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.Plants have evolved symbiotic associations with soil microorganisms to facilitate their mineral nutrition. An example is the specific interaction of different species of the Leguminosae (legumes) with the nitrogen-fixing soil bacteria from the Rhizobiaceae family (rhizobia). This symbiosis leads to the de novo formation of a root organ, the nodule, hosting nitrogen-fixing rhizobia that feed the host plant with ammonium. Another example is the widespread association of plants with fungi from the order of Glomales leading to the formation of arbuscular endomycorrhiza that extends the plant root system and facilitates nutrient uptake. The initial stages of rhizobial and mycorrhizal interactions share certain common molecular mechanisms (Albrecht et al., 1999;Kistner and Parniske, 2002). Because mycorrhizas are more common and ancient, the rhizobial symbiosis might have acquired existing mechanisms from them.Two major types of legume nodules are distinguished (Crespi and Gálvez, 2000): the indeterminate type, formed by e.g. Medicago truncatula, pea (Pisum sativum), broad bean (Vicia faba), white clover (Trifolium repens), or Galega orientalis, and the determinate type, formed by e.g. Lotus japonicus or soybean (Glycine max). Indeterminate nodules have a complex structure composed of different central tissues surrounded by a cortex (Vasse et al., 1990). The persistent apical meristem is zone I. In zone II, postmeristematic cells gradually differentiate and become infected with rhizobia, encapsulated in a membrane envelope. Interzone II-III is characterized by amyloplast accumulation and major transcriptional changes in both plant and bacterial cells. The proximal zone III is compos...

CLE Peptides Control Medicago truncatula Nodulation Locally and Systemically

“…Medicago truncatula 'Jemalong A17' and 'J5' as well as nin, bit1-1, nsp1, nsp2, dmi1, dmi2, dmi3, and sunn-1 mutants (Catoira et al, 2000;Oldroyd and Long, 2003;Marsh et al, 2007;Middleton et al, 2007) and pENOD11:GUS transgenic seeds (Journet et al, 2001) were grown and inoculated as described (Mergaert et al, 2003). Sinorhizobium meliloti 1021, Sm1021 pHC60-GFP (Cheng and Walker, 1998), Sm1021 pQE81-dsRedT3 (Bevis and Glick, 2002), Sm2011 pBHRmRFP (Smit et al, 2005), Sm2011 pHC60-GFP (Cheng and Walker, 1998), and Sm2011 pMH682-Gmi6390 (Roche et al, 1991) were grown at 28°C in yeast extract broth medium (Vervliet et al, 1975) supplemented with 10 mg L 21 tetracycline for the Sm1021 pHC60-GFP, Sm1021 pQE81-dsRedT3, Sm2011 pBHR-mRFP, Sm2011 pHC60-GFP, and Sm1021 pMH682-Gmi6390 strains.…”

Bridging Model and Crop Legumes through Comparative Genomics

“…Motif analysis identified three major gene families from this putative legume-specific gene set, including F-box-related proteins, Pro-rich proteins, and Cys cluster proteins. In particular, the more than 300 Cys cluster proteins, with predicted similarity to defensins, represent approximately 1% of expressed genes in Medicago, primarily from nodules (Fedorova et al, 2002;Mergaert et al, 2003;Graham et al, 2004). Work is under way to characterize these putative legumespecific genes using both forward and reverse genetic tools (K. VandenBosch, personal communication).…”

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“…Growth of Medicago truncatula line Jemalong J5, Sinorhizobium meliloti 1021, and plant inoculation were as described (Mergaert et al, 2003). For each nodulation stage, nodules from 30 plants were harvested.…”