Genomic Inventory and Transcriptional Analysis of Medicago truncatula Transporters

Transporters move hydrophilic substrates across hydrophobic biological membranes and play key roles in plant nutrition, metabolism, and signaling and, consequently, in plant growth, development, and responses to the environment. To initiate and support systematic characterization of transporters in the model legume Medicago truncatula, we identified 3,830 transporters and classified 2,673 of these into 113 families and 146 subfamilies. Analysis of gene expression data for 2,611 of these transporters identified 129 that are expressed in an organ-specific manner, including 50 that are nodule specific and 36 specific to mycorrhizal roots. Further analysis uncovered 196 transporters that are induced at least 5-fold during nodule development and 44 in roots during arbuscular mycorrhizal symbiosis. Among the nodule- and mycorrhiza-induced transporter genes are many candidates for known transport activities in these beneficial symbioses. The data presented here are a unique resource for the selection and functional characterization of legume transporters.Transporters are membrane-spanning proteins that selectively transport hydrophilic solutes across hydrophobic membranes. They are present and required in all cellular membranes, including the cell or plasma membrane that separates cellular contents from the external environment and membranes of the various subcellular organelles. By transporting metabolites and nonmetabolites, such as inorganic ions, transporters play integral roles in cell metabolism, ion homeostasis, osmoregulation, signaling, and other processes. Transporters move solutes not only within cells but also between cells, tissues, and organs of complex, multicellular organisms such as higher plants. Therefore, they help to coordinate metabolic, physiological, and developmental processes in higher plants and other organisms.Transporter proteins/complexes contain multiple membrane-spanning domains that form an aqueous pore in the membrane, which enables movement of selected solutes from one side of the membrane to the other. Membrane-spanning domains are hydrophobic in nature, or at least partially so, which enables them to interact with the phospholipid bilayer of membranes. Many transporters contain hydrophobic α -helical segments that span the membrane, while others contain β -barrel transmembrane domains (TMD). Computer programs have been developed to identify putative membrane-spanning α -helices (Hoffman and Stoffel, 1993; Hirokawa et al., 1998; Tusnady and Simon, 2001) and β -barrels (Koebnik et al., 2000; Valavanis et al., 2006), which facilitate de novo prediction of putative membrane proteins, including transporters. Databases of known, characterized transport proteins aid in the identification and classification of transporters in new species via sequence similarity. Perhaps the most comprehensive of these is the Transporter Classification Database (TCDB; Saier et al., 2006), which was created to serve as a repository of functionally characterized transporters. It also serves to categorize new transporters into families and subfamilies based on molecular, evolutionary, and functional properties. At present, it consists of approximately 3,000 transporters classified in more than 500 families (www.tcdb.org).The legume family is second only to the grass family in importance to humans as a source of food, feed for livestock, and raw materials for industry (Graham and Vance, 2003). Legumes are the lynch pin of sustainable agriculture, because they supply their own nitrogen by “fixing” it (reducing N₂ to NH₃) in a symbiotic association with bacteria called rhizobia. This mutually beneficial association provides legumes and subsequent crops with a free and renewable source of usable nitrogen (Udvardi and Day, 1997). Legumes also establish symbiosis with mycorrhizal fungi that help the plant mine phosphorous and other nutrients from the soil (Smith and Read, 2008).Symbiotic nitrogen fixation (SNF) in root nodule cells of legumes is carried out by rhizobia that are completely surrounded by a plant membrane called the symbiosome membrane (SM), which forms a nitrogen-fixing organelle, the symbiosome, within the plant cytoplasm. Infected cortical cells of nodules contain thousands of symbiosomes, each containing one or a few bacteria. Infected plant cells, interspersed with noninfected cells, constitute the central tissue of nodules, which is surrounded by uninfected tissue that restricts gas exchange with the soil, and phloem and xylem, which import and export nutrients from the nodule, respectively. In exchange for ammonium produced by bacterial nitrogenase and released to the plant, rhizobia receive reduced carbon (principally dicarboxylic acids such as malate) and every other nutrient required for bacterial cell growth and maintenance (Udvardi and Day, 1997). Exchange of nutrients between the plant cell cytoplasm and rhizobia is mediated by a variety of transporters in the SM, some of which are induced during nodule development (Benedito et al., 2008). Transporters perform many other important roles in nodules, such as short- and long-distance transport of nutrients between plant cells and tissues and between the nodule and other organs, processes facilitated by proteins of the plant cell plasma membrane. On the other hand, transporters on the membranes of organelles such as mitochondria, plastids, and peroxisomes facilitate the movement of metabolites between cellular compartments, which is crucial for nodule metabolism and SNF.In the arbuscular mycorrhizal (AM) symbiosis, the fungal symbionts inhabit the root cortex, where they obtain carbon from the plant, and in exchange they deliver mineral nutrients, particularly phosphorus and nitrogen, to the root. Mineral nutrient transfer between symbionts occurs at a specialized symbiotic interface between branched hyphae, called arbuscules, and the cortical cells that they inhabit (Parniske, 2008). The interface is delimited by a plant-derived membrane called the periarbuscular membrane, which is continuous with the plasma membrane but contains some unique proteins, including novel inorganic phosphate (Pi) transporters (Harrison et al., 2002; Paszkowski et al., 2002). These transporters are required to transfer Pi that is released from the arbuscule into the cortical cell. It is assumed, but not yet shown directly, that nitrogen, and possibly other mineral nutrients such as zinc, is also transferred between the symbionts at this membrane interface (Smith and Read, 2008). However, the transport proteins involved are currently unknown. Likewise, transporters involved in carbon transfer to the fungal symbiont have not been identified. While it is expected that the periarbuscular membrane will contain additional transport activities, only a handful of transporters residing in this membrane have been identified to date.Although inroads have been made in the characterization of individual transporters in a variety of legume species, no systematic work has been done to identify and characterize all the transporters in any one species. Three legume species, Medicago truncatula, Glycine max (soybean), and Lotus japonicus, have been the subject of extensive cDNA and genomic DNA sequencing over the past few years (Young et al., 2003, 2005; Sato et al., 2007, 2008), making them interesting model systems for whole-genome analysis of transporters. The genome sequence of M. truncatula is being annotated by the International Medicago Genome Annotation Group (IMGAG), which described 38,335 genes in its version 2.0 of the genome sequence (http://www.medicago.org/genome/downloads/Mt2/). Additional resources relevant to Medicago functional genomics include the Medicago Gene Expression Atlas (http://bioinfo.noble.org/gene-atlas/v2), which provides developmental expression data for the majority of Medicago genes (Benedito et al., 2008), and a Tnt1 transposon-insertion mutant population with insertions in the majority of genes, which enables efficient forward and reverse genetics (Tadege et al., 2005, 2008). To facilitate systematic functional analysis of transporters in Medicago, and especially those involved in nitrogen-fixing and AM symbioses, we have identified and categorized 2,673 transporter genes and analyzed the expression patterns of 2,604 of these. The results of this work are presented here.     

Spatial effects and rare outcrossing events in Medicago truncatula (Fabaceae)

In order to elucidate the mechanisms underlying the large amount of RAPD polymorphism found in 1990 in a population of the selfing annual Medicago truncatula GAERTN. (Fabaceae), we have analysed most of the individuals (n = 363) from the same population 6 years later using microsatellite loci. We confirm the result of the earlier study, namely that this population is very polymorphic and highly subdivided, with approximately 37% of the variance distributed among subpopulations, only 50 m apart one from another. We use standard F-statistics analyses, linkage disequilibria, minimum spanning network, multilocus assignment tests and spatial autocorrelation analyses to test the hypotheses that spatial structure and outcrossing events are involved in maintaining the large amount of genetic diversity at the level of each subpopulation. Interestingly, fine-scale spatial structure could be observed in only one subpopulation suggesting that other mechanisms are acting elsewhere. To the best of our knowledge, this is the first study of fine spatial genetic structure in a predominantly selfing species.     

Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.     

Microsynteny between pea and Medicago truncatula in the SYM2 region

The crop legume pea (Pisum sativum) is genetically well characterized. However, due to its large genome it is not amenable to efficient positional cloning strategies. The purpose of this study was to determine if the model legume Medicago truncatula, which is a close relative of pea, could be used as a reference genome to facilitate the cloning of genes identified based on phenotypic and genetic criteria in pea. To this end, we studied the level of microsynteny between the SYM2 region of pea and the orthologous region in M. truncatula. Initially, a marker tightly linked to SYM2 was isolated by performing differential RNA display on near-isogenic pea lines. This marker served as the starting point for construction of a BAC physical map in M. truncatula. A fine-structure genetic map, based on eight markers from the M. truncatula physical map, indicates that the two genomes in this region share a conserved gene content. Importantly, this fine structure genetic map clearly delimits the SYM2-containing region in pea and the SYM2-orthologous region in M. truncatula, and should provide the basis for cloning SYM2. The utility of the physical and genetic tools in M. truncatula to dissect the SYM2 region of pea should have important implications for other gene cloning experiments in pea, in particular where the two genomes are highly syntenic within the region of interest.     

Identification and molecular characterization of Medicago truncatula NRT2 and NAR2 families

Nitrate transporters received little attention to legumes probably because these species are able to adapt to N starvation by developing biological N₂ fixation. Still it is important to study nitrate transport systems in legumes because nitrate intervenes as a signal in regulation of nodulation probably through nitrate transporters. The aim of this work is to achieve a molecular characterization of nitrate transporter 2 (NRT2) and NAR2 (NRT3) families to allow further work that would unravel their involvement in nitrate transport and signaling. Browsing the latest version of the Medicago truncatula genome annotation (v4 version) revealed three putative NRT2 members that we have named MtNRT2.1 (Medtr4g057890.1), MtNRT2.2 (Medtr4g057865.1) and MtNRT2.3 (Medtr8g069775.1) and two putative NAR2 members we named MtNAR2.1 (Medtr4g104730.1) and MtNAR2.2 (Medtr4g104700.1). The regulation and the spatial expression profiles of MtNRT2.1, the coincidence of its expression with that of MtNAR2.1 and MtNAR2.2 and the size of the encoded protein with 12 transmembrane (TM) spanning regions strongly support the idea that MtNRT2.1 is a nitrate transporter with a major contribution to the high‐affinity transport system (HATS), while a very low level of expression characterized MtNRT2.2. Unlike MtNRT2.1, MtNRT2.3 showed a lower level of expression in the root system but was expressed in the shoots and in the nodules thus suggesting an involvement of the encoded protein in nitrate transport inside the plant and/or in nitrate signaling pathways controlling post‐inoculation processes that govern nodule functioning.     

转录因子E2F1抑制CREG表达调控体外培养的人血管平滑肌细胞分化

为探讨转录因子E2F1在血管平滑肌细胞(vascular smooth muscle cells,VSMCs)表型转化中的作用及其对E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes,CREG)表达调控的分子机制,应用生物信息学方法,定位人CREG(hCREG)基因启动子并确定转录因子E2F1在hCREG启动子区的结合位点,PCR方法克隆并构建hCREG基因启动子绿色荧光报告基因载体,以hCREG启动子区E2F1结合位点为模板,化学合成E2F1寡聚脱氧核苷酸(ODN)和错配E2F1ODN,利用转录因子"诱骗(Decoy)"策略,用E2F1ODN转染体外培养的VSMCs以阻断E2F1与hCREG基因启动子区的结合,蛋白质印迹(Western blot)分析检测阻断前后细胞内hCREG蛋白、报告基因绿色荧光蛋白(green fluorescent protein,GFP)和平滑肌细胞分化标志蛋白SMα-actin表达变化.结果显示:分化表型HITASY细胞中E2F1表达下调伴出核转位,而增殖表型的HITASY细胞中E2F1蛋白表达明显增加且定位于核内.进一步应用FuGene6瞬时转染E2F1ODN和错配E2F1ODN于体外培养HITASY细胞中,蛋白质印迹分析发现,转染E2F1ODN后,HITASY细胞中hCREG、SMα-actin和GFP表达均较未阻断组及错配组细胞明显增加.上述研究结果证实,E2F1是hCREG基因转录的重要调控因子,能够直接结合于hCREG启动子区阻遏hCREG表达,参与hCREG蛋白对VSMCs表型转化的调控作用.     

The CRE1 Cytokinin Pathway Is Differentially Recruited Depending on Medicago truncatula Root Environments and Negatively Regulates Resistance to a Pathogen

Cytokinins are phytohormones that regulate many developmental and environmental responses. The Medicago truncatula cytokinin receptor MtCRE1 (Cytokinin Response 1) is required for the nitrogen-fixing symbiosis with rhizobia. As several cytokinin signaling genes are modulated in roots depending on different biotic and abiotic conditions, we assessed potential involvement of this pathway in various root environmental responses. Phenotyping of cre1 mutant roots infected by the Gigaspora margarita arbuscular mycorrhizal (AM) symbiotic fungus, the Aphanomyces euteiches root oomycete, or subjected to an abiotic stress (salt), were carried out. Detailed histological analysis and quantification of cre1 mycorrhized roots did not reveal any detrimental phenotype, suggesting that MtCRE1 does not belong to the ancestral common symbiotic pathway shared by rhizobial and AM symbioses. cre1 mutants formed an increased number of emerged lateral roots compared to wild-type plants, a phenotype which was also observed under non-stressed conditions. In response to A. euteiches, cre1 mutants showed reduced disease symptoms and an increased plant survival rate, correlated to an enhanced formation of lateral roots, a feature previously linked to Aphanomyces resistance. Overall, we showed that the cytokinin CRE1 pathway is not only required for symbiotic nodule organogenesis but also affects both root development and resistance to abiotic and biotic environmental stresses.     

Spatial and Temporal Patterns in Assemblages of Temperate Reef Fish

SYNOPSIS. For reef fish in temperate marine regions, such componentsof local assemblage diversity (i.e., within a reef) as speciesrichness, total fish density, and rank order of abundance canremain relatively constantthrough time. Long-term data (17 years)for assemblages on 2 reefs in Southern California revealed that,despite high turnover in rare species, overall species richnesswas affected only moderately by major oceanographicdisturbances.This resilience of the assemblage is in marked contrast to hightemporal variation in densities exhibited by many local populationsof individual species, and it suggests that measurements ofdiversity to indicate status of an assemblage should be usedwith caution. Here we consider various processes and factors,together with the spatial and temporal scales over which theyoperate, that can influence local diversity (and its estimation)of reef fishes. Mechanisms that can "buffer" local diversityof reef fishes include dispersal of young that inter-connectssubpopulations, high "inertia" in relative abundance and populationstructures (especially for long-lived species), and broad ecologicalrequirements of many species. These considerations suggest thatthe effect of disturbances on local diversity of reef fisheswill depend in part on the magnitude, duration, frequency andspatial scale of the perturbation. While long-term data arefew, available information suggests that, due to life historycharacteristics of the fish and the spatial and temporal scalesat which disturbances are likely to occur, assemblages of temperatemarine reef fish might be relatively resilient to environmentalperturbations