A Krüppel-like transcription factor gene is involved in salt stress responses in Medicago spp.

Legume plants are able to fix nitrogen in symbiotic association with rhizobia and, like many crops, are sensitive to high salt conditions. However, very few molecular markers can be associated to stress tolerance in legume crops. A Kruppel-like transcription factor, Mtzpt2-1, required for the formation of the nitrogen-fixing region, confers salt tolerance to yeast cells. Here, legume responses to salt stresses were studied using alfalfa and its close relative Medicago truncatula, a model legume species. Salt stress induces the Mszpt2-1 gene both in roots and root harbouring nodules. In addition, Sinorhizobium meliloti strains tolerating up to 700 mM NaCl, were used in nodulation assays to assess salt tolerance of the symbiotic response of M. truncatula. Few nodules, mainly in the upper part of the root, could be detected in plants treated with 200 mM NaCl, suggesting that nodule initiation was particularly sensitive to salt stress. We have also defined for M. truncatula the threshold of NaCl tolerance after which recovery of stressed plants is irreversible under laboratory conditions. After analysing several times of salt treatment (150 mM NaCl), M. truncatula 108R plants stressed for 7 days could not recover (less than 5%), whereas a 4-day treatment allowed at least 75% recovery. Transgenic M. truncatula plants expressing Mtzpt2-1 in antisense configuration are more sensitive to `recover' from salt stress than the wild type. These results identify Mtzpt2-1 as a molecular marker potentially linked to stress tolerance in M. truncatula and suggest its participation in a transcriptional program induced in these plants to cope with salt stress.Facultad de Ciencias ExactasInstituto de Biotecnología y Biología Molecula

Functional Genomic Analysis of Global Regulator NoIR in Sinorhizobium meliloti

NolR is a regulator of nodulation genes present in species belonging to the genera Rhizobium and Sinorhizobium. The expression of the nolR gene in Sinorhizobium meliloti AK631 was investigated in relation to stage of growth, availability of nutrients, and different environmental stimuli using the nol

Two Classes of the Cdh1-Type Activators of the Anaphase-Promoting Complex in Plants: Novel Functional Domains and Distinct Regulation

The Cdc20 and Cdh1/Fzr proteins are the substrate-specific activators of the anaphase-promoting complex (APC). In Medicago truncatula, the MtCcs52A and MtCcs52B proteins represent two subgroups of the Cdh1-type activators, which display differences in their cell cycle regulation, structure, and function. The ccs52A transcripts are present in all phases of the cell cycle. By contrast, expression of ccs52B is restricted to late G2-phase and M-phase, and its induced overexpression in BY2 cells inhibited mitosis. MtCcs52A is active in Schizosaccharomyces pombe and binds to the S. pombe APC, whereas MtCcs52B does not because of differences in the N-terminal region. We identified a new functional domain, the Cdh1-specific motif conserved in the Cdh1 proteins that, in addition to the C-box and the terminal Ile and Arg residues, was essential for the activity and required for efficient binding to the APC. Moreover, we demonstrate that cyclin-dependent kinase phosphorylation sites adjacent to the C-box may regulate the interaction with the APC. In the different plant organs, the expression of Mtccs52A and Mtccs52B displayed differences and indicated the involvement of the APC in differentiation processes

Enod40, a Short Open Reading Frame–Containing mRNA, Induces Cytoplasmic Localization of a Nuclear RNA Binding Protein in Medicago truncatula

In eukaryotes, diverse mRNAs containing only short open reading frames (sORF-mRNAs) are induced at specific stages of development. Their mechanisms of action may involve the RNA itself and/or sORF-encoded oligopeptides. Enod40 genes code for highly structured plant sORF-mRNAs involved in root nodule organogenesis. A novel RNA binding protein interacting with the enod40 RNA, MtRBP1 (for Medicago truncatula RNA Binding Protein 1), was identified using a yeast three-hybrid screening. Immunolocalization studies and use of a MtRBP1-DsRed2 fluorescent protein fusion showed that MtRBP1 localized to nuclear speckles in plant cells but was exported into the cytoplasm during nodule development in enod40-expressing cells. Direct involvement of the enod40 RNA in MtRBP1 relocalization into cytoplasmic granules was shown using a transient expression assay. Using a (green fluorescent protein)/MS2 bacteriophage system to tag the enod40 RNA, we detected in vivo colocalization of the enod40 RNA and MtRBP1 in these granules. This in vivo approach to monitor RNA–protein interactions allowed us to demonstrate that cytoplasmic relocalization of nuclear proteins is an RNA-mediated cellular function of a sORF-mRNA

Genomic organization and evolutionary insights on GRP and NCR genes, two large nodule-specific gene families in Medicago truncatula.

Deciphering the mechanisms leading to symbiotic nitrogen-fixing root nodule organogenesis in legumes resulted in the identification of numerous nodule-specific genes and gene families. Among them, NCR and GRP genes encode short secreted peptides with potential antimicrobial activity. These genes appear to form large multigenic families in Medicago truncatula and other closely related legume species, whereas no similar genes were found in databases of Lotus japonicus and Glycine max. We analyzed the genomic organization of these genes as well as their evolutionary dynamics in the M. truncatula genome. A total of 108 NCR and 23 GRP genes have been mapped that were often clustered in the genome. These included 29 new NCR and 17 new GRP genes. Reverse transcription-polymerase chain reaction analyses of the novel genes confirmed their exclusive nodule-specific expression similar to the previously identified members. Protein alignments and phylogenetic analyses revealed traces of several duplication events in the history of GRP and NCR genes. Moreover, microsyntenic evidences between M. truncatula and L. japonicus validated the hypothesis that these genes are specific for the inverted repeat-lacking clade of hologalegoid legumes, which allowed dating the appearance of these two gene families during the evolution of legume plants