The Arabidopsis HUELLENLOS gene, which is essential for normal ovule development, encodes a mitochondrial ribosomal protein.

The HUELLENLOS (HLL) gene participates in patterning and growth of the Arabidopsis ovule. We have isolated the HLL gene and shown that it encodes a protein homologous to the L14 proteins of eubacterial ribosomes. The Arabidopsis genome also includes a highly similar gene, HUELLENLOS PARALOG (HLP), and genes for both cytosolic (L23) and chloroplast ribosome L14 proteins. Phylogenetic analysis shows that HLL and HLP differ significantly from these other two classes of such proteins. HLL and HLP fusions to green fluorescent protein were localized to mitochondria. Ectopic expression of HLP complemented the hll mutant, indicating that HLP and HLL share redundant functions. We conclude that HLL and HLP encode L14 subunits of mitochondrial ribosomes. HLL mRNA was at significantly higher levels than HLP mRNA in pistils, with the opposite pattern in leaves. This differential expression can explain the confinement of effects of hll mutations to gynoecia and ovules. Our elucidation of the nature of HLL shows that metabolic defects can have specific effects on developmental patterning.

NOZZLE regulates proximal-distal pattern formation, cell proliferation and early sporogenesis during ovule development in Arabidopsis thaliana.

With the characterisation of the NOZZLE gene we aim at a better understanding of the molecular and genetic mechanism underlying pattern formation and growth control during floral organogenesis. Our data indicate that NOZZLE links these processes during ovule development. In the ovule primordium NOZZLE plays a central role in the formation of the nucellus through antagonizing the activities of BELL, AINTEGUMENTA and INNER NO OUTER, all encoding putative transcription factors, in the prospective nucellar region. We provide evidence that NOZZLE and BELL are chalaza identity genes that share overlapping functions in establishing the prospective chalaza of the ovule. In addition, NOZZLE plays a role in controlling the cell number and by this means the length of the funiculus, again through antagonizing AINTEGUMENTA and INNER NO OUTER function. NOZZLE is also required for the development of the integuments. We show that during the initial phase of this process NOZZLE is transcriptionally regulated by AINTEGUMENTA and INNER NO OUTER. NOZZLE thus represents a downstream target of these two genes in the integument development pathway.

INNER NO OUTER regulates abaxial- adaxial patterning in Arabidopsis ovules.

The Arabidopsis INNER NO OUTER (INO) gene is essential for formation and asymmetric growth of the ovule outer integument. INO encodes a member of the newly described YABBY family of putative transcription factors that contain apparent Cys(2)-Cys(2) zinc-finger domains and regions of similarity to the high mobility group (HMG) transcription factors. In wild-type plants, INO is expressed specifically on one side of the central region of each ovule primordium in the cells that give rise to the outer integument. Alterations in the INO expression pattern in mutant backgrounds implicate INO as a positive regulator of its own expression, and ANT, HLL, BEL1, and SUP as direct or indirect negative regulators that help to establish the spatial pattern of INO expression. We hypothesize that INO is necessary for polarity determination in the central part of the ovule. Maintenance of polarity in other parts of ino ovules indicates the existence of additional regulators and provides further evidence that the ovule is a compound structure.

Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana.

Sexual reproduction is a salient aspect of plants, and elaborate structures, such as the flowers of angiosperms, have evolved that aid in this process. Within the flower the corresponding sex organs, the anther and the ovule, form the male and female sporangia, the pollen sac and the nucellus, respectively. However, despite their central role for sexual reproduction little is known about the mechanisms that control the establishment of these important structures. Here we present the identification and molecular characterization of the NOZZLE (NZZ) gene in the flowering plant Arabidopsis thaliana. In several nzz mutants the nucellus and the pollen sac fail to form. It indicates that NZZ plays an early and central role in the development of both types of sporangia and that the mechanisms controlling these processes share a crucial factor. In addition, NZZ may have an early function during male and female sporogenesis as well. The evolutionary aspects of these findings are discussed. NZZ encodes a putative protein of unknown function. However, based on sequence analysis we speculate that NZZ is a nuclear protein and possibly a transcription factor.

The molecular and genetic control of ovule development.

A genetic approach has resulted in an extensive framework for the methodical analysis of ovule development. The most recent progress was accomplished in the areas of primordium formation and integument morphogenesis. Furthermore, systematic screens have identified a number of gametophytic mutations disrupting several distinct steps of embryo sac ontogenesis.

Pattern formation and growth during floral organogenesis: HUELLENLOS and AINTEGUMENTA are required for the formation of the proximal region of the ovule primordium in Arabidopsis thaliana.

Our understanding of the molecular mechanisms that regulate and integrate the temporal and spatial control of cell proliferation during organ ontogenesis, particularly of floral organs, continues to be primitive. The ovule, the progenitor of the seed, of Arabidopsis thaliana has been used to develop an effective model system for the analysis of plant organogenesis. A typical feature of a generalized ovule is the linear arrangement of at least three distinct elements, the funiculus, chalaza and nucellus, along a proximal-distal axis. This pattern is supposed to be established during the early proliferative phase of ovule development. We provide genetic evidence that the young ovule primordium indeed is a composite structure. Two genes, HUELLENLOS and AINTEGUMENTA have overlapping functions in the ovule and differentially control the formation of the central and proximal elements of the primordium. The results indicate that proximal-distal pattern formation in the Arabidopsis ovule takes place in a sequential fashion, starting from the distal end. Furthermore, we show that HUELLENLOS also regulates the initiation and/or maintenance of integument and embryo sac ontogenesis and interestingly prevents inappropriate cell death in the young ovule.

The molecular and genetic basis of ovule and megagametophyte development.

The formation of ovules is a key step in the plant life cycle which alternates between a diploid and haploid generation, the sporophyte and the gametophyte. The transitions between the two generations in the female occur in the ovule, the site of meiosis, female gametogenesis and double fertilization. The intimate association of sporophytic and gametophytic tissues in the ovule allows an investigation of their cellular interactions during ovule and seed development. In Arabidopsis, several sporophytically acting loci which primarily affect the morphogenesis of the integuments have been identified. Early acting genes may respond to positional information leading to a region-specific initiation of morphogenesis, whereas later acting genes affect cellular aspects of integument growth. Much less is known about the mechanisms controlling the development of the megagametophyte. Comparative morphology and genetic analyses suggests that regulatory mechanisms ensure a tight coordination of independently controlled cellular processes with cell specification and differentiation.

The STUD gene is required for male-specific cytokinesis after telophase II of meiosis in Arabidopsis thaliana.

During male meiosis in wild-type Arabidopsis the pollen mother cell (PMC) undergoes two meiotic nuclear divisions in the absence of cell division. Only after telophase II is a wall formed which partitions the PMC into four microspores. Each microspore undergoes two subsequent mitotic divisions to produce one vegetative cell and two sperm cells in the mature pollen grain. In this paper we describe the isolation and the phenotypic characterization of mutations in the STUD (STD) gene, which is specifically required for male-specific cytokinesis after telophase II of meiosis. Although the male meiotic nuclear divisions are normal in std mutant plants, no walls are formed resulting in a tetranucleate microspore. Despite the absence of cell division in the PMC, postmeiotic development in the coenocytic microspore proceeds relatively normally, resulting in the formation of large pollen grains which contain four vegetative nuclei and up to eight sperm cells. Interestingly, these enlarged pollen grains which contain multiple vegetative nuclei and extra sperm cells behave as single male gametophytes, producing only single pollen tubes and resulting in partial male fertility in std mutant plants. Characterization of the process of pollen development and pollen function in std mutants thus reveals two different types of developmental regulation. Each of the four nuclei found in a std microspore following meiosis is capable of independently undergoing the complete mitotic cell division (including cytokinesis) which the single nucleus of a wild-type microspore would normally undertake. The ability of the four meiotic products to independently continue through mitosis does not depend on their division into separate cells, but is controlled by some subcellular component found within the coenocytic microspore. By contrast, the mature std pollen grain functions as a unit and produces only a single pollen tube despite the presence of multiple nuclei within the vegetative cell, suggesting that this process is controlled at the cellular level independently of the extra subcellular components.

Dissection of sexual organ ontogenesis: a genetic analysis of ovule development in Arabidopsis thaliana.

Understanding organogenesis remains a major challenge in biology. Specification, initiation, pattern formation and cellular morphogenesis, have to be integrated to generate the final three-dimensional architecture of a multicellular organ. To tackle this problem we have chosen the ovules of the flowering plant Arabidopsis thaliana as a model system. In a first step towards a functional analysis of ovule development, we performed a large-scale genetic screen and isolated a number of sterile mutants with aberrant ovule development, We provide indirect genetic evidence for the existence of proximal-distal pattern formation in the Arabidopsis ovule primordium. The analysis of the mutants has identified genes that act at an intermediate regulatory level and control initiation of morphogenesis in response to proximal-distal patterning. A second group of genes functions at a subordinate control level and regulates general cellular processes of morphogenesis. A large group of male and female sterile mutants shows defects restricted to early or late gametogenesis. In addition, we propose that the mature ovule obtains its overall curved shape by at least three different processes that act in only one domain of the ovule.

Dachsous encodes a member of the cadherin superfamily that controls imaginal disc morphogenesis in Drosophila.

Mutations in the dachsous gene of Drosophila lead to striking defects in the morphogenesis of the thorax, legs, and wings. The dachsous gene has been cloned and shown to encode a huge transmembrane protein that is a member of the cadherin superfamily, similar to the fat gene reported previously. Both the Dachsous and Fat proteins contain large tandem arrays of cadherin domains--27 and 34, respectively--as compared with 4 cadherin domains in classic vertebrate cadherins. In addition, Dachsous and Fat each has a cytoplasmic domain with sequence similarity to the cytoplasmic beta-catenin-binding domain of classic vertebrate cadherins. The dachsous gene is expressed in the ectoderm of embryos, whereas its expression in larvae is restricted to imaginal discs and specific regions of the brain. The phenotypes of, and genetic interactions between dachsous and fat are consistent with a model in which cell proliferation and morphogenesis of imaginal structures depends on the coupled equilibria between homo- and heterophilic interactions of the Dachsous and Fat cadherin proteins.

Genetic Evidence for a Long-Range Activity That Directs Pollen Tube Guidance in Arabidopsis.

The fertilization process of plants is governed by different kinds of cell-cell interactions. In higher plants, these interactions are required both for recognition of the pollen grain by the female reproductive system and to direct the growth of the pollen tube inside the ovary. Despite many years of study, the signaling mechanisms that guide the pollen tube toward its target, the ovule, are largely unknown. Two distinct types of principles, mechanical and chemotropic, have been suggested to account for the directed growth of the pollen tube. The first of these two types of models implies that the guidance of the pollen tube depends on the architecture and chemical properties of the female reproductive tissues, whereas the latter suggests that the ovule provides a signal for the target-directed growth of the pollen tube. To examine such a role for the ovules, we analyzed the growth path of pollen tubes in mutants defective in ovule development in Arabidopsis. The results presented here provide unique in vivo evidence for an ovule-derived, long-range activity controlling pollen tube guidance. A morphological comparison of the ovule mutants used in this study indicates that within the ovule, the haploid embryo sac plays an important role in this long-range signaling process.

Molecular genetics of cell interactions in Arabidopsis.

Many events in plant development are regulated by the interactions of neighboring cells. We are interested in determining what sorts of molecules act as signals and/or receptors in these interactions and how these mechanisms relate to those used in animals and fungi. We are presently working on two different types of systems to try to address this question. In one case we are starting at the molecular level and characterizing a family of receptor protein kinase genes which seem natural candidates for mediating cellular interactions. By analyzing the expression patterns of these genes as well as the phenotypes of transgenic plants bearing altered genes we hope to determine what roles these proteins play in plant development. In the second case we are starting from the organismic level and using genetics to identify genes essential to a whole range of cellular interactions which are required for proper male gametophyte development during reproduction. These interactions involve both recognition of the pollen grain to verify that it is from the correct species and also a transfer of positional information from the female to the male which first allows the pollen tube to determine the polarity of the stigmatic cell on which it has germinated and later provides 'guidance' for the elongating tube to find the ovule.

Molecular genetics of aristaless, a prd-type homeo box gene involved in the morphogenesis of proximal and distal pattern elements in a subset of appendages in Drosophila.

Viable aristaless (al) mutations of Drosophila affect pattern elements at both ends of the proximodistal axis in a subset of adult appendages. The al gene has been cloned and identified by P-element-mediated germ-line transformation with a genomic DNA fragment, which rescues a lethal mutation of al as well as aspects of the adult al phenotype. The al gene contains a prd-type homeo domain and a Pro/Gln-rich domain and, hence, probably encodes a transcription factor. Its transcript distribution in third-instar imaginal discs closely corresponds to the anlagen of the tissues that later become visibly affected in adult al mutants. The striking similarity of a bimodal al expression in different imaginal discs indicates that al is under the control of a "prepattern," which is shared at least among antennal, leg, and wing discs. The al gene is also transcribed during embryogenesis. Apart from a function in the ontogeny of specific larval head and tail organs, its embryonic transcript pattern suggests a possible role in early imaginal disc development.

The Min DNA inversion enzyme of plasmid p15B of Escherichia coli 15T-: a new member of the Din family of site-specific recombinases.

Plasmid p15B is a bacteriophage P1-related resident of Escherichia coli 15T-. Both genomes contain a segment in which DNA inversion occurs, although this part of their genomes is not identical. This DNA segment of p15B was cloned in a multicopy vector plasmid. Like its parent, the resulting plasmid, pAW800, undergoes complex multiple DNA inversions: this DNA inversion system is therefore called Min. The min gene, which codes for the p15B Min DNA invertase, can complement the P1 cin recombinase gene. The Min inversion system is thus a new member of the Din family of site-specific recombinases to which Cin belongs. The DNA sequence of the min gene revealed that Min is most closely related to the Pin recombinase of the e14 defective viral element on the E. coli K12 chromosome. Like other members of the Din family, the min gene contains a recombinational enhancer element which stimulates site-specific DNA inversion 300-fold.