Lectin genes from the legume Medicago truncatula

We report the cloning and characterization of two lectin genes from Medicago truncatula, designated Mtlec1 and Mtlec2. The two genes show a high degree of homology and apparently belong to a small multigene family. Mtlec1 appears to encode a functional lectin with 277 amino acids, whereas Mtlec2 is probably non-functional, since a frameshift mutation (insertion of two nucleotides) leads to premature translation termination after only 98 amino acids. The deduced amino acid sequence of the polypeptide MtLEC1 suggests that this lectin is a metalloprotein with Glc/Man specificity.     

Molecular systematics of the Old World Astragalus (Fabaceae) as inferred from nrDNA ITS sequence data

This study represents a nuclear rDNA ITS-based phylogenetic analyses of a greater sampling of the Old WorldAstragalus compared to our previous work (212 vs. 134 taxa). Phylogenetic relationships among 212 species (213 accessions) of the Old WorldAstragalus, including newly segregated monotypic genusPodlechiella, the two aneuploid New WorldAstragalus, and five related genera, were inferred from analyses of nuclear rDNA ITS sequences using maximum parsimony. A total of 658 nucleotide sites and four binary characters for indels were analyzed. The results of phylogenetic analyses suggest sect.Phyllolobium, comprising mostly the Chinese species, is placed outside of the so-calledAstragalus s. str. and is a well-supported monophyletic group. The monotypic annual segregate genusThlaspidium (≡Astragalus sect.Thlaspidium, A. thlaspi), is clearly nested withinAstragalus s. str. Among the many sections analyzed here, only sects.Cenanthrum, Caraganella, Eremophysa, Incani, Laxiflori, andLotidium are strongly supported as monophyletic. Our analysis, in agreement with previous studies, shows that the North American euploidAstragalus species are scattered throughout the Old World groups of the genus.     

Phylogeny of the New World diploid cottons (Gossypium L., Malvaceae) based on sequences of three low-copy nuclear genes

American diploid cottons (Gossypium L., subgenus Houzingenia Fryxell) form a monophyletic group of 13 species distributed mainly in western Mexico, extending into Arizona, Baja California, and with one disjunct species each in the Galapagos Islands and Peru. Prior phylogenetic analyses based on an alcohol dehydrogenase gene (AdhA) and nuclear ribosomal DNA indicated the need for additional data from other molecular markers to resolve phylogenetic relationships within this subgenus. Toward this end, we sequenced three nuclear genes, the anonymous locus A1341, an alcohol dehydrogenase gene (AdhC), and a cellulose synthase gene (CesA1b). Independent and combined analyses resolved clades that are congruent with current taxonomy and previous phylogenies. Our analyses diagnose at least two long distance dispersal events from the Mexican mainland to Baja California, following a rapid radiation of the primary lineages early in the diversification of the subgenus. Molecular data support the proposed recognition of a new species closely related to Gossypium laxum that was recently collected in Mexico.     

Inferring phylogeny at low taxonomic levels: utility of rapidly evolving cpDNA and nuclear ITS loci

Plant molecular systematic studies of closely related taxa have relied heavily on sequence data from nuclear ITS and cpDNA. Positive attributes of using ITS sequence data include the rapid rate of evolution compared to most plastid loci and availability of universal primers for amplification and sequencing. On the other hand, ITS sequence data may not adequately track organismal phylogeny if concerted evolution and high rDNA array copy number do not permit identification of orthologous copies. Shaw et al. (American Journal of Botany 92: 142-166) recently identified nine plastid regions that appear to provide more potentially informative characters than many other plastid loci. In the present study, sequences of these loci and ITS were obtained for six taxonomic groups in which phylogenetic relationships have been difficult to establish using other data. The relative utility of these regions was compared by assessing the number of parsimony informative characters, character congruence, resolution of inferred trees, clade support, and accuracy. No single locus emerged as the best in all lineages for any of these measures of utility. Results further indicated that in preliminary studies, sampling strategy should include at least four exemplar taxa. The importance of sampling data from independent distributions is also discussed.     

The taxonomic status of Astragalus mokiacensis (Fabaceae), a species endemic to the southwestern United States

Astragalus mokiacensis has been a problematic species since it was first described in 1877. Every major revision delimited this taxon differently. A principal components analysis of morphological data from herbarium specimens was used to determine the affinities between type specimens and extant populations ofA. mokiacensis. The taxon recently recognized asA. lentiginosus var.trumbullensis is morphologically similar to the lectotype ofAstragalus mokiacensis. Astragalus lentiginosus var.trumbullensis is herein recognized as a lowelevation minor variant and considered a synonym ofA. mokiacensis. A taxonomic key and complete synonymy are included.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

A new Astragalus (Leguminosae) from Argentina, notable for placentation of its seeds

From the Patagonian region in ArgentinaAstragalus neocarpus is described and illustrated. The false septum of its fruit is formed by union of the seed-funicles with the endocarp.

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Se describe e ilustraAstragalus neocarpus de la región Patagónica de Argentina, su principal característica distintiva es la morfología del fruto, donde el falso tabique que divide en dos a la cavidad está formado por la unión de largos funículos de las semillas con el endocarpo.

     

Ankyrin protein kinases: a novel type of plant kinase gene whose expression is induced by osmotic stress in alfalfa

Interaction between Medicago spp. and Sinorhizobium meliloti leads to the development of a novel organ, the root nodule. A gene, Msapk1, encoding a novel type of plant protein kinase containing a N-terminal region with an ankyrin domain, was identified and shown to be expressed both in S. meliloti-infected and spontaneous nodules in alfalfa. This gene is not exclusively associated to nodulation since its expression was detected in other plant organs. Several genes coding for ankyrin protein kinases (APKs) were detected in various plants and animals. Three closest A. thaliana homologues of Msapk1 were identified in databases and two of them were shown to express differentially in various organs using gene-specific RT-PCR. In contrast, Southern analysis suggests that a single-copy gene exists in diploid M. truncatula. By screening a M. truncatula BAC library the Mtapk1 genomic region was isolated and sequenced. Two neighbouring genes showing homologies to previously identified sequences in data banks were detected in the vicinity of the Mtapk1 gene and compared to similar regions of the three Atapk genes. The distribution of exons/introns was the same for all expressed genes of both species although Mtapk1 contained larger introns. Upon osmotic stress Msapk1 expression was induced in roots of alfalfa starting from three hours up to two days of treatment. These data suggest that Msapk1, involved in alfalfa osmotic stress responses, belongs to a novel class of plant protein kinases.     

Molecular systematics of the genus Astragalus L. (Fabaceae): Phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacers and chloroplast gene ndhF sequences

Comparative sequencing of internal transcribed spacers (ITS) and 5.8S gene of nuclear ribosomal DNA was carried out to examine phylogenetic relationships among subgenera and sections of Old World Astragalus as well as the recent segregate genera Barnebyella and Ophiocarpus. For a subset of these taxa (43 accessions), the nrDNA ITS data were supplemented by sequences from the chloroplast ndhF gene. Phylogenetic trees resulting from separate analyses of the nrDNA ITS and ndhF sequences were in conflict mainly on the position and relationships of Ophiocarpus aitchisonii, Astragalus hemsleyi, A. grammocalyx, A. coelicolor, A. capito, A. epiglottis and A. annularis. Excluding these taxa, phylogenetic analysis of a combined nrDNA ITS-ndhF data matrix was also conducted, so that in the resulting tree, most clades were more resolved and better statistically supported than those were in the separate analyses. Our results indicate that the monotypic segregate genera Barnebyella (= A. migpo), Ophiocarpus (= A. ophiocarpus) and morphologically isolated annual species A. dipelta (= Didymopelta turkestanica), A. schmalhausenii (= Sewerzowia turkestanica) and A. vicarius (= S. vicaria) are clearly nested within Astragalus. Our results confirm earlier studies that shows A. vogelii is allied with the genera Colutea and Oxytropis rather than with any Astragalus species. It is therefore excluded from Astragalus and elevated to the new generic rank and named as Podlechiella Maassoumi and Kazempour Osaloo. None of the eight traditionally recognized Astragalus subgenera Epiglottis, Trimeniaeus, Phaca, Hypoglottis, Calycophysa, Tragacantha, Cercidothrix and Calycocystis are monophyletic. Similarly, the monophyly of Podlechs new subgenera Trimeniaeus, Astragalus and Cercidothrix is not supported. Among the many species-rich sections analyzed here, only Cenanthrum, Chronopus, Laxiflori, Lotidium, Incani and Amodendron are monophyletic.     

Master: a novel family of PIF/Harbinger-like transposable elements identified in carrot (Daucus carota L.)

Members of a novel Master family of class II transposons were identified in the carrot genome. Two elements, 2.5 kb long DcMaster1 and 4.4 kb long DcMaster-a, are characterized by 22 bp imperfect terminal inverted repeats and by 3 bp target site duplications. GenBank search revealed that related elements are also present in Medicago truncatula, including a 5.1 kb element MtMaster-a. Both DcMaster-a and MtMaster-a contain open reading frames encoding for putative transposases with the complete DDE domain typical for plant class II transposable elements belonging to PIF/Harbinger superfamily, where the Master elements form a distinct group. Less than 10 copies of the DcMaster element containing the DDE domain are present in genomes of carrot and other Apiaceae, but more copies with internal deletions or insertions may occur. DcMaster elements were associated with putative coding regions in 8 of 14 identified insertion sites. PCR amplification of carrot genomic DNA using a primer complementary to TIRs of DcMaster gave products <400 bp in size. We speculate that these may all represent a MITE-like family of transposable elements that we named Krak, present in the carrot genome in at least 3,600 copies. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers DQ250792 to DQ250807 and DQ353734 to DQ353752.     

Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants

A novel, constitutively expressed gene, designated MtHP, was isolated from the model legume species Medicago truncatula. Sequence analysis indicates that MtHP most likely belongs to the PR10 multi-gene family. The MtHP promoter was fused to a -glucuronidase gene to characterize its expression in different plant species. Transient assay by microprojectile bombardment and hairy root transformation by Agrobacterium rhizogenes revealed GUS expression in leaf, stem, radicle and root in M. truncatula. Detailed analysis in transgenic Arabidopsis plants demonstrated that the promoter could direct transgene expression in different tissues and organs at various developmental stages; its expression pattern was similar to that of CaMV35S promoter, and the level of expression was higher than the reporter gene driven by CaMV35S promoter. Deletion analysis revealed that even a 107 bp fragment of the promoter could still lead to a moderate level of expression. The promoter was further characterized in white clover (Trifolium repens), a widely grown forage legume species. Strong constitutive expression was observed in transgenic white clover plants. Compared with CaMV35S promoter, the level of GUS activity in transgenic white clover was higher when the transgene was driven by MtHP promoter. Thus, the promoter provides a useful alternative to the CaMV35S promoter in plant transformation for high levels of constitutive expression.     

Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes

Phylogenetic utility of two nuclear genes (GBSSI-2 and DHAR) was explored in genus Fragaria in order to clarify phylogenetic relationships among taxa and to elucidate the origin of the polyploid species. Orthology of the amplified products was assessed by several methods. Our results strongly suggest the loss of one GBSSI duplicated copy (GBSSI-1) in the Fragariinae subtribe. Phylogenetic analyses provided new insights into the evolutionary history of Fragaria, such as evidence supporting the presence of three main diploid genomic pools in the genus and demonstrating the occurrence of independent events of polyploidisation. In addition, the data provide evidence supporting an allopolyploid origin of the hexaploid F. moschata, and the octoploids F. chiloensis, F. iturupensis and F. virginiana. Accordingly, a new pattern summarizing our present knowledge on the Fragaria evolutionary history is proposed. Additionally, sequence analyses also revealed relaxed constraints on homoeologous copies at high ploidy level, as demonstrated by deletion events within DHAR coding sequences of some allo-octoploid haplotypes.     

Conserved and novel miRNAs in the legume <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> in response to stress

MicroRNAs (miRNAs) are small RNA molecules recognized as important regulators of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data. We are interested in the identification of miRNAs in Phaseolus vulgaris (common bean) to uncover different plant strategies to cope with adverse conditions and because of its relevance as a crop in developing countries. Here we present the identification of conserved and candidate novel miRNAs in P. vulgaris present in different organs and growth conditions, including drought, abscisic acid treatment, and Rhizobium infection. We also identified cDNA sequences in public databases that represent the corresponding miRNA precursors. In addition, we predicted and validated target mRNAs amongst reported EST and cDNAs for P. vulgaris. We propose that the novel miRNAs present in common bean and other legumes, are involved in regulation of legume-specific processes including adaptation to diverse external cues. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isoenzymes and flow cytometry for the assessment of true-to-typeness of calluses and cell suspensions of barrel medic prior to regeneration

A successful exploitation of in vitro tools for breeding and for the understanding of gene functioning requires the regeneration of true-to-type plants and experiments were therefore performed with several genotypes of Medicago truncatula (J5, TRV25, TR122) to characterize mother plants and regenerating tissues. Each sample was assessed by flow cytometry and, whatever the genotype and regeneration pathway, a divergent phenotype was systematically linked to an abnormal nuclear DNA content. All samples assessed were classed according to their flow cytometry profiles into normal (true-to-type) material, aneuploids, endoreduplicated tissues, tetraploids, mixoploids and senescent tissues. Deviating calluses failed to regenerate or gave rise to infertile, non-viable plants. In turn, all tissues with non true-to-type flow cytometry profiles were examined in terms of isoenzyme banding patterns compared to the mother plants. Esterases, Peroxidases and Leucine aminopeptidase appeared to be the best isozyme systems to show differences between the original genotypes but also between diverging materials and the mother plants. Interestingly, such differences were more often qualitative (presence or absence of bands) than quantitative (i.e. differences in colour intensity of bands) thereby making easier an accurate distinction between genotypes. Peroxidases were prone to variation with culture medium and tissue age. The results stressed the importance of using more than one approach when undertaking the characterisation of materials as, for some of the genotypes analysed, differences compared to the respective mother plants could be shown with flow cytometry that were not reflected in a different banding pattern with isoenzymes.