, and digested in a glycine‐SDS gel. The resulting peptides were subsequently extracted as previously described (Thal et al ., ) and resuspended in 20 μl of 5% [v/v] ACN, 0.1% [v/v] TFA. LC‐MS/MS was primarily performed as described in (Thal et al ., ) with minor differences.…”

“…The resulting peptides were subsequently extracted as previously described (Thal et al ., ) and resuspended in 20 μl of 5% [v/v] ACN, 0.1% [v/v] TFA. LC‐MS/MS was primarily performed as described in (Thal et al ., ) with minor differences. Peptide solution (1 μl) was loaded on a 2‐cm C18 reversed phase trap column (Acclaim PepMap100, diameter: 100 μm, granulometry: 5 μm, pore size: 100 Å; Thermo Fisher Scientific, Waltham, MA, USA; https://www.thermofisher.com/order/catalog/product/164567#/164567) and further separated on a 50‐cm C18 reversed phase analytical column (Acclaim PepMap100, diameter: 75 μm, granulometry: 3 μm, pore size: 100 Å; Thermo Fisher Scientific).…”

What is hot in plant mitochondria?

“…It took 10 years before a combination of more sensitive mass spectrometers, additional data bases, better search engines and, perhaps, more amenable mitochondria allowed the identification of 1060 proteins in potato tuber mitochondria (POM) (Salvato et al ). At ICPMB in Wroclaw, two posters reported the identification of 1655 and 1800 proteins in POM and bean root mitochondria, respectively (Thal et al Poster 87, Møller et al Poster 93). This raises the question of how many different proteins you can expect to find in mitochondria from one tissue under a given set of condition?…”

MULocDeep: A deep-learning framework for protein subcellular and suborganellar localization prediction with residue-level interpretation

“…We published a dataset of the experimentally extracted mitochondrial proteome from three species (Mito3 dataset) and used it to test the performance of subcellular localization prediction by different methods ( Table 2). We also collected a comprehensive dataset for 45 Table 2).…”

“…First, mitochondria were isolated from an Arabidopsis thaliana cell culture, Solanum tuberosum tubers, and Vicia faba roots. Then, the mitochondrial proteins of the three species were identified and quantified using shotgun mass spectrometry according to Thal et al 45 . The generated raw files were analysed with the Proteome Discoverer software (Thermo Fisher Scientific, Dreieich, Germany) using the Mascot (Matrix Science, London, UK) search engine against in-house protein sequence databases, depending on the analyzed species: Arabidopsis thaliana, Solanum tuberosum and Vicia faba spectra were queried against the TAIR 10 database, the Solanum tuberosum protein database and the Medicago truncatula protein database 4.0, respectively.…”