Recent Advances in Medicago truncatula Genomics

Legume rotation has allowed a consistent increase in crop yield and consequently in human population since the antiquity. Legumes will also be instrumental in our ability to maintain the sustainability of our agriculture while facing the challenges of increasing food and biofuel demand. Medicago truncatula and Lotus japonicus have emerged during the last decade as two major model systems for legume biology. Initially developed to dissect plant-microbe symbiotic interactions and especially legume nodulation, these two models are now widely used in a variety of biological fields from plant physiology and development to population genetics and structural genomics. This review highlights the genetic and genomic tools available to the M. truncatula community. Comparative genomic approaches to transfer biological information between model systems and legume crops are also discussed

Recent Advances in \u3cem\u3eMedicago truncatula\u3c/em\u3e Genomics

Legume rotation has allowed a consistent increase in crop yield and consequently in human population since the antiquity. Legumes will also be instrumental in our ability to maintain the sustainability of our agriculture while facing the challenges of increasing food and biofuel demand. Medicago truncatula and Lotus japonicus have emerged during the last decade as two major model systems for legume biology. Initially developed to dissect plant-microbe symbiotic interactions and especially legume nodulation, these two models are now widely used in a variety of biological fields from plant physiology and development to population genetics and structural genomics. This review highlights the genetic and genomic tools available to the M. truncatula community. Comparative genomic approaches to transfer biological information between model systems and legume crops are also discussed

The molecular genetic linkage map of the model legume Medicago truncatula: an essential tool for comparative legume genomics and the isolation of agronomically important genes

BACKGROUND: The legume Medicago truncatula has emerged as a model plant for the molecular and genetic dissection of various plant processes involved in rhizobial, mycorrhizal and pathogenic plant-microbe interactions. Aiming to develop essential tools for such genetic approaches, we have established the first genetic map of this species. Two parental homozygous lines were selected from the cultivar Jemalong and from the Algerian natural population (DZA315) on the basis of their molecular and phenotypic polymorphism. RESULTS: An F2 segregating population of 124 individuals between these two lines was obtained using an efficient manual crossing technique established for M. truncatula and was used to construct a genetic map. This map spans 1225 cM (average 470 kb/cM) and comprises 289 markers including RAPD, AFLP, known genes and isoenzymes arranged in 8 linkage groups (2n = 16). Markers are uniformly distributed throughout the map and segregation distortion is limited to only 3 linkage groups. By mapping a number of common markers, the eight linkage groups are shown to be homologous to those of diploid alfalfa (M. sativa), implying a good level of macrosynteny between the two genomes. Using this M. truncatula map and the derived F3 populations, we were able to map the Mtsym6 symbiotic gene on linkage group 8 and the SPC gene, responsible for the direction of pod coiling, on linkage group 7. CONCLUSIONS: These results demonstrate that Medicago truncatula is amenable to diploid genetic analysis and they open the way to map-based cloning of symbiotic or other agronomically-important genes using this model plant

Comparison of Vacuum MALDI and AP-MALDI Platforms for the Mass Spectrometry Imaging of Metabolites Involved in Salt Stress in Medicago truncatula

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is routinely used to determine the spatial distributions of various biomolecules in tissues. Recently, there has been an increased interest in creating higher resolution images using sources with more focused beams. One such source, an atmospheric pressure (AP) MALDI source from MassTech, has a laser capable of reaching spatial resolutions of 10 μm. Here, the AP-MALDI source coupled with a Q Exactive HF Orbitrap platform is compared to the commercial MALDI LTQ Orbitrap XL system using Medicago truncatula root nodules. AP-MALDI parameters, such as the S-lens value, capillary temperature, and spray voltage, were optimized on the Q Exactive-HF platform for optimal detection of plant metabolites. The performance of the two systems was evaluated for sensitivity, spatial resolution, and overall ability to detect plant metabolites. The commercial MALDI LTQ Orbitrap XL was superior regarding the number of compounds detected, as at least two times more m/z were detected compared to the AP-MALDI system. However, although the AP-MALDI source requires a spatial resolution higher than 10 μm to get the best signal, the spatial resolution at 30 μm is still superior compared to the 75 μm spatial resolution achieved on the MALDI platform. The AP-MALDI system was also used to investigate the metabolites present in M. truncatula roots and root nodules under high salt and low salt conditions. A discriminative analysis with SCiLS software revealed m/z ions specific to the control and salt conditions. This analysis revealed 44 m/z ions present at relatively higher abundances in the control samples, and 77 m/z enriched in the salt samples. Liquid chromatography-tandem MS was performed to determine the putative molecular identities of some of the mass ions enriched in each sample, including, asparagine, adenosine, and nicotianamine in the control samples, and arginine and soyasaponin I in the salt treated samples

Agrobacterium-Mediated Transient Gene Expression and Silencing: A Rapid Tool for Functional Gene Assay in Potato

Potato is the third most important food crop worldwide. However, genetic and genomic research of potato has lagged behind other major crops due to the autopolyploidy and highly heterozygous nature associated with the potato genome. Reliable and technically undemanding techniques are not available for functional gene assays in potato. Here we report the development of a transient gene expression and silencing system in potato. Gene expression or RNAi-based gene silencing constructs were delivered into potato leaf cells using Agrobacterium-mediated infiltration. Agroinfiltration of various gene constructs consistently resulted in potato cell transformation and spread of the transgenic cells around infiltration zones. The efficiency of agroinfiltration was affected by potato genotypes, concentration of Agrobacterium, and plant growth conditions. We demonstrated that the agroinfiltration-based transient gene expression can be used to detect potato proteins in sub-cellular compartments in living cells. We established a double agroinfiltration procedure that allows to test whether a specific gene is associated with potato late blight resistance pathway mediated by the resistance gene RB. This procedure provides a powerful approach for high throughput functional assay for a large number of candidate genes in potato late blight resistance

Potential regulatory phosphorylation sites in a Medicago truncatula plasma membrane proton pump implicated during early symbiotic signaling in roots

AbstractIn plants and fungi the plasma membrane proton pump generates a large proton-motive force that performs essential functions in many processes, including solute transport and the control of cell elongation. Previous studies in yeast and higher plants have indicated that phosphorylation of an auto-inhibitory domain is involved in regulating pump activity. In this report we examine the Medicago truncatula plasma membrane proton pump gene family, and in particular MtAHA5. Yeast complementation assays with phosphomimetic mutations at six candidate sites support a phosphoregulatory role for two residues, suggesting a molecular model to explain early Nod factor-induced changes in the plasma membrane proton-motive force of legume root cells

A network-based comparative framework to study conservation and divergence of proteomes in plant phylogenies

Comparative functional genomics offers a powerful approach to study species evolution. To date, the majority of these studies have focused on the transcriptome in mammalian and yeast phylogenies. Here, we present a novel multi-species proteomic dataset and a computational pipeline to systematically compare the protein levels across multiple plant species. Globally we find that protein levels diverge according to phylogenetic distance but is more constrained than the mRNA level. Module-level comparative analysis of groups of proteins shows that proteins that are more highly expressed tend to be more conserved. To interpret the evolutionary patterns of conservation and divergence, we develop a novel network-based integrative analysis pipeline that combines publicly available transcriptomic datasets to define co-expression modules. Our analysis pipeline can be used to relate the changes in protein levels to different species-specific phenotypic traits. We present a case study with the rhizobia-legume symbiosis process that supports the role of autophagy in this symbiotic association

Symbiotic Nitrogen Fixation and the Challenges to Its Extension to Nonlegumes

Access to fixed or available forms of nitrogen limits the productivity of crop plants and thus food production. Nitrogenous fertilizer production currently represents a significant expense for the efficient growth of various crops in the developed world. There are significant potential gains to be had from reducing dependence on nitrogenous fertilizers in agriculture in the developed world and in developing countries, and there is significant interest in research on biological nitrogen fixation and prospects for increasing its importance in an agricultural setting. Biological nitrogen fixation is the conversion of atmospheric N2 to NH3, a form that can be used by plants. However, the process is restricted to bacteria and archaea and does not occur in eukaryotes. Symbiotic nitrogen fixation is part of a mutualistic relationship in which plants provide a niche and fixed carbon to bacteria in exchange for fixed nitrogen. This process is restricted mainly to legumes in agricultural systems, and there is considerable interest in exploring whether similar symbioses can be developed in nonlegumes, which produce the bulk of human food. We are at a juncture at which the fundamental understanding of biological nitrogen fixation has matured to a level that we can think about engineering symbiotic relationships using synthetic biology approaches. This minireview highlights the fundamental advances in our understanding of biological nitrogen fixation in the context of a blueprint for expanding symbiotic nitrogen fixation to a greater diversity of crop plants through synthetic biology.Biotechnology and Biological Sciences Research Council (Great Britain) (Grants BB/L011484/1 and BB/L011476/1)National Science Foundation (U.S.) (Grant 1331098

Presence of three mycorrhizal genes in the common ancestor of land plants suggests a key role of mycorrhizas in the colonization of land by plants

• The colonization of land by plants fundamentally altered environmental conditions on earth. Plant–mycorrhizal fungus symbiosis likely played a key role in this process by assisting plants to absorb water and nutrients from soil. • Here, in a diverse set of land plants, we investigated the evolutionary histories and functional conservation of three genes required for mycorrhiza formation in legumes and rice ( Oryza sativa ), DMI1 , DMI3 and IPD3 . • The genes were isolated from nearly all major plant lineages. Phylogenetic analyses showed that they had been vertically inherited since the origin of land plants. Further, cross-species mutant rescue experiments demonstrated that DMI3 genes from liverworts and hornworts could rescue Medicago truncatula dmi3 mutants for mycorrhiza formation. Yeast two-hybrid assays also showed that bryophyte DMI3 proteins could bind to downstream-acting M. trunculata IPD3 protein. Finally, molecular evolutionary analyses revealed that these genes were under purifying selection for maintenance of their ancestral functions in all mycorrhizal plant lineages. • These results indicate that the mycorrhizal genes were present in the common ancestor of land plants, and that their functions were largely conserved during land plant evolution. The evidence presented here strongly suggests that plant–mycorrhizal fungus symbiosis was one of the key processes that contributed to the origin of land flora.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78704/1/j.1469-8137.2009.03137.x.pd

Shifts in evolutionary lability underlie independent gains and losses of root-nodule symbiosis in a single clade of plants

11 Pág.Root nodule symbiosis (RNS) is a complex trait that enables plants to access atmospheric nitrogen converted into usable forms through a mutualistic relationship with soil bacteria. Pinpointing the evolutionary origins of RNS is critical for understanding its genetic basis, but building this evolutionary context is complicated by data limitations and the intermittent presence of RNS in a single clade of ca. 30,000 species of flowering plants, i.e., the nitrogen-fixing clade (NFC). We developed the most extensive de novo phylogeny for the NFC and an RNS trait database to reconstruct the evolution of RNS. Our analysis identifies evolutionary rate heterogeneity associated with a two-step process: An ancestral precursor state transitioned to a more labile state from which RNS was rapidly gained at multiple points in the NFC. We illustrate how a two-step process could explain multiple independent gains and losses of RNS, contrary to recent hypotheses suggesting one gain and numerous losses, and suggest a broader phylogenetic and genetic scope may be required for genome-phenome mapping.This work was supported by DOE grant DE-SC0018247 to M.K., R.G., P.S., and D.S. and a UFBI grant (University of Florida). We thank Katharina Pawlowski for reviewing our scoring of actinorhizal symbiosis and for related discussions. We thank Colin Hughes and other members of the Legume Phylogeny Working Group for reviewing and helping to resolve taxonomy issues in Leguminosae. We thank Mark Whitten, Kelly Balmant, Chris Dervinis, Joshua Dieringer, and Henry Schmidt for help with specimen sampling.Peer reviewe