Expressions of ECE-CYC2 clade genes relating to abortion of both dorsal and ventral stamens in Opithandra (Gesneriaceae)

Background  

ECE-CYC2 clade genes known in patterning floral dorsoventral asymmetry (zygomorphy) in Antirrhinum majus are conserved in the dorsal identity function including arresting the dorsal stamen. However, it remains uncertain whether the same mechanism underlies abortion of the ventral stamens, an important morphological trait related to evolution and diversification of zygomorphy in Lamiales sensu lato, a major clade of predominantly zygomorphically flowered angiosperms. Opithandra (Gesneriaceae) is of particular interests in addressing this question as it is in the base of Lamiales s.l., an early representative of this type zygomorphy.     

Trans-activation of an artificial dTam3 transposable element in transgenic tobacco plants

In Antirrhinum majus only autonomous Tam3 transposons have been characterized. We investigated whether an artificial dTam3 element, with a deletion in the presumptive transposase coding region, can be trans-activated in tobacco by an activator Tam3 element, which was immobilized by the deletion of one inverted repeat. A phenotypic assay based on restored hygromycin resistance demonstrates that a dTam3 element harbouring a bacterial plasmid can be trans-activated with a low frequency. Molecular analysis confirms that the dTam3 element has been excised from the HPTII marker gene. Reintegration of the dTam3 element into the tobacco genome is detected only in one out of six hygromycin-resistant plants analysed. PCR analysis of empty donor sites shows that excision of the dTam3 element in tobacco results in rearrangements (deletions and additions), that have been shown to be characteristic of Tam3 excision in the original host Antirrhinum majus. This trans-activation assay allowed us to establish that, in contrast to what has been detected in Antirrhinum majus, a periodical temperature shift down to 15°C does not enhance dTam3 transposition in regenerating tobacco calli.     

A putative autonomous 20.5 kb-CACTA transposon insertion in an <Emphasis Type="Italic">F3'H</Emphasis> allele identifies a new CACTA transposon subfamily in <Emphasis Type="Italic">Glycine max</Emphasis>

Background  

The molecular organization of very few genetically defined CACTA transposon systems have been characterized thoroughly as those of Spm/En in maize, Tam1 of Antirrhinum majus Candystripe1 (Cs1) from Sorghum bicolor and CAC1 from Arabidopsis thaliana, for example. To date, only defective deletion derivatives of CACTA elements have been described for soybean, an economically important plant species whose genome sequence will be completed in 2008.     

The backstage of the ABC model: The Antirrhinum majus contribution

At the beginning of the 1990s, a simple genetic model that explained flower development was presented based on Arabidopsis thaliana and Antirrhinum majus floral homeotic mutants. According to this model, which is a milestone in plant development studies, flower development can be explained by three classes of genes (A, B and C), each one controlling the identity of organs in two adjacent whorls. Intriguingly, more than 20 years later, there are still some unanswered questions, in particular regarding the universality of the class A-function genes. Class A genes are well characterised in A. thaliana, but so far no A mutants have been described in other plant species nor in Antirrhinum majus. Here, we retrace the story that led to the proposal of the ABC model focusing on the contribution of A. majus to this model. Although fewer groups are still using A. majus as a model system, this plant was a master contributor to our comprehension of the molecular networks controlling flower development.     

Evidence for correlation between mutability of an unstable anthocyanin-governing locus and abundance of anthocyanin

Summary The pal-rec gene of Antirrhinum majus suppresses anthocyanin except in those cell lines where pal-rec has mutated to Pal, so that anthocyanin-coloured flecks appear on whitish petals. Antirrhinum majus families of very high and very low anthocyanin content (Dark and Pale) were obtained and crossed with two pal-rec pal-rec lines, one with consistently high and the other consistently low mutability. Mutable offspring from Dark parents tended to show higher mutability than those from Pale parents in crosses with either mutable line, providing evidence for an association between intense pigmentation and high mutability. Such an association is discussed in the context of relationship between precursor availability for conversion by a gene product and initiation of activity of that gene.     

SQUAMOSA‐PROMOTER BINDING PROTEIN 1 initiates flowering in Antirrhinum majus through the activation of meristem identity genes

The degree to which developmental genetic pathways are conserved across distantly related organisms is a major question in biology. In Arabidopsis thaliana (L.) Heynh., inflorescence development is initiated in response to a combination of external and internal floral inductive signals that are perceived across the whole plant, but are integrated within the shoot apical meristem. Recently, it was demonstrated that SQUAMOSA‐PROMOTER BINDING PROTEIN (SBP)‐box proteins regulate A. thaliana flowering time by mediating signals from the autonomous and photoperiod pathways, and by directly activating key genes involved in inflorescence and floral meristem identity, including FRUITFULL (FUL), APETALA1 (AP1) and LEAFY (LFY). In the distantly related core eudicot species Antirrhinum majus L., paralogous SBP‐box proteins SBP1 and SBP2 have likewise been implicated in regulating the AP1 ortholog SQUAMOSA (SQUA). To test the hypothesis that SBP‐box genes are also involved in the floral induction of A. majus, we used a reverse genetic approach to silence SBP1. SBP1‐silenced lines are late to nonflowering, and show reduced apical dominance. Furthermore, expression and sequence analyses suggest that the SBP1‐mediated transition to flowering occurs through the positive regulation of FUL/LFY homologs. Together, these data outline the utility of virus‐induced gene silencing in A. majus, and provide new insight into the conservation of flowering time genetic pathways across core eudicots.     

Rapid linkage disequilibrium decay in the <Emphasis Type="Italic">Lr10</Emphasis> gene in wild emmer wheat (<Emphasis Type="Italic">Triticum dicoccoides</Emphasis>) populations

Introduction  

Recombination is a key evolutionary factor enhancing diversity. However, the effect of recombination on diversity in inbreeding species is expected to be low. To estimate this effect, recombination and diversity patterns of Lr10 gene were studied in natural populations of the inbreeder species, wild emmer wheat (Triticum dicoccoides). Wild emmer wheat is the progenitor of most cultivated wheats and it harbors rich genetic resources for disease resistance. Lr10 is a leaf rust resistance gene encoding three domains: a coiled-coil, nucleotide-binding site, and leucine-rich repeat (CC-NBS-LRR).     

Dominance relationships between alleles at the pallida locus in Antirrhinum majus

Flowers of the snapdragon, Antirrhinum majus, carrying various alleles at the pallida locus were examined for anthocyanin intensity. All the alleles were found to exert an effect in all combinations, with varying degrees of dominance.     

Functional conservation and maintenance of expression pattern of <Emphasis Type="Italic">FIDDLEHEAD</Emphasis>-like genes in Arabidopsis and Antirrhinum

In Arabidopsis, loss of function of the epidermis-specific FDH gene coding for a putative -ketoacyl-CoA synthase results in ectopic organ fusions in mutants. Corresponding mutants are not available for Antirrhinum majus, however, organ fusions can be induced in both species by chloroacetamide inhibitors of -ketoacyl-CoA synthases using a chemical genetics approach. We isolated the ortholog of FDH from Antirrhinum majus, the ANTIRRHINUM FIDDLEHEAD (AFI) gene, and showed that AFI complements fdh when expressed in the epidermis under control of the FDH promoter. Like FDH, the AFI gene exhibits protodermis- and epidermis-specific expression, and its promoter directs the expression of reporter genes to the epidermis in transgenic Antirrhinum and Arabidopsis. We demonstrate down-regulation of the FDH promoter in the epidermis of the ovary septum, thereby supporting the assumption that FDH-like genes may directly facilitate the cell–cell interactions that need to occur during carpel fusion and pollen tube growth. Up-regulation of FDH in the stomium, on the other hand, provides evidence for its possible involvement in cell separation during anther dehiscence. Down-regulation of the FDH and AFI promoters in the septum is observed in transgenic Arabidopsis but not in Antirrhinum plants. This probably reflects differences in the ontogeny of the ovary septum between the two species. We also show that epidermis-specific FDH-like genes may not be able to efficiently elongate fatty acid chains when misexpressed in seeds.     

Natural transformation of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> as a tool - Optimizing the procedure

Background  

Vibrio cholerae gains natural competence upon growth on chitin. This allows the organism to take up free DNA from the environment and to incorporate it into its genome by homologous recombination.     

An improved protocol for<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Antirrhinum majus</Emphasis> L.

Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.Communicated by G. Jürgens     

Comparative analysis of right element mutant <Emphasis Type="Italic">lox</Emphasis> sites on recombination efficiency in embryonic stem cells

Background  

Cre-mediated site-specific integrative recombination in mouse embryonic stem (ES) cells is a useful tool for genome engineering, allowing precise and repeated site-specific integration. To promote the integrative reaction, a left element/right element (LE/RE) mutant strategy using a pair of lox sites with mutations in the LE or RE of the lox sequence has previously been developed. Recombination between LE and RE mutant lox produces a wild-type lox P site as well as an LE+RE double mutant lox site, which has mutations in both sides and less affinity to Cre, resulting in stable integration. We previously demonstrated successful integrative recombination using lox 71 (an LE mutant) and lox 66 (an RE mutant) in ES cells. Recently, other LE/RE mutant lox sites showing higher recombination efficiency in Escherichia coli have been reported. However, their recombination efficiency in mammalian cells remains to be analyzed.     

PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the <Emphasis Type="Italic">Arabidopsis</Emphasis> genome

Background  

The large serine recombinase phiC31 from broad host range Streptomyces temperate phage, catalyzes the site-specific recombination of two recognition sites that differ in sequence, typically known as attachment sites attB and attP. Previously, we characterized the phiC31 catalytic activity and modes of action in the fission yeast Schizosaccharomyces pombe.     

<Emphasis Type="Italic">TaMSH7</Emphasis>: A cereal mismatch repair gene that affects fertility in transgenic barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Background  

Chromosome pairing, recombination and DNA repair are essential processes during meiosis in sexually reproducing organisms. Investigating the bread wheat (Triticum aestivum L.) Ph2 (Pairing homoeologous) locus has identified numerous candidate genes that may have a role in controlling such processes, including TaMSH7, a plant specific member of the DNA mismatch repair family.     

Die Mutabilit?t der Plastiden vonAntirrhinum Majus L.Sippe 50

Summary Experiments to raise the frequency of plastid-mutations inAntirrhinum majus have been ineffective when proplastids were X-rayed in egg-cells or were treated in meristems of growing plants by infiltration of three different chemicals.

---

---

Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Immobilized copies with a nearly intact structure of the transposon Tam3 in Antirrhinum majus: implications for the cis-element related to the transposition

 In this study we have focused on two copies of the transposon Tam3 isolated from an Antirrhinum majus plant which has flower variegation due to the excision of Tam3 from the nivea locus. These two copies possess a high homology, over 95%, to an active Tam3 element found in the nivea ^{recurrence:Tam3} allele. Although somatic excision of the Tam3 copy from the nivea locus can be detected at 15°C by Southern blotting, neither of the two copies showed any sign of the excision. Both of the immobilized copies were also found in five varieties from different A. majus sources, all of which contain common fragments. The results suggest that the two copies have been fixed in the genomes of many A. majus varieties. Structural differences between these immobilized copies and the known active copy were mainly observed in the subterminal regions, including the terminal inverted repeats. The immobility of the two Tam3 copies might be due to mutations within the end regions of essential cis-elements in Tam3 transposition, as reported for Ac and En/Spm. Received: 30 June 1997 / Accepted: 5 August 1997     

The <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> MPN229 gene encodes a protein that selectively binds single-stranded DNA and stimulates Recombinase A-mediated DNA strand exchange

Background  

Mycoplasma pneumoniae has previously been characterized as a micro-organism that is genetically highly stable. In spite of this genetic stability, homologous DNA recombination has been hypothesized to lie at the basis of antigenic variation of the major surface protein, P1, of M. pneumoniae. In order to identify the proteins that may be involved in homologous DNA recombination in M. pneumoniae, we set out to characterize the MPN229 open reading frame (ORF), which bears sequence similarity to the gene encoding the single-stranded DNA-binding (SSB) protein of other micro-organisms.     

Genetic fine-mapping of <Emphasis Type="Italic">DIPLOSPOROUS</Emphasis> in <Emphasis Type="Italic">Taraxacum</Emphasis> (dandelion; Asteraceae) indicates a duplicated <Emphasis Type="Italic">DIP</Emphasis>-gene

Background  

DIPLOSPOROUS (DIP) is the locus for diplospory in Taraxacum, associated to unreduced female gamete formation in apomicts. Apomicts reproduce clonally through seeds, including apomeiosis, parthenogenesis, and autonomous or pseudogamous endosperm formation. In Taraxacum, diplospory results in first division restitution (FDR) nuclei, and inherits as a dominant, monogenic trait, independent from the other apomixis elements. A preliminary genetic linkage map indicated that the DIP-locus lacks suppression of recombination, which is unique among all other map-based cloning efforts of apomeiosis to date. FDR as well as apomixis as a whole are of interest in plant breeding, allowing for polyploidization and fixation of hybrid vigor, respectively. No dominant FDR or apomixis genes have yet been isolated. Here, we zoom-in to the DIP-locus by largely extending our initial mapping population, and by analyzing (local) suppression of recombination and allele sequence divergence (ASD).     

Scarless and site-directed mutagenesis in <Emphasis Type="Italic">Salmonella enteritidis</Emphasis>chromosome

Background  

A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome.