Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events including abiotic/biotic stress responses, innate immunity, hormone signaling and cell specificity in plants. The MAPK-mediated stress and ethylene signaling are recently known to be involved in nitogen-fixing symbiotic interactions; however, the biological role of MAPK for nodule development in legume plants is largely unknown. We here elucidated that MtMKK5-MtMPK3/6 cascade negatively regulate the nitrogen fixing nodule formation in Medicago truncatula. MtMKK5, an ortholog of SIMKK, overexpression significantly reduces the nodule formation in M. truncatula roots. MtMKK5 directly activates MtMPK3/6 by phosphorylation on the TEY motif within the activation loop in the cytoplasm, which might link to EFD as a negative regulator for nodule formation. EFD has a putative MAPK phosphorylation Thr residue and could be a target of the activated MtMPK3/6 in the nucleus. Consistently, a MAPK specific inhibitor U0126 enhances nodule formation and confers similar nodule phenotypes to the efd-1 mutant such as lower proliferation and differentiation to symbiotic tissues. Our works thus reveal a key negative signaling module mediated by MtMKK5-MtMPK3/6-EFD for symbiotic nitrogen fixing nodule organogenesis.

Legume and rhizobia symbiosis plays an important role in conversion of atmospheric dinitrogen to ammonia. On a global scale, this interaction represents a key entry point for reduced nitrogen into the biosphere, and as a consequence this symbiosis is important in both natural and agricultural systems. Symbiotic development of nodule organ is triggered by chito-oligosaccharide signals (Nod factors) from the bacterium which are perceived by the legume root. Understanding the molecular and cellular processes that underlie Nod factor perception is one focus of legume biology. Although forward genetics has proved to be an important tool to identify key players in Nod factor perception, we still know relatively little regarding the functional networks of genes and proteins that connect the earliest steps of Nod factor perception to immediate downstream outcomes. To elucidate genes and proteins that link Nod factor perception to cellular and physiological responses we are taking a discovery-based strategy based on whole transcriptome profiling using RNA-seq analysis in the roots of Medicago truncatula in response to Sinorhizobium meliloti. Functional characterization of a number of candidate genes is currently in progress to further examine their role in nodulation such as generating transgenic plants

Legume and rhizobia symbiosis plays an important role in conversion of atmospheric dinitrogen to ammonia. On a global scale, thin interaction represent a key entry point for reduced nitrogen into the biosphere, and as a consequence this symbiosis in important in both natural and agricultural systems. Symbiotic development of nodule organ in triggered by chito-oligosaccharide signals(Nod factors) from the bacterium which are perceived by the legume root. Understanding the molecular and cellular processes that underlie Nod factor perception is one focus of legume biology. Although forward genetics has proved to be an important tool to elucidate key players in Nod factor perception, we still know relatively little regarding the functional networks of genes and proteins that connect the earliest steps of Nod factor perception to immediate downstream outcomes. To identify genes and proteins that link Nod factor perception to cellular and physiological responses we are taking a discovery-based strategy on large-scale transcriptome profiling using RNA sequencing in the roots of Medicago truncatula in response to Sinorhizobium meliloti. Functional characterization of a number of candidate genes is currently in progress to further examine their role in nodulation.

Cross-species translation of genomic information may play a crucial role in applying biological knowledge gained from one species to other genomes. To screen and identify a broad range of abiotic stress-responsive genes, we employed a diverse array of resources, including Arabidopsis databases (http://www.arabidopsis.org), expression profiling data and previously reported literatures. As a result, a total of 1,377 genes were identified and classified into 18 different functional criteria based on biological processes of gene ontology. The gene set was translated into M. truncatula, which is a representative model system in the Fabaceae, by identifying orthologous genes between these two genomes with a combination of tBlastx and BlastP analyses. It is shown that approximately 82% of genes were estimated to be translated between the two genomes below the E-value of 10-30. These orthologous loci were used to construct comparative maps by developing a user-friendly analysis platform, resulting in a total of 52 synteny blocks. Furthermore, to discover central genes by which control responses to the abiotic stresses, a combination of AraNet (http://www.functionanet.org) and the Cytoscape program was used for the gene network analysis. The analysis resulted in the identification of 240 potential key genes. We anticipate that these genes may impact molecular breeding programs by discovering trait-associated SNPs followed by marker development.

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F2 population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence-tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy, with primers designed to anneal in conserved exon regions and amplify across intron regions. Polymorphisms were significantly more frequent in intron vs exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in Medicago sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar, and establishes the basis for a “Medicago” composite map.

Cross-species translation of genomic information may play a pivotal role in applying biological knowledge gained from one species to other genomes. Abiotic stress-responsive genes in Arabidopsis have been translated to a legume model system, Medicago truncatula. A total of 1,370 Arabidopsis genes were identified by searching TAIR database, expression profiling data and literatures. For purposes of cross-genome identification of orthologous genes, tBlastX or BlastP were employed between these two model systems. Candidate genes potentially associated with abiotic stress responses were classified into 18 functional criteria and corresponding genomic locations were analyzed by Circos program. To do this, user-friendly bioinformatic analysis platform was established. In order to discover abiotic stress-associated genes, gene network and/or interactome analyses were conducted using a combination of AraNet web-based platform and CytoScape program. As a result, we could identify 240 key genes that appeared to play an important role within central gene networks. We anticipate that these genes may impact molecular breeding programs by developing them into genetic markers and discovering trait-associated nucleotide variations.

Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events including abiotic/biotic stress responses, innate immunity, hormone signaling and cell specificity in plants. The MAPK-mediated stress and ethylene signaling are recently known to be involved in nitogen-fixing symbiotic interactions; however, the biological role of MAPK for nodule development in legume plants is largely unknown. We here elucidated that MtMKK5-MtMPK3/6 cascade negatively regulate the nitrogen fixing nodule formation in Medicago truncatula. MtMKK5, an ortholog of SIMKK, overexpression significantly reduces the nodule formation in M. truncatula roots. MtMKK5 directly activates MtMPK3/6 by phosphorylation on the TEY motif within the activation loop in the cytoplasm, which might link to EFD as a negative regulator for nodule formation. EFD has a putative MAPK phosphorylation Thr residue and could be a target of the activated MtMPK3/6 in the nucleus. Consistently, a MAPK specific inhibitor U0126 enhances nodule formation and confers similar nodule phenotypes to the efd-1 mutant such as lower proliferation and differentiation to symbiotic tissues. Our works thus reveal a key negative signaling module mediated by MtMKK5-MtMPK3/6-EFD for symbiotic nitrogen fixing nodule organogenesis.

To tolerate environmentally adverse conditions such as cold, drought, and salinity, plants often synthesize and accumulate proline in cells as compatible osmolytes. δ1 -Pyrroline-5-carboxylate synthetase(P5CS) catalyzes the rate-limiting step of proline biosynthesis from glutamate. Two complete genes, MtP5CS1 and MtP5CS2, were isolated from the model legume Medicago truncatula by cDNA cloning and bacterial artificial chromosome library screening. Nucleotide sequence analysis showed that both genes consisted of 20 exons and 19 introns. Alignment of the predicted amino acid sequences revealed high similarities with P5CS proteins from other plant species. The two MtP5CS genes were expressed in response to high salt and low temperature treatments. Semi-quantitative reverse transcription-polymerase chain reaction showed that MtP5CS1 was expressed earlier than MtP5CS2, indicating differential regulation of the two genes. To evaluate the reverse genetic effects of nucleotide changes on MtP5CS function, a Targeting Induced Local Lesions in Genomes approach was taken. Three mutants each were isolated for MtP5CS1 and MtP5CS2, of which a P5CS2 nonsense mutant carrying a codon change from arginine to stop was expected to bring translation to premature termination. These provide a valuable genetic resource with which to determine the function of the P5CS genes in environmental stress responses of legume crops.