Proteome reference maps of the Lotus japonicus nodule and root

Abstract

Legume symbiosis with rhizobia results in the formation of a specialized organ, the root nodule, where atmospheric dinitrogen is reduced to ammonia. In Lotus japonicus (Lotus), several genes involved in nodule development or nodule function have been defined using biochemistry, genetic approaches, and high throughput transcriptomics. We have employed proteomics to further understand nodule development. Two developmental stages representing nodules prior to nitrogen fixation (white) and mature nitrogen fixing nodules (red) were compared with roots. In addition, the proteome of a spontaneous nodule formation mutant (snf1) was determined. From nodules and roots, 780 and 790 protein spots from 2D gels were identified and approximately 45% of the corresponding unique gene accessions were common. Including a previous proteomics set from Lotus pod and seed, the common gene accessions were decreased to 7%. Interestingly, an indication of more pronounced post translational modifications in nodules than in roots was determined. Between the two nodule developmental stages, higher levels of pathogen related 10 proteins, HSP's, and proteins Accepted Article www.proteomics-journal.com Page 3 ProteomicsThis article is protected by copyright. All rights reserved involved in redox processes were found in white nodules, suggesting a higher stress level at this developmental stage. In contrast, protein spots corresponding to nodulins such as leghemoglobin, asparagine synthetase, sucrose synthase, and glutamine synthetase were prevalent in red nodules. The distinct biochemical state of nodules was further highlighted by the conspicuous presence of several nitrilases, ascorbate metabolic enzymes and putative rhizobial effectors. IntroductionLegumes are the third largest plant family with more than 20 000 species [1] and most of these are able to interact with rhizobia and develop symbiotic root nodules when available soil nitrogen is low. The bacteroids, differentiated rhizobia inside nodule cells, contain nitrogenase that converts atmospheric dinitrogen into ammonia/ammonium, which is used by the legume. In return, bacteroids are supplied with carbohydrates derived from photosynthesis. Within the nodule cells bacteroids are enclosed in a specialized plant membrane termed the symbiosome membrane separating bacteroids from the plant cytosol. A distinctive feature of a functional nodule is the red color originating from plant leghemoglobin, which is involved in buffering the oxygen level to avoid inactivation of the oxygen sensitive nitrogenase [2,3]. Formation of the nodule organ housing the bacteroids is an inducible process initiated when rhizobial lipochitin-oligosaccharide signal molecules are recognized by plant Nod factor receptors [4][5][6][7]. Plant genes such as SymRK and CCaMK including spontaneous nodule formation 1 (snf1), pivotal for developing a functional nodule, have been identified using forward genetics [8][9][10][11]. As a result, pathways involved in nodule development and resources for global transcriptom...

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

“…The mass range was set to m/z 700–5000 and different laser intensities and numbers of laser exposures to obtain an optimal spectrum were used. The obtained MS and MS/MS spectra were combined and searched against an in‐house Lotus database version 2.5 as reported (Dam et al ., ) for protein identification. For the peptidase, the MS spectra for wild type and the Ljgnt II mutants were compared and some of the MS/MS spectra corresponding to N ‐glycopeptides were manually annotated.…”

Biotic Interactions in the Rhizosphere: A Diverse Cooperative Enterprise for Plant Productivity

“…In fungi, 208,883 putative secretory proteins have been identified (Choi et al, 2010), but only a small fraction of them have been studied, and so far, nobody knows the biological/ecological role of most of them. On the other hand, there are many reports describing the use of plant mutants, pathogenic bacteria, and symbiotic microbes that highlight the vast diversity of proteins or even the function of a group of proteins during plant-microbe interaction (De-la-Peña et al, 2008;Afroz et al, 2013;Alberton et al, 2013;Dam et al, 2014). These and other proteomic reports can be used to study proteins important to plant productivity.…”

The Lotus japonicus Genome

“…Proteomics is a large scale study of the proteome and four major steps, i.e., protein extraction, protein/peptide separation, mass spectrometry, and data searches, are required prior to data analysis. For Lotus tissues, we have successfully used a SDS/phenol protein extraction followed by ammonium acetate precipitation and further removal of interfering compounds by acetone washes (Dam et al 2009;Nautrup-Pedersen et al 2010;Dam et al 2014). For plants, protein extraction is challenging due to interfering compounds such as polyphenols, terpenes, and organic acids that are abundant in green tissues and, thus, several protein extraction protocols have been developed Xie et al 2007;Jellouli et al 2010).…”

“…For plants, protein extraction is challenging due to interfering compounds such as polyphenols, terpenes, and organic acids that are abundant in green tissues and, thus, several protein extraction protocols have been developed Xie et al 2007;Jellouli et al 2010). In Lotus, the protein separation/ digestion strategy was used to create proteome reference maps of seed, pod, nodule, and root using 2D gels (Nautrup-Pedersen et al 2010;Dam et al 2014) together with subcellular protein extraction of seed globulin-, plant cytosolic-, and symbiosome membrane proteins (Dam et al 2009(Dam et al , 2014Nautrup-Pedersen et al 2010;Wienkoop and Saalbach 2003) and, furthermore, the digestion/peptide methodology has been initiated for nodules and roots. After extraction, the protein fraction is redissolved and proteins are either separated/digested or digested/separated.…”

“…Lotus with the sequenced diploid genome and Lotus retrotransposon 1 (LORE1) mutant population, currently with more than 80,000 lines, is an excellent model plant to study symbiosis . Furthermore, proteomics of the soybean cytosolic nodule fraction together with the plant and bacterial fractions of Medicago nodules were performed (Oehrle et al 2008;Larrainzar et al 2007) and the intersection of homologous proteins identified between nodule proteomics studies was determined (Dam et al 2014). Currently, proteomics is less used to study symbiosis; however, comparative proteomics of wild-type nodules and nodulation mutants can be essential to identify proteins affected/delayed in the functional nodule formation.…”