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.


INTRODUCTION
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 sub-cellular organelles. By transporting metabolites and non-metabolites, 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 Presently, it consists of ~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 N 2 to NH 3 ) in a symbiotic association with bacteria called rhizobia. This mutuallybeneficial 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 non-infected 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 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 P and N, 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  SOSUI integrates multiple properties, including hydropathy, amphiphilicity, amino acid charges, and sequence length to predict protein topology in the membrane.

Identification of putative transporters
Among the 38,335 IMGAG-annotated gene products ( Figure 1A), 44% were predicted by TMPred to contain at least one TMD, while 32% and 21% were predicted to have one or more TMD by HMMTOP and SOSUI, respectively. In total, 18,684 proteins were predicted to contain at least one TMD by one or more of the three programs. 7,438 proteins were predicted to contain two or more TMD by at least one program, of which 2,405 were identified by all three programs ( Figure 1B). Additionally, all 38,335 IMGAG proteins were compared by sequence homology to proteins of the Transporter Classification Database (TCDB) (Saier et al., 2006). This approach was used to identify potential transporters not recognized by any of the TMD prediction algorithms, as well as to guide transporter classification. Among the IMGAG-predicted proteins, 2,039 (5.3%) showed significant similarity to a TCDB sequence. Of these, 1,114 proteins were also found to contain at least two (2+) TMD by at least two prediction programs ( Figure 1C).  Figure 1D). TMD prediction was avoided as a method to select putative transporters encoded by ESTs because many of these sequences are incomplete.

Transporter classification
Medicago proteins with similarity to TCDB proteins were classified into families and,  Table 3 and a complete list is given in Table S2.
Our analysis of transporters encoded by genomic DNA was based on IMGAG version 2.0 annotation of the genome, which discarded seven IMGAG version 1.0 gene models encoding putative transporters represented by probesets on the Affymetrix Medicago GeneChip, including a putative ammonium transporter gene known to be expressed during mycorrhizal symbiosis (Gomez et al., 2009). Therefore, we included these seven putative transporter genes in our subsequent analyses of gene expression.

Analysis of transporter gene expression
The Affymetrix Medicago GeneChip contains 50,900 Medicago truncatula probesets corresponding to most of the gene transcripts in this species. A script was written in Perl to map probesets to IMGAG version 2.0 gene sequences (see Materials and Methods). In this way, Affymetrix probesets were assigned to 18,909 IMGAG-annotated genes and 21,284 ESTs (TCs and singlets) not represented by genomic sequence ( Figure 1A). Of the 2,673 genes encoding TCDB-classified transporters, 2,604 were represented by probesets on the Affymetrix Medicago GeneChip (Table 2 and S3). Affymetrix probesets matched seven additional transporter genes predicted by IMGAG v.1 but not IMGAG v.2. We included these in our analyses and assigned them confidence level "X" to make them easy to identify in the tables.
Expression data for putative transporter genes were retrieved from the Medicago Gene Atlas (Benedito et al., 2008), including data from all major organ systems (Table S4), a nodule developmental series, and mycorrhizal roots (Table S5). Based on presence/absence calls (at least two present calls within three biological replicates) obtained from chip analysis, 94% of transporter genes were expressed in at least one organ, with each organ expressing about 60% of all transporter genes. Only 4.5% of transporter genes (129) were organ-specific (i.e. active in a single organ type), while 29% of genes (823) were expressed in more than one, but not all organ types. The majority of transporter genes (61%) were expressed in all organs analyzed, although at different levels (Table S4).

Transporter gene expression during root symbioses
Regulation of transporter gene expression during development of two different root symbioses was investigated: nitrogen-fixing and arbuscular mycorrhizal symbioses.
Gene expression data for developing and nitrogen-fixing root nodules were obtained from showing >100 fold-change in expression (Table S5). Table 4 shows 37 genes that were induced >50-fold during nodule development. Figure 2 shows membrane transporters highly induced in, or specific to nodules.  (Tables 5 and S5). Classification into a specific family/subfamily was given a confidence score from 1-3, based on whether or not additional information supported the results of TCDB analysis, as described in the results section. Proteins with two or more TMD that did not match proteins in the TCDB, but for which additional information pointed to possible transporter function were in two or more organs, approximately 4.5% (129) were expressed in an organ-specific (Table   S4). Presumably, these play specialized roles in organ development, differentiation, and/or function, and they represent interesting targets for future functional analysis. However, because of our interest in beneficial plant-microbe interactions, we focused most of our attention on genes induced during nodule development and SNF or during AM symbiosis. 87% of all transporter genes were differentially-expressed during nodule development and SNF, of which 196 were induced more than 5-fold compared to non-inoculated roots, and 25 were induced >100-fold. (Table S5). 886 genes were differentially-expressed during AM symbiosis, of which 44 were induced more than 5-fold compared to non-inoculated roots. In the following paragraphs, we discuss some of these genes in the context of what is known about transport and transporters in the two types of symbiosis.   (Table 4). An unrelated, nodule-specific SM protein of soybean called GmZIP1

Transporters involved in N 2 fixation symbiosis
(TC 2.A.5), which transports zinc has also been characterized (Moreau et al., 2002). Inhibition of zinc transport into isolated symbiosomes by an antibody to GmZIP1 implicates the transporter in zinc supply to the bacteroids (Moreau et al., 2002). We identified 23 ZIP family members in Medicago, six of which were induced more than 2-fold during nodule development (Table S5).  (Table 4).
A P-type H + -ATPase (TC 3.A.3) and possibly other proton pumps energize the SM, which drives many secondary transport processes on this membrane (Udvardi and Day, 1989; Fedorova et al., 1999). However, the specific isoforms responsible remain to be identified. We found a nodule-specific P-type ATPase in Medicago, which is an interesting candidate for this activity (Table 4).  (Table 4), although it is not the most similar in sequence to the CgAUX1.

ATP for plant membrane energization and metabolism in nodule cells is provided
Apart from interesting candidates for known transport functions in nodules, our analysis of Medicago transporter gene expression identified many nodule-induced or nodule-specific transporters that presumably carry out novel transport functions in nodules. Clearly, both sets of transporters warrant further work in future to characterize their biochemical and physiological roles in nodules.

Transporters involved in mycorrhizal symbiosis
The number of differentially induced transporters in mycorrhizal roots in comparison to control roots was much smaller than that observed during nodulation, with only 33 transporters showing > 5 fold-change induction in mycorrhizal roots. This was probably due to a dilution effect, since whole root systems were sampled, in contrast to nodule samples, which were excised from nodulated roots and compared to non-nodulated control roots. Nonetheless, some transporters showed a massive induction (>100 fold-change) in mycorrhizal roots. Statistical test revealed 658 transporters differentially expressed in mycorrhizal roots (Table S5).
One of the greatest benefits of mycorrhizal symbiosis for the plants is an increased phosphate uptake, mediated by the AM fungi, which deliver Pi directly to the root cortex. Plant  (Table S5). In our study, we found evidence for an additional fungalderived transporter belonging to the Iron/Lead Transporter Superfamily (ILT, 9.A.10) that is induced 33-fold in mycorrhizal roots (Table 4).

Perspectives
Our search for transporters in Medicago uncovered and classified 2,673 putative transporter genes, many of which are induced during symbiosis with nitrogen-fixing rhizobia or AM fungi. Given the importance of these symbioses to plant nutrition and sustainable agriculture, it will be interesting to characterize the function of many of the symbiosis-induced transporters. Systematic analysis of transporters in Medicago will be aided not only by the results presented here, but also by the availability of Tnt1 insertion lines of Medicago (Tadege et al., IMGAG v.2 predicted proteins were analyzed for sequence similarity to transporters of the TC database (TCDB, http://www.tcdb.org/index.php), using BLASTP with an e-value cutoff ≤ e-3.

Transporter identification and sequence analyses
All sequences with two or more predicted TMD or with significant similarity to TCDB proteins were selected for manual curation.
Expressed sequence tags (tentative consensuses and singlets) of the Medicago truncatula ATPase, extrusion and exchanger) were retrieved for further analyses.

Mapping of IMGAG v.2 predicted genes onto the Medicago Affymetrix GeneChip
Affymetrix        *Note that the total number of MTGI sequences is higher than shown in Table 1