Genetic Control of Flower Development by Homeotic Genes in Antirrhinum majus.

Homeotic mutants have been useful for the study of animal development. Such mutants are also known in plants. The isolation and molecular analysis of several homeotic genes in Antirrhinum majus provide insights into the underlying molecular regulatory mechanisms of flower development. A model is presented of how the characteristic sequential pattern of developing organs, comprising the flower, is established in the process of morphogenesis.

MADS-box genes in plant ontogeny and phylogeny: Haeckel's 'biogenetic law' revisited.

Data currently accumulating with impressive speed indicate that the molecular evolution of MADS-box genes was a decisive aspect of the morphological evolution of plants. Studies on MADS-box genes in diverse plant species thus help us to understand the emergence of morphological novelties, such as the flower, in evolution. This furthers our understanding of the relationship between ontogeny and phylogeny, which has been a controversial issue since Ernst Haeckel published his 'biogenetic law' more than a century ago.

Isolation of Y chromosome-specific sequences from Silene latifolia and mapping of male sex-determining genes using representational difference analysis.

The genomic subtraction method representational difference analysis (RDA) was used to identify male-specific restriction fragments in the dioecious plant Silene latifolia. Male-specific restriction fragments are linked to the male sex chromosome (the Y chromosome). Four RDA-derived male-specific restriction fragments were used to identify polymorphisms in a collection of X-ray-generated mutant plants with either hermaphroditic or asexual flowers. Some of the mutants have cytologically detectable deletions in the Y chromosome that were correlated with loss of male-specific restriction fragments. One RDA-derived probe detected a restriction fragment present in all mutants, indicating that each has retained Y chromosomal DNA. The other three probes detected genomic fragments that were linked in a region deleted in some hermaphroditic and some asexual mutants. Based on the mutant phenotypes and the correlation of cytologically visible deletions with loss of male-specific restriction fragments, these markers were assigned to positions on the Y chromosome close to the carpel suppression locus. This RDA mapping also revealed a Y-linked locus, not previously described, which is responsible for early stamen development.

Isolation and characterization of new MIKC*-Type MADS-box genes from the moss Physcomitrella patens.

MADS-box genes encode for a large family of transcription-regulating proteins, which were isolated from all groups of eukaryotic organisms. The plant-specific MIKC-type MADS-box genes have been intensively analyzed for their roles in controlling developmental processes. Well-known are the MADS-box genes acting as homeotic selector genes in the differentiation of whorls of floral organs in seed plants. The MADS-box gene family has also been studied in non-flowering plants, such as lycophytes, pteridophytes, and bryophytes. The analysis of MADS-box genes in the moss Physcomitrella patens led to the identification of a new class of MIKC-type genes, designated as MIKC*-type genes. The MIKC*-type genes possess a number of structural features which clearly distinguish them from the already known MIKC-type genes. Recently, orthologues of the Physcomitrella MIKC*-type genes were found in Arabidopsis thaliana, demonstrating the conservation of these genes in tracheophytes. Here, we report the isolation of two new MIKC*-type MADS-box genes from Physcomitrella. Structural features and expression patterns of these genes were analyzed. The contribution of our findings to a better understanding of the evolution of MIKC*-type genes in land plants is discussed.

Molecular biology of flower development in Antirrhinum majus (snapdragon).

In recent years, isolation of several genes affecting flower development in Antirrhinum majus made this species a major model system to study this important developmental process. Genes like SQUAMOSA and FLORICAULA are involved in determination of the floral meristem. Their mutation results in the development of bract-forming shoots at positions where normally flowers would develop. The phenotypes obtained upon mutation of the genes found to affect floral organogenesis fall into three major categories. In each category, always the floral organs in two adjacent whorls become homeotically transformed. Based on this observation a simple genetic model has been proposed to explain the establishment of floral organ identity in the four concentric whorls of the flower. The model hypothesizes the independent induction of two developmental pathways specifying floral organ identity after the formation of sepals as the basic type of organ following induction of a floral meristem. One of these pathways is under the control of the PLENA gene, the other is controlled by the DEFICIENS and GLOBOSA genes. These genes, as well as SQUAMOSA, encode transcription factors sharing a conserved DNA binding domain: the MADS-box. In vitro DNA-binding studies complemented with molecular genetic analysis of the respective mutants show that the DEF and GLO proteins may act together in the form of a heterodimer in the regulation of their target genes as well as in autoregulation. The possible interactions between other MADS-box proteins and their role in flower development is under current investigation.

Plant transposons: contributors to evolution?

A spectrum of different hypotheses has been presented by various authors, from plant transposable elements as major agents in evolution to the very opposite, transposons as mainly selfish DNA constituting a genetic burden for the organisms. The following review will focus on: (1) a short survey of the two main different assessments of transposable elements (TEs) concerning the origin of species (selfish vs useful DNA); (2) the significance of the hierarchy of gene functions and redundancies for TE activities (selfish in non-redundant parts of the genome, but as a source of variability in the rest); (3) the relevance of the results of TE research in Zea mays and Antirrhinum majus for species formation in the wild (contrast between artificial and natural selection); (4) three areas of research where a synthesis between the two different evaluations of TEs seems possible: regressive evolution, the origin of ecotypes and the origin of cultivated plants; and (5) some possible prospects regarding TE-induced species formation in the angiosperms in general, i.e., the basic difference between systematic and genetic species concepts and the conceivable origin of a large part of angiosperm morphospecies owing to loss of function and further mutations by TE activities.

Structural characterization, chromosomal localization and phylogenetic evaluation of two pairs of AGAMOUS-like MADS-box genes from maize.

In order to gain a better understanding of the development and evolution of cereal flowers, we have cloned and sequenced two MADS-box genes from maize and the partial cDNA of a third one. One of the genomic clones was identified as ZAG2 (Zea AGAMOUS 2), while the other has a very similar structure and the potential to encode a protein which shares 94% sequence identity with the putative ZAG2 gene product. The cDNA reveals considerable similarity to ZAG1. Phylogenetic evaluation of the sequence information, as well as chromosomal localization, both suggest that we have identified two pairs of AGAMOUS-like MADS-box genes which were created during duplication of chromosomal segments or complete chromosomes.

Characterization of three GLOBOSA-like MADS-box genes from maize: evidence for ancient paralogy in one class of floral homeotic B-function genes of grasses.

Floral homeotic B-function genes are involved in specifying the identity of petals and stamens during flower development in higher eudicotyledonous plants. Monocotyledonous plants belonging to the grass family (Poaceae) have very similar B-function genes, except that these genes specify lodicules rather than petals. All B-function genes known so far are members of the MADS-box gene family encoding transcription factors. In some eudicot model systems such as Arabidopsis and Antirrhinum, the B-function is provided by heterodimeric protein complexes encoded by one DEF- and one GLO-like gene. In several different lineages of flowering plant species, however, more than one DEF- or GLO-like gene is found. A known example is the monocot model system rice, which contains two GLO-like genes, termed OSMADS2 and OSMADS4. Duplications of floral homeotic genes may have played a critical role in the diversification of floral homeotic functions and thus the evolution of flowers. In order to date the gene duplication event that gave rise to these two genes, we cloned cDNAs of three different GLO-like genes from maize, a distant relative of rice within the Poaceae family. Phylogeny reconstructions and chromosomal mapping indicate that one of these genes, named ZMM16, is orthologous to OSMADS2, and that the other two, ZMM18 and ZMM29, are probably orthologous to OSMADS4. The gene duplication which gave rise to OSMADS2- and OSMADS4-like genes occurred probably after the split of the lineages that resulted in extant Liliaceae and Poaceae, but before the separation of the lineages that gave rise to extant maize and rice about 50 MYA. Northern and in situ hybridization studies demonstrated that the maize genes are expressed in lodicules, stamens and carpels throughout spikelet development in male and female inflorescences. The GLO-like genes from rice have very similar patterns of mRNA accumulation. In addition, ZMM16 shows also weak expression in vegetative organs. Conservation of the expression in lodicules and stamens is in perfect agreement with a floral homeotic B-function of the GLO-like genes in grasses. The conserved expression in carpels is discussed. Moreover, circumstantial evidence for a functional diversification of GLO-like genes in grasses is provided.

Molecular characterisation of the Arabidopsis SBP-box genes.

The Arabidopsis thaliana SPL gene family represents a group of structurally diverse genes encoding putative transcription factors found apparently only in plants. The distinguishing characteristic of the SPL gene family is the SBP-box encoding a conserved protein domain of 76 amino acids in length, the SBP-domain, which is responsible for the interaction with DNA. We present here characterisation of 12 members of the SPL gene family. These genes show highly diverse genomic organisations and are found scattered over the Arabidopsis genome. Some SPL genes are constitutively expressed, while transcriptional activity of others is under developmental control. Based on phylogenetic reconstruction, gene structure and expression patterns, they can be divided into subfamilies. In addition to the Arabidopsis SPL genes, we isolated and determined the sequences of three SBP-box genes from Antirrhinum majus and seven from Zea mays.

Molecular analysis of tap2, an anther-specific gene from Antirrhinum majus.

Deficiens is a floral homeotic gene of Antirrhinum majus, mutation of which results in transformation of petals to sepals and stamens to carpels. In a search for putative target genes, controlled by this regulatory locus, cDNA clones representing genes, that are expressed in wild type but not in the deficiens mutant flowers, were isolated by differential screening. The molecular structure and the expression pattern of one of these genes, tap2, is described. Tap2 is transiently and tissue-specifically expressed in the tapetum of the anthers. It encodes a 131 amino-acids-long protein with a hydrophobic N-terminus, displaying all characteristic features of a signal peptide. This indicates that the TAP2 protein may be secreted.

MADS-box genes are involved in floral development and evolution.

MADS-box genes encode transcription factors in all eukaryotic organisms thus far studied. Plant MADS-box proteins contain a DNA-binding (M), an intervening (I), a Keratin-like (K) and a C-terminal C-domain, thus plant MADS-box proteins are of the MIKC type. In higher plants most of the well-characterized genes are involved in floral development. They control the transition from vegetative to generative growth and determine inflorescence meristem identity. They specify floral organ identity as outlined in the ABC model of floral development. Moreover, in Antirrhinum majus the MADS-box gene products DEF/GLO and PLE control cell proliferation in the developing flower bud. In this species the DEF/GLO and the SQUA proteins form a ternary complex which determines the overall "Bauplan" of the flower. Phylogenetic reconstructions of MADS-box sequences obtained from ferns, gymnosperms and higher eudicots reveal that, although ferns possess already MIKC type genes, these are not orthologous to the well characterized MADS-box genes from gymnosperms or angiosperms. Putative orthologs of floral homeotic B- and C-function genes have been identified in different gymnosperms suggesting that these genes evolved some 300-400 million years ago. Both gymnosperms and angiosperms also contain a hitherto unknown sister clade of the B-genes, which we termed Bsister. A novel hypothesis will be described suggesting that B and Bsister might be involved in sex determination of male and female reproductive organs, respectively.

Plant transposable elements: their role in evolution.

Transposable elements (TE) are natural constituents of plant genomes. However, their presence only becomes apparent if they become dislodged from their resident positions in the genome and transpose into another gene, thereby inducing a mutation. Such TE-induced mutations are somatically unstable because they revert to wild type and hence reconstitute the expression of the mutated gene. The frequent somatic excision of the TE results in a variegated phenotype. Since this instability is inherited in a Mendelian manner the variegated phenotype is nuclear determined. By this criterion TE have been shown to occur in more than 30 species belonging to different families and genera. Many questions arise when dealing with TE: their structure and functions, and the biological significance of the activity of elements in the differentiation of a normal plant or in the evolution of plant genes.

[Antibiotic resistance factors and "jumping" genes (author's transl)]. / Antibiotika-Resistenz-Faktoren und "springende" Gene.

Antibiotics are natural products inhibiting the growth of bacteria. In research they play an important role in studying the details of bacterial macromolecular synthesis. However, their major importance lies in application, i.e., in treatment of infectious bacterial diseases and as additives to livestock feed. The application of antibiotics, however, presents serious problems: the development and spread of antibiotic-resistant bacterial strains which impede therapy.

Technical advance: display and isolation of transposon-flanking sequences starting from genomic DNA or RNA.

Insertion mutagenesis using transposons has become a powerful tool for the isolation of genes involved in any given biochemical or developmental pathway. We describe here ligation-mediated PCR techniques for the isolation of sequences flanking the transposable elements En/Spm, Mu1 and Cin4 in Zea mays and Arabidopsis thaliana. Two versions of this transposon insertion display (TID) method use biotinylated linkers or biotinylated primers to rapidly isolate transposon-flanking sequences starting from digested genomic DNA. TID protocols have been employed to clone several genes from En/Spm insertion mutants of Arabidopsis. A novel procedure, expression TID (ETID), is also introduced, which provides a direct approach for the isolation of transposon insertions that tag transcribed portions of genes. ETID uses RNA as a starting material and exploits 5' RACE PCR to identify transposon copies that form parts of gene transcripts. The detection of several En/Spm insertion mutations in Arabidopsis illustrates the power of this method. ETID offers important advantages for the isolation of mutant alleles of novel genes that are expressed in specific tissues in plants and animals.

Deletions and an inversion induced by a resident IS1 of the lactose transposon Tn951.

DNA-DNA filter binding tests, "Southern" blotting experiments and DNA heteroduplex analysis clearly show that Tn951 contains an IS1 element. This IS1-951 sequence is peculiar in that it does not contain the PstI cleavage site which is usually observed on E. coli derived IS1 elements. Nonetheless, IS1-951 induces deletions. This process is temperature dependent. One instance of an IS1-951 induced inversion was observed, the structure of which is compatible with the current models of transposition of IS elements.

Transposition in plants: a molecular model.

A molecular model for transposition of plant transposable elements is described. This process may occur via excision and re-integration of the element. Excision generates DNA sequence diversity which suggests the participation of DNA repair enzymes in the healing of the donor molecule.

HOMOLOGY-DEPENDENT GENE SILENCING IN PLANTS.

Homology-dependent gene silencing phenomena in plants have received considerable attention, especially when it was discovered that the presence of homologous sequences not only affected the stability of transgene expression, but that the activity of endogenous genes could be altered after insertion of homologous transgenes into the genome. Homology-mediated inactivation most likely comprises at least two different molecular mechanisms that induce gene silencing at the transcriptional or posttranscriptional level, respectively. In this review we discuss different mechanistic models for plant-specific inactivation mechanisms and their relationship with repeat-specific silencing phenomena in other species.

IS2-61 and IS2-611 arise by illegitimate recombination from IS2-6.

A more stable derivative of IS2-6 has been isolated, which had lost 54 bp of the 108 bp long insert characteristic of IS2-6. This new allele of IS2, IS2-61, segregates the remaining 54 bp to yield allele IS2-611. DNA sequence analysis shows that the segregation products of IS2-6 arise by recA-independent, illegitimate recombination at 9 bp long direct sequence repetitions.