The Mitochondrial Complexome of Medicago truncatula.
ABSTRACT: Legumes (Fabaceae, Leguminosae) are unique in their ability to carry out an elaborate endosymbiotic nitrogen fixation process with rhizobia proteobacteria. The symbiotic nitrogen fixation enables the host plants to grow almost independently of any other nitrogen source. Establishment of symbiosis requires adaptations of the host cellular metabolism, here foremost of the energy metabolism mainly taking place in mitochondria. Since the early 1990s, the galegoid legume Medicago truncatula Gaertn. is a well-established model for studying legume biology, but little is known about the protein complement of mitochondria from this species. An initial characterization of the mitochondrial proteome of M. truncatula (Jemalong A17) was published recently. In the frame of this study, mitochondrial protein complexes were characterized using Two-dimensional (2D) Blue native (BN)/SDS-PAGE. From 139 detected spots, the "first hit" (=most abundant) proteins of 59 spots were identified by mass spectrometry. Here, we present a comprehensive analysis of the mitochondrial "complexome" (the "protein complex proteome") of M. truncatula via 2D BN/SDS-PAGE in combination with highly sensitive MS protein identification. In total, 1,485 proteins were identified within 158 gel spots, representing 467 unique proteins. Data evaluation by the novel GelMap annotation tool allowed recognition of protein complexes of low abundance. Overall, at least 36 mitochondrial protein complexes were found. To our knowledge several of these complexes were described for the first time in Medicago. The data set is accessible under http://www.gelmap.de/medicago/. The mitochondrial protein complex proteomes of Arabidopsis available at http://www.gelmap.de/arabidopsis/ and Medicago are compared.
Project description:Legume crops present important agronomical and environmental advantages mainly due to their capacity to reduce atmospheric N2 to ammonium via symbiotic nitrogen fixation (SNF). This process is very sensitive to abiotic stresses such as drought, but the mechanism underlying this response is not fully understood. The goal of the current work is to compare the drought response of two legumes with high economic impact and research importance, Medicago truncatula and Glycine max, by characterizing their root nodule proteomes. Our results show that, although M. truncatula exhibits lower water potential values under drought conditions compared to G. max, SNF declined analogously in the two legumes. Both of their nodule proteomes are very similar, and comparable down-regulation responses in the diverse protein functional groups were identified (mainly proteins related to the metabolism of carbon, nitrogen, and sulfur). We suggest lipoxygenases and protein turnover as newly recognized players in SNF regulation. Partial drought conditions applied to a split-root system resulted in the local down-regulation of the entire proteome of drought-stressed nodules in both legumes. The high degree of similarity between both legume proteomes suggests that the vast amount of research conducted on M. truncatula could be applied to economically important legume crops, such as soybean.
Project description:Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ?94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.
Project description:BACKGROUND: Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula. RESULTS: This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene. CONCLUSIONS: This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.
Project description:Grain legumes are highly valuable plant species, as they produce seeds with high protein content. Increasing seed protein production and improving seed nutritional quality represent an agronomical challenge in order to promote plant protein consumption of a growing population. In this study, we used the genetic diversity, naturally present in Medicago truncatula, a model plant for legumes, to identify genes/loci regulating seed traits. Indeed, using sequencing data of 162 accessions from the Medicago HAPMAP collection, we performed genome-wide association study for 32 seed traits related to seed size and seed composition such as seed protein content/concentration, sulfur content/concentration. Using different GWAS and postGWAS methods, we identified 79 quantitative trait nucleotides (QTNs) as regulating seed size, 41 QTNs for seed composition related to nitrogen (i.e. storage protein) and sulfur (i.e. sulfur-containing amino acid) concentrations/contents. Furthermore, a strong positive correlation between seed size and protein content was revealed within the selected Medicago HAPMAP collection. In addition, several QTNs showed highly significant associations in different seed phenotypes for further functional validation studies, including one near an RNA-Binding Domain protein, which represents a valuable candidate as central regulator determining both seed size and composition. Finally, our findings in M. truncatula represent valuable resources to be exploitable in many legume crop species such as pea, common bean, and soybean due to its high synteny, which enable rapid transfer of these results into breeding programs and eventually help the improvement of legume grain production.
Project description:Rhizobium and allied bacteria form symbiotic nitrogen-fixing nodules on legume roots. Plant hormones appear to play a role in nodule formation. We treated Medicago truncatula roots with auxin transport inhibitors (ATIs) N-(1-naphthyl)phthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA) to induce the formation of pseudonodules. We compared the transcriptional responses of M. truncatula roots treated with ATIs to roots inoculated with Sinorhizobium meliloti. The transcriptional response of M. truncatula roots 1 and 7 days after ATI treatment were opposite to roots treated with S. meliloti. Three independent biological replicates were performed for each treatment (buffer, ATI and wild-type bacteria) at 21 days after treatment.
Project description:Nitrogen is one of the most essential plant nutrients and one of the major factors limiting crop productivity. Having the goal to perform a more sustainable agriculture, there is a need to maximize biological nitrogen fixation, a feature of legumes. To enhance our understanding of the molecular mechanisms controlling the interaction between legumes and rhizobia, the symbiotic partner fixing and assimilating the atmospheric nitrogen for the plant, researchers took advantage of genetic and genomic resources developed across different legume models (e.g., Medicago truncatula, Lotus japonicus, Glycine max, and Phaseolus vulgaris) to identify key regulatory protein coding genes of the nodulation process. In this study, we are presenting the results of a comprehensive comparative genomic analysis to highlight orthologous and paralogous relationships between the legume genes controlling nodulation. Mining large transcriptomic datasets, we also identified several orthologous and paralogous genes characterized by the induction of their expression during nodulation across legume plant species. This comprehensive study prompts new insights into the evolution of the nodulation process in legume plant and will benefit the scientific community interested in the transfer of functional genomic information between species.
Project description:Medicago truncatula belongs to the legume family and forms symbiotic associations with nitrogen fixing bacteria, the rhizobia. During these interactions, the plants develop root nodules in which bacteria invade the plant cells and fix nitrogen for the benefit of the plant. Despite massive infection, legume nodules do not develop visible defence reactions, suggesting a special immune status of these organs. Some factors influencing rhizobium maintenance within the plant cells have been previously identified, such as the M. truncatula NCR peptides whose toxic effects are reduced by the bacterial protein BacA. In addition, DNF2, SymCRK, and RSD are M. truncatula genes required to avoid rhizobial death within the symbiotic cells. DNF2 and SymCRK are essential to prevent defence-like reactions in nodules after bacteria internalization into the symbiotic cells. Herein, we used a combination of genetics, histology and molecular biology approaches to investigate the relationship between the factors preventing bacterial death in the nodule cells. We show that the RSD gene is also required to repress plant defences in nodules. Upon inoculation with the bacA mutant, defence responses are observed only in the dnf2 mutant and not in the symCRK and rsd mutants. In addition, our data suggest that lack of nitrogen fixation by the bacterial partner triggers bacterial death in nodule cells after bacteroid differentiation. Together our data indicate that, after internalization, at least four independent mechanisms prevent bacterial death in the plant cell. These mechanisms involve successively: DNF2, BacA, SymCRK/RSD and bacterial ability to fix nitrogen.
Project description:Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad-spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen-associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae-specific PRR ELONGATION FACTOR-THERMO UNSTABLE RECEPTOR (EFR), conferring recognition of the bacterial EF-Tu protein, from Arabidopsis thaliana to the legume Medicago truncatula. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF-Tu-derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR-Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.
Project description:Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project 'GLIP': Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project 'IFCOSMO': Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.
Project description:Zinc (Zn) is an essential nutrient for plants that is involved in almost every biological process. This includes symbiotic nitrogen fixation, a process carried out by endosymbiotic bacteria (rhizobia) living within differentiated plant cells of legume root nodules. Zn transport in nodules involves delivery from the root, via the vasculature, release into the apoplast and uptake into nodule cells. Once in the cytosol, Zn can be used directly by cytosolic proteins or delivered into organelles, including symbiosomes of infected cells, by Zn efflux transporters. Medicago truncatula MtMTP2 (Medtr4g064893) is a nodule-induced Zn-efflux protein that was localized to an intracellular compartment in root epidermal and endodermal cells, as well as in nodule cells. Although the MtMTP2 gene is expressed in roots, shoots, and nodules, mtp2 mutants exhibited growth defects only under symbiotic, nitrogen-fixing conditions. Loss of MtMTP2 function resulted in altered nodule development, defects in bacteroid differentiation, and severe reduction of nitrogenase activity. The results presented here support a role of MtMTP2 in intracellular compartmentation of Zn, which is required for effective symbiotic nitrogen fixation in M. truncatula.