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.