A novel family in Medicago truncatula consisting of more than 300 nodule-specific genes coding for small, secreted polypeptides with conserved cysteine motifs

Abstract

Transcriptome analysis of Medicago truncatula nodules has led to the discovery of a gene family named NCR (nodule-specific cysteine rich) with more than 300 members. The encoded polypeptides were short (60-90 amino acids), carried a conserved signal peptide, and, except for a conserved cysteine motif, displayed otherwise extensive sequence divergence. Family members were found in pea (Pisum sativum), broad bean (Vicia faba), white clover (Trifolium repens), and Galega orientalis but not in other plants, including other legumes, suggesting that the family might be specific for galegoid legumes forming indeterminate nodules. Gene expression of all family members was restricted to nodules except for two, also expressed in mycorrhizal roots. NCR genes exhibited distinct temporal and spatial expression patterns in nodules and, thus, were coupled to different stages of development. The signal peptide targeted the polypeptides in the secretory pathway, as shown by green fluorescent protein fusions expressed in onion (Allium cepa) epidermal cells. Coregulation of certain NCR genes with genes coding for a potentially secreted calmodulin-like protein and for a signal peptide peptidase suggests a concerted action in nodule development. Potential functions of the NCR polypeptides in cell-to-cell signaling and creation of a defense system are discussed.

Figure 1

The NCR multigene family of Medicago. A, Southern hybridization of genomic DNA digested with EcoRI (E) or HindIII (H) from M. truncatula and alfalfa (Medicago sativa) with the NCR001 probe revealing multiple hybridizing bands. MtR108, M. truncatula line R108; MtJ5, M. truncatula line Jemalong J5; MsA2, alfalfa subsp. varia line A2. The HindIII digestion was partial. B, Alignment of the 42 most abundantly expressed NCRs (minimum 10 EST hits) forming group A with four and group B with six Cys residues. Identical residues conserved in at least 60% of the sequences are in black, whereas the similar amino acids are in gray background. The signal peptides are underlined. In the consensus sequence, “h” stands for a hydrophobic residue (I, L, V, or M), and “b” stands for a basic residue (R or K). The conserved Cys are numbered as described in the text.

Figure 2

NCR homologs and similar polypeptides in other species. A, NCR homologs in the galegoid group of legumes. The signal peptides and the conserved residues are indicated as in Figure 1. Mt, M. truncatula; Ps, pea; Vf, broad bean; Go, G. orientalis; Tr, white clover. B, Proteins with similar structure to NCRs. Sequences indicated with asterisks were found in BLAST searches with NCR polypeptides as query. Accession numbers are indicated. The conserved Cys are shaded. SP, Signal. A, Self-incompatibility (SCR) proteins; B, NCR proteins; C, pollen coat proteins (PCPs), defensin proteins, γ-thionin proteins, and p322 proteins (the latter are incorrectly designated in databases as proteinase inhibitors); D, scorpion (Buthus martensii and Thyus serrulatus) neurotoxins; E, avirulence protein Avr9. Ser proteinase inhibitors were omitted from this alignment because of their large diversity (Laskowski and Kato, 1980). Br, Brassica rapa; Bo, Brassica oleracea; Mt, M. truncatula; Bm, Buthus martensii; Ts, Tityus serrulatus; Ps, pea; Ms, alfalfa; Gm, soybean; Lj, L. japonicus; St, Solanum tuberosum; Cv, Cladosporium fulvum. C, Cys motifs conserved in SCR-like, defensin-like, NCR-like, and neurotoxin polypeptides. Conserved Cys are in black shading, and conserved spacings are in gray shading.

Figure 3

Subcellular localization of GFP fusion proteins in onion epidermal cells. A to C, Confocal sections of the GFP control. D to F, Confocal sections of the NCR084-GFP fusion. Sections were through the cortex (A and D), the vacuole (B and E), and the nucleus (C and F). TVS, Trans-vacuolar strands, containing cytosol and endoplasmic reticulum (ER); V, vacuole; N, nucleus.

Figure 4

Expression profiles of NCR genes in M. truncatula. A, Heat map of macroarray hybridizations. NCR (in blue), nodulin (in black), and CaM-like and signal peptide peptidase (SPP; in green) genes are presented in the rows and hybridization experiments in the columns indicating the plant line, the bacterial strain, and the age of the nodules between brackets, if different from 3 weeks after inoculation. Black corresponds to mean level of transcript accumulation, whereas different intensities from black to red or to green indicate increasing or decreasing expression levels. B, Expression analysis by reverse transcriptase (RT)-PCR for the constitutive gene Mtc27, a CaM-like gene (TC51594), the nodule-expressed SPP genes (amplified by common primers), and the NCR genes 001, 099, 084, 053, 094, and 007. Lanes are amplifications of cDNA from: 1, roots; 2, roots treated with Nod factor; 3, Sm41-induced nodule primordia 7 d postinoculation (dpi); 4, nodules at 13 dpi; 5, nodules at 20 dpi; 6, nodules at 29 dpi; 7, cotyledons; 8, hypocotyls; 9, petioles; 10, stems; 11, apical meristems; 12, young leaves; 13, mature leaves; 14, closed flowers; 15, open flowers; 16, water control. C, Heat map of in silico expression. Red shading means relative fraction of ESTs found in the different libraries, and the histogram indicates the total number of ESTs for each NCR gene. The libraries and genes are ordered according to the nodule developmental stage from which they were prepared or in which they were expressed, respectively. Blue, Very early; light blue, early; green, medium; yellow, late; and red, very late. The libraries were: N1, MtBB; N2, R108Mt; N3, GVN; N4, GVSN; N5, nodulated root; M, MtBC; O, other than nodule libraries.

Figure 5

Localization of NCR001 and NCR084 transcripts in M. truncatula nodules. A and D, Hybridization of M. truncatula nodule sections with the NCR084 antisense probe. B, Hybridization of the antisense leghemoglobin Lb1 probe. C and E, Hybridization of the antisense NCR001 probe. Hybridization signals are observed as black dots. The different nodule zones in the sections are indicated with arrows. The red arrows point to amyloplasts in D and E. Bars = 200 μm.

Figure 6

Alignment of SPPs. Underlined accession numbers correspond to the nodule specific TCs. The sequence of TC44385 is partial at the N terminus and that of TC44387 and TC52930 at the C terminus. The most conserved residues in the SPP family (Weihofen et al., 2002) are indicated with an asterisk, and the two aspartic acids of the active site are marked with an underlined asterisk. ER retention signals are boxed. Conserved residues are indicated as in Figure 1. Mt, M. truncatula; At, Arabidopsis; Hs, human (Homo sapiens).

Figure 7

Phylogeny of Leguminosae based on the rbcL sequence. The tree is adopted from Doyle (1998), and only the branch of the Papilionoideae subfamily relevant to the discussion is shown. The galegoid clade is boxed, and the genera, where NCR family members were found, are underlined. The nodule types are symbolized by a circle for the round-shaped determinate nodules and by an oval form for the meristem containing elongated indeterminate nodules.