Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome.

In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modified Ds transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.

Comparative sequence analysis reveals extensive microcolinearity in the lateral suppressor regions of the tomato, Arabidopsis, and Capsella genomes.

A 57-kb region of tomato chromosome 7 harboring five different genes was compared with the sequence of the Arabidopsis genome to search for microsynteny between the genomes of these two species. For all five genes, homologous sequences could be identified in a 30-kb region located on Arabidopsis chromosome 1. Only two inversion events distinguish the arrangement of the five genes in tomato from that in Arabidopsis. Inversions were not detected when the arrangement of the five Arabidopsis genes was compared with the arrangement in the orthologous region of Capsella, a plant closely related to Arabidopsis. These results provide evidence for microcolinearity between closely and distantly related dicotyledonous species. The degree of microcolinearity found can be exploited to localize orthologous genes in Arabidopsis and tomato in an unambiguous way.

A novel transposon tagging element for obtaining gain-of-function mutants based on a self-stabilizing Ac derivative.

A novel tagging system AcREH, designed for obtaining gain-of-function mutations, was prepared on the basis of a self-stabilizing Ac transposon derivative. The transposable element, DsAT, was constructed in a way that it can activate transcription of neighboring genes by two 35S promoters and/or by four tandem repeats of the enhancer fragment of this promoter. DsAT revealed somatic excision in the first generation of the tobacco transformants. The element exhibited germinal excision to the next generation, as demonstrated by PCR and Southern hybridization analysis. In spite of the structure of the element, which may inhibit the expression of the transposase gene, the frequency of germinal excision was comparable to or higher than those so far reported, suggesting the applicability of the element for gene tagging.

Genetic control of branching in Arabidopsis and tomato.

The patterns of axillary bud formation and the growth characteristics of side-shoots determine to a large extent the form of plants. Characterization of mutants in the monopodial plant Arabidopsis thaliana and in the sympodial tomato, as well as cloning of some of the respective genes, contributes to a better understanding of side-shoot development. Genes have been identified that influence the initiation of axillary meristems and the pattern of their subsequent development.

The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family.

The ability of the shoot apical meristem to multiply and distribute its meristematic potential through the formation of axillary meristems is essential for the diversity of forms and growth habits of higher plants. In the lateral suppressor mutant of tomato the initiation of axillary meristems is prevented, thus offering the unique opportunity to study the molecular mechanisms underlying this important function of the shoot apical meristem. We report here the isolation of the Lateral suppressor gene by positional cloning and show that the mutant phenotype is caused by a complete loss of function of a new member of the VHIID family of plant regulatory proteins.

Expression of genes for a defensin and a proteinase inhibitor in specific areas of the shoot apex and the developing flower in tomato.

Two genes that are highly expressed in the tomato shoot apex have been cloned by differential hybridization. One of the deduced polypeptides (AT1) shows significant similarities to class II proteinase inhibitors, while the other (AT2) displays similarities to defensins. Transcripts of both genes are also detectable in the developing flower and are present only in minor amounts in other tissues tested. In situ hybridization analysis revealed that both genes are expressed in non-overlapping subsets of cells in the shoot apex, as well as in the developing flower. The potential use of these genes as markers for certain cell types and the possible biological function of the encoded proteins are discussed.

Genetic and physical mapping of the lateral suppressor (ls) locus in tomato.

Tomato plants homozygous for the recessive lateral suppressor (ls) mutation show a number of phenotypic abnormalities among which the lack of lateral meristem initiation during vegetative growth and the absence of petals on the flower are the most prominent. As a first step towards the isolation of the Ls gene by means of map-based cloning, we have determined its position on the restriction fragment length polymorphism (RFLP) map of tomato. RFLP analysis of 527 F2 plants segregating for the ls allele allowed us to define an interval of 0.8 cM in which the Ls gene is located. Analysis of the physical distance between the two flanking RFLP markers by pulsed field gel electrophoresis revealed that they lie no further than 375 kb apart. Knowledge of the physical distance together with the availability of a tomato yeast artificial chromosome (YAC) library, makes it feasible to isolate the Ls gene by a map-based cloning approach.

Isolation and characterization of the tomato homeobox gene THOM1.

We have screened a cDNA library prepared from tomato (Lycopersicon esculentum Mill. cv. VFNT Cherry) shoot tips for homeobox-containing clones. The isolated cDNA clone THOM1 (tomato homeobox gene 1) contains a homeobox, a leucine zipper motif and and an acidic region. These features support the hypothesis that the THOM1 gene product acts as a transcriptional activator. THOM1 is highly expressed in the vegetative shoot apical meristem, the floral meristem and axillary meristems. Young derivatives of these meristems show similar levels of THOM1 transcripts which decrease with increasing age of the respective tissue. The THOM1 gene is located in the centromere region of chromosome 1 and belongs to a small gene family of the tomato genome.

A self-stabilizing Ac derivative and its potential for transposon tagging.

With the goal of developing a self-stabilizing transposon tagging system, a derivative of the maize transposable element Ac (Ds303) harbouring a deletion between bp 246 and 736 has been introduced into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation. The deletion removes the major transcription start site, 84 bp of the putative Ac promoter and part of the untranslated leader. Transpositions from the T-DNA, where Ds303 was inserted between the mannopine synthase 1' promoter and the GUS gene, were observed in four independent transgenic plants analysed. After transposition the transposed Ds303 elements were stably integrated in the genome and could not transactivate a tester Ds element. However, somatic and germinal transpositions of the transposed Ds303 elements occurred in the presence of a transposase source.

Transgenic tomato lines containing Ds elements at defined genomic positions as tools for targeted transposon tagging.

We have introduced a genetically marked Dissociation transposable element (DsHPT) into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation. Probes for the flanking regions of the T-DNA and transposed DsHPT elements were obtained with the inverse polymerase chain reaction (IPCR) technique and used in RFLP linkage analyses. The RFLP map location of 11 T-DNAs carrying DsHPT was determined. The T-DNAs are distributed on 7 of the 12 tomato chromosomes. To explore the feasibility of gene tagging strategies in tomato using DsHPT, we examined the genomic distribution of DsHPT receptor sites relative to the location of two different, but very closely linked, T-DNA insertion sites. After crosses with plants expressing Ac transposase, the hygromycin phosphotransferase (HPT) marker on the Ds element and the excision markers beta-glucuronidase (GUS) and Basta resistance (BAR) facilitated the identification of plants bearing germinally transposed DsHPT elements. RFLP mapping of 21 transposed DsHPT elements originating from the two different T-DNA insertions revealed distinct patterns of reintegration sites.

The pattern of histone H4 expression in the tomato shoot apex changes during development.

Two histone H4 cDNA clones were isolated from a tomato (Lycopersicon esculentum Mill.) shoot-tip cDNA library using a heterologous probe from barley (Hordeum vulgare L.). Both cDNAs, which are 81% identical in the coding region, are polyadenylated and belong to a small gene family in the tomato genome. Histone H4 message is abundant in young tissues and rare in older tissues. In the shoot apical meristem, the distribution of H4-expressing cells changes during development. In a juvenile vegetative apex, H4 message is detectable in the central region and the peripheral parts of the meristem. In a mature vegetative apical meristem, H4-expressing cells are localized in the peripheral zone extending into the provascular strands and the rib meristem whereas the central zone is almost devoid of H4 mRNA. After floral transition, H4 mRNA is found throughout the floral meristem, indicating a second change in the pattern of H4 expression. The observed changes in H4 expression are indicative of changes in the distribution of mitotic activity in the shoot apical meristem during plant development. In addition, H4-expressing cells were found to occur frequently in clusters, which may indicate a partial synchronization of cell divisions in the shoot apex.

Structural and functional analysis of the Bz2 locus of Zea mays: characterization of overlapping transcripts.

Analysis of the transcription pattern of the Bz2 locus revealed that overlapping transcripts are derived from opposite DNA strands. The most abundant transcript (sense transcript) has an open reading frame coding for a protein of 241 amino acids, whilst in the antisense orientation no open reading frame has been detected; the antisense transcripts are detected only in those tissues that show high levels of sense transcript. Particle gun experiments indicate that the sense transcript is sufficient to provide the Bz2 function. The promoter driving the sense transcript contains the elements usually found in front of eukaryotic genes. In addition an element with similarity to the C1 and R binding sites identified in the Bz1 promoter is found. Further upstream in the promoter region a transposon-like insertion has been identified. This element has features similar to members of the Ac/Ds transposable element family. The putative Bz2 protein shows similarity to various other plant proteins and to an Escherichia coli protein. All related proteins have in common the fact that they are involved in stress responses.