Genome-wide identification of nodule-specific transcripts in the model legume Medicago truncatula

Abstract

The Medicago truncatula expressed sequence tag (EST) database (Gene Index) contains over 140,000 sequences from 30 cDNA libraries. This resource offers the possibility of identifying previously uncharacterized genes and assessing the frequency and tissue specificity of their expression in silico. Because M. truncatula forms symbiotic root nodules, unlike Arabidopsis, this is a particularly important approach in investigating genes specific to nodule development and function in legumes. Our analyses have revealed 340 putative gene products, or tentative consensus sequences (TCs), expressed solely in root nodules. These TCs were represented by two to 379 ESTs. Of these TCs, 3% appear to encode novel proteins, 57% encode proteins with a weak similarity to the GenBank accessions, and 40% encode proteins with strong similarity to the known proteins. Nodule-specific TCs were grouped into nine categories based on the predicted function of their protein products. Besides previously characterized nodulins, other examples of highly abundant nodule-specific transcripts include plantacyanin, agglutinin, embryo-specific protein, and purine permease. Six nodule-specific TCs encode calmodulin-like proteins that possess a unique cleavable transit sequence potentially targeting the protein into the peribacteroid space. Surprisingly, 114 nodule-specific TCs encode small Cys cluster proteins with a cleavable transit peptide. To determine the validity of the in silico analysis, expression of 91 putative nodule-specific TCs was analyzed by macroarray and RNA-blot hybridizations. Nodule-enhanced expression was confirmed experimentally for the TCs composed of five or more ESTs, whereas the results for those TCs containing fewer ESTs were variable.

Figure 1

Distribution of nodule-specific TCs by functional categories. Classification was performed for 137 nodule-specific TCs with strong statistical similarity to GenBank protein sequences (E values lower than 10⁻⁸).

Figure 2

Comparison of the deduced amino acid sequences of M. truncatula nodule-specific calmodulin-like proteins (encoded by TC35910, TC35911, TC35912, TC41252, TC34223, TC37063-s, and TC37063-l), typical M. truncatula calmodulins (encoded by TC31994 and TC35885), and calmodulins from Medicago sativa (GenBank accession no. X52398), bean (AAD10245), T. pyriformis (P02598), and T. gondii (Y08373). Comparisons are referenced to the calmodulin-like protein encoded by TC35910. Dots represent identical amino acids. Amino acids shaded in gray possess similar physico-chemical properties. EF hand domains, as predicted by PSORT, are shown in boxes. Underlined amino acids are essential for Ca²⁺ binding. The circled amino acids in the N-terminal portion of the polypeptides are the last ones in the predicted cleavable signal peptide. Gaps in the sequences (indicated by dashes) are introduced to maintain maximum sequence similarity.

Figure 3

Comparison of the deduced amino acid sequences of several TCs encoding CCPs with CCPs from other legumes: pea ENOD3 (GenBank accession no. P25225), broad bean CCP2 and CCP5 (GenBank accession nos. AJ243463 and AJ243466), and hypothetical protein of G. orientalis (GenBank accession no. CAB51773). Comparisons are made against pea ENOD3. Dots represent identical amino acids. Amino acids shaded in gray share similar physico-chemical properties. Gaps (indicated by dashes) are introduced to maintain maximum sequence similarity. Conserved Cys clusters are shown in boxes. The circled amino acids are the last ones in the predicted cleavable N-terminal signal sequence. Underlined amino acid in TC39082 is the last one in the predicted signal peptide that is not cleaved.

Figure 4

Northern-blot analysis of selected nodule-specific TCs. Twenty micrograms of total RNA from nodules (N), senescent nodules (SN), roots (R), leaves (L), flowers (F), and pods (P) was separated by gel electrophoresis, transferred onto a nitrocellulose membrane, and hybridized with radioactively labeled cDNA inserts. Inserts represent the clones that belong to nine TCs identified in silico as nodule-specific. Transcript size (kb) is estimated from its electrophoretic mobility. Radioactivity with 28S RNA probe quantified by the AMBIS Radioanalytic Image System (Scanalytics, Billerica, MA) demonstrates the comparative RNA loading.