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H Lörz

Publications and source records attributed to H Lörz.

At least 19 recordsLinked to original sources

Site-specific recombination in Zea mays.

The elimination of marker genes after selection is recommended for the commercial use of genetically modified plants. We compared the applicability of the two site-specific recombination systems Cre/lox and Flp/FRT for marker gene elimination in maize plants. The selection marker gene pat surrounded by two identically directed lox or FRT sites was introduced into maize. Sexual crossing with plants harboring the corresponding constitutively expressed recombinase led to the precise and complete excision of the lox-flanked marker gene in the F1 progeny, whereas Flp-mediated recombination of FRT sequences occurred rarely. Further examination of site-specific integration was done by biolistic bombardment of immature embryos harboring only one lox site with a lox.uidA sequence with results indicating directed integration.

Bacteriophage P1↗

Construction of a BAC library of Rosa rugosaThunb. and assembly of a contig spanning Rdr1, a gene that confers resistance to blackspot.

A BAC library to serve as a general tool for the physical mapping and positional cloning of rose genes has been constructed from Rosa rugosa DNA. With 27,264 clones the library contains 5.2 genome equivalents. The library was used to assemble a contig of BAC clones spanning Rdr1, a locus that confers resistance to blackspot. For this purpose fine-scale mapping of the target locus was achieved by bulked segregant analysis using 816 AFLP primer combinations. The target region around Rdr1 comprises about 400 kb and is covered by a minimum of six BAC clones. Furthermore, the detection of at least five resistance gene analogs of the TIR-NBS-LRR family on the contig indicates the presence of a cluster of resistance genes around Rdr1. These results will not only allow the isolation and identification of Rdr1 in the near future, but also provide the tools for the physical mapping and positional cloning of other horticulturally interesting genes in roses.

Base Sequence↗

Molecular characterisation of two novel maize LRR receptor-like kinases, which belong to the SERK gene family.

Genes encoding two novel members of the leucine-rich repeat receptor-like kinase (LRR-RLK) superfamily have been isolated from maize (Zea mays L.). These genes have been named ZmSERK1 and ZmSERK2 since features such as a putative leucine zipper (ZIP) and five leucine rich repeats in the extracellular domain, a proline-rich region (SPP) just upstream of the transmembrane domain and a C-terminal extension (C) after the kinase domain identify them as members of the SERK (somatic embryogenesis receptor-like kinase) family. ZmSERK1 and ZmSERK2 are single-copy genes and show 79% identity among each other in their nucleotide sequences. They share a conserved intron/exon structure with other members of the SERK family. In the maize genome, ZmSERK1 maps to position 76.9 on chromosome arm 10L and ZmSERK2 to position 143.5 on chromosome arm 5L, in regions generally not involved in duplications. ZmSERK1 is preferentially expressed in male and female reproductive tissues with strongest expression in microspores. In contrast, ZmSERK2 expression is relatively uniform in all tissues investigated. Both genes are expressed in embryogenic and non-embryogenic callus cultures.

Amino Acid Sequence↗

Transposition of the maize transposable element Ac in barley (Hordeum vulgare L.).

Transposition of the maize autonomous element Ac (Activator) was investigated in barley (Hordeum vulgare L.) with the aim of developing a transposon tagging system for the latter. The Ac element was introduced into meristematic tissue of barley by microprojectile bombardment. Transposon activity was then examined in the resulting transgenic plants. Multiple excision events were detected in leaf tissue of all plant lines. The mobile elements generated empty donor sites with small DNA sequence alterations, similar to those found in maize. Reintegration of Ac at independent genomic loci in somatic tissue was demonstrated by isolation of new element-flanking regions by AIMS-PCR (amplification of insertion-mutagenized sites). In addition, transmission of transposed Ac elements to progeny plants was confirmed. The results indicate that the introduced Ac element is able to transpose in barley. This is a first step towards the establishment of a transposon tagging system in this economically important crop.

Base Sequence↗

ZmES genes encode peptides with structural homology to defensins and are specifically expressed in the female gametophyte of maize.

All four members of a gene family, which are highly expressed in the cells of the female gametophyte (ZmES1--4: Zea mays embryo sac), were isolated from a cDNA library of maize egg cells. High expression of ZmES genes in the synergids around the micropylar region was detected in thin sections of maize ovaries. Single-cell RT--PCR analyses with the various cells of the female gametophyte confirmed the expression in synergids and also showed expression in the egg cell and central cell, and low expression in the antipodals. The expression of the whole gene family is suppressed after fertilization of the embryo sac, and expression in two-cell or later embryo stages or other tissues of maize could not be detected. In order to investigate ZmES mRNA gradients in the highly polarized and vacuolized cells of the maize embryo sac, a whole-mount in situ protocol with isolated single cells was developed: as for total RNA, ZmES transcripts are uniformly distributed in the cytoplasm of egg cell, synergids and central cell. ZmES genes encode small, cysteine-rich proteins with an N-terminal signal peptide, probably for translocation into the embryo sac cell wall. The four ZmES proteins display high sequence identity with each other, and the proposed tertiary structure of the mature peptides is similar to that of plant and animal defensins. The function of ZmES1-4 during the fertilization process is discussed.

Amino Acid Sequence↗

Heterologous expression of genes mediating enhanced fungal resistance in transgenic wheat.

Three cDNAs encoding the antifungal protein Ag-AFP from the fungus Aspergillus giganteus, a barley class II chitinase and a barley type I RIP, all regulated by the constitutive Ubiquitin1 promoter from maize, were expressed in transgenic wheat. In 17 wheat lines, stable integration and inheritance of one of the three transgenes has been demonstrated over four generations. The formation of powdery mildew (Erysiphe graminis f. sp. tritici) or leaf rust (Puccinia recondita f. sp. tritici) colonies was significantly reduced on leaves from afp or chitinase II- but not from rip I-expressing wheat lines compared with non-transgenic controls. The increased resistance of afp and chitinase II lines was dependent on the dose of fungal spores used for inoculation. Heterologous expression of the fungal afp gene and the barley chitinase II gene in wheat demonstrated that colony formation and, thereby, spreading of two important biotrophic fungal diseases is inhibited approximately 40 to 50% at an inoculum density of 80 to 100 spores per cm2.

Animals↗

The maize MADS box gene ZmMADS3 affects node number and spikelet development and is co-expressed with ZmMADS1 during flower development, in egg cells, and early embryogenesis.

MADS box genes represent a large gene family of transcription factors with essential functions during flower development and organ differentiation processes in plants. Addressing the question of whether MADS box genes are involved in the regulation of the fertilization process and early embryo development, we have isolated two novel MADS box cDNAs, ZmMADS1 and ZmMADS3, from cDNA libraries of maize (Zea mays) pollen and egg cells, respectively. The latter gene is allelic to ZAP1. Transcripts of both genes are detectable in egg cells and in in vivo zygotes of maize. ZmMADS1 is additionally expressed in synergids and in central and antipodal cells. During early somatic embryogenesis, ZmMADS1 expression is restricted to cells with the capacity to form somatic embryos, and to globular embryos at later stages. ZmMADS3 is detectable only by more sensitive reverse transcriptase-PCR analyses, but is likewise expressed in embryogenic cultures. Both genes are not expressed in nonembryogenic suspension cultures and in isolated immature and mature zygotic embryos. During flower development, ZmMADS1 and ZmMADS3 are co-expressed in all ear spikelet organ primordia at intermediate stages. Among vegetative tissues, ZmMADS3 is expressed in stem nodes and displays a gradient with highest expression in the uppermost node. Transgenic maize plants ectopically expressing ZmMADS3 are reduced in height due to a reduced number of nodes. Reduction of seed set and male sterility were observed in the plants. The latter was due to absence of anthers. Putative functions of the genes during reproductive and vegetative developmental processes are discussed.

Amino Acid Sequence↗

[Effects of the physical configuration of plasmid DNA and other factors on production of fertile transgenic wheat plants].

The applicability of hyperosmotic treatment and different configurations of plasmid DNA for stable transformation of wheat mediated by particle bombardment was investigated. Hyperosmotic treatment increased the frequency of transient expression and had also a positive effect on stable transformation. Denaturation of plasmid DNA prior to bombardment led to dramatic reduction of transient expression. However, there were no marked differences between single-stranded and double-stranded DNA in stable transformation. Single-stranded plasmid DNA, double-stranded plasmid DNA in linear state and double-stranded plasmid DNA in circular state could all be used to produce transgenic wheat plants. A total of 26 independent transgenic plants of winter wheat genotype Florida and 4 independent transgenic plants of spring wheat genotype Veery were obtained. Most transgenic plants have set seeds. T2 seeds of some spring wheat transgenic plants have also been harvested.

Aminobutyrates↗

Novel ribosomal genes from maize are differentially expressed in the zygotic and somatic cell cycles.

We have isolated cDNAs representing more than 50 different genes from libraries of unfertilised egg cells and zygotes of maize, expression of which is up- or downregulated after in vitro fertilisation (IVF). Among the cDNAs isolated are seven which encode proteins that are probably involved in translation and two encoding proteins probably involved in DNA replication. The latter genes are strongly induced on fertilisation. This indicates that zygotic gene activation (ZGA) - the switch from maternal to embryonic control of development - occurs in the zygote shortly after fertilisation in higher plants - earlier than in animal systems so far investigated. Four novel transcripts for ribosomal proteins (S21A, S21B, L39, P0) involved in ribosome biosynthesis and translation were analysed in more detail. The expression of all four genes correlates with cell division activity and is strongly induced during the G1 phase of the somatic cell cycle. A different mode of regulation operates in the first embryonic cell cycle: relatively large amounts of transcript are stored in the unfertilised egg cells, and by 18 h after IVF, two ribosomal genes are induced while a third is downregulated. These results indicate that using the combination of single-cell culture techniques with novel molecular methods, it is possible to isolate and study numerous genes expressed in female gametes and zygotes of higher plants. The detailed analysis of the four ribosomal protein genes demonstrates that the zygotic and somatic cell cycles are differentially regulated.

Amino Acid Sequence↗

A transcript encoding translation initiation factor eIF-5A is stored in unfertilized egg cells of maize.

Differential screening of cDNA libraries of unfertilized egg cells and in vitro zygotes of maize resulted in the isolation of more than 50 different genes whose expression is up- or down-regulated after in vitro fertilization (IVF). Among these genes, we identified a cDNA encoding the eukaryotic translation initiation factor eIF-5A. This highly conserved factor is thought to be necessary for selective mRNA stabilization and translation. It is also the only known protein that contains the unusual amino acid hypusine which is required for biological activity. High transcript amounts are stored in the egg cell, which is, in terms of metabolism, relatively inactive. Upon fertilization transcript amounts decrease, in contrast to metabolically inactive embryos in which the transcript cannot be detected and transcript levels increase upon germination. The expression pattern during the first embryonic cell cycle is also different from that observed during the somatic cell cycle: egg cells in the G0 phase contain high transcript levels, while arrested suspension cells contain few transcripts. In the somatic cell cycle, eif-5A is strongly induced during the G1 phase and transcripts are continuously degraded during the S, G2 and M phases until new induction during the G1 phase of the next cycle. eif-5A, a member of a small gene family in maize, is expressed in most maize tissues investigated. Based on our results, we suggest that the unfertilized egg cell of maize, although relatively inactive regarding its metabolism, is prepared for selective mRNA translation that is quickly triggered after fertilization. We also suggest that the regulation of eif-5A in the first embryonic cell cycle is different from the somatic cell cycle.

Amino Acid Sequence↗

Isolation of viable egg cells of rape (Brassica napus L.).

The isolation of viable egg cells of rape (Brassica napus L.) has been achieved from microdissected ovules. The non-gametic cells of the embryo sac, synergids and central cells have also been isolated. Their morphology corresponded to that of these cells in situ, making a discrimination from isolated sporophytic cells possible. Two hours after isolation the egg cells were still viable. Viable egg cells have been reproducibly isolated with a frequency of 25% per dissected ovule.

Brassica↗

Isolation of a full-length cDNA encoding calreticulin from a PCR library of in vitro zygotes of maize.

A full-size cDNA clone (1614 bp) encoding calreticulin was isolated from a PCR-based cDNA library of maize in vitro zygotes. Calreticulin is a major Ca2+ storage protein located mainly in the lumen of the endoplasmic reticulum but also in the nucleus and/or cytoplasm of some cells. A differential screening between cDNA libraries originating from 104 in vitro zygotes (18 h after in vitro fertilization) and 128 unfertilized egg cells was performed to isolate newly expressed genes or genes expressed more abundantly after fertilization. The expression of the isolated cDNA clone is enhanced after fertilization and strongly correlated to cell division. Sequence comparison to a shorter maize calreticulin cDNA isolated from a PCR based cDNA library construction from a few plant cells [12]. It is further shown that calreticulins in maize are probably transcribed from a small gene family differentially expressed in abundance in diverse tissues. The deduced amino acid sequence encodes an acidic protein (pI 4.17) of 48 kDa sharing 77-92% and 50-54% homology to other plant and animal calreticulins, respectively. The described calreticulin gene represents to our knowledge the first cDNA clone isolated from a RT/PCR cDNA library originating from only a few plant cells and is the first gene isolated from zygotes of higher plants.

Amino Acid Sequence↗

Isolation and characterization of a Tritordeum cDNA encoding S-adenosylmethionine decarboxylase that is circadian-clock-regulated.

Sequence analysis of the two cDNA clones 47/11 and 50A which were isolated by differential screening of an explant cDNA library obtained from the monocot Tritordeum (hexaploid hybrid of diploid wild barley and tetraploid wheat lines) reveals that both clones include the same open reading frame (ORF). The sequence of this ORF shows a high degree of similarity with dicot S-adenosylmethionine decarboxylase (SAMDC) gene sequences and contains regions highly conserved in all known SAMDC sequences. It is further shown that the sequence represented by the cDNA clones 47/11 and 50A is derived from the wild barley (Hordeum chilense) genome, where it is present as a single-copy gene. Northern analyses indicate the corresponding transcript to accumulate in response to wounding and the transcript level changes with a circadian rhythm, having a beak in the middle of the light period. The periodicity continues in constant light, but is changed in constant darkness.

Adenosylmethionine Decarboxylase↗

In vitro fertilisation of maize by single egg and sperm cell protoplast fusion mediated by high calcium and high pH.

We present evidence for the fusion of isolated single maize egg and sperm cell protoplasts in a mannitol solution (400-430 mosmol/kg H2O) containing 0.05 M CaCl2 at pH 11.0, followed by cell division of the fusion products. These findings allow the performance of in vitro fertilisation of higher plants by combining single gametes as in lower plant and animal systems. Further, our findings open new avenues for investigating the basic mechanisms of adhesion and fusion of higher plant gametes and eventually for examining processes that inhibit polyspermy in higher plants.

Botany↗

Fertile transgenic wheat from microprojectile bombardment of scutellar tissue.

A reproducible transformation system for hexaploid wheat was developed based on particle bombardment of scutellar tissue of immature embryos. Particle bombardment was carried out using a PDS 1000/He gun. Plant material was bombarded with the plasmid pDB1 containing the beta-glucuronidase gene (uidA) under the control of the actin-1 promoter of rice, and the selectable marker gene bar (phosphinothricin acetyltransferase) under the control of the CaMV 35S promoter. Selection was carried out using the herbicide Basta (Glufosinate-ammonium). From a total number of 1050 bombarded immature embryos, in seven independent transformation experiments, 59 plants could be regenerated. Putative transformants were screened for enzyme activity by the histochemical GUS assay using cut leaf material and by spraying the whole plants with an aqueous solution of the herbicide Basta. Twelve regenerants survived Basta spraying and showed GUS-activity. Southern-blot analysis indicated the presence of introduced foreign genes in the genomic DNA of the transformants and both marker genes were present in all plants analysed. To date, four plants have been grown to maturity and set seed. Histochemically stained pollen grains showed a 1:1 segregation of the uidA gene in all plants tested. A 3:1 segregation of the introduced genes was demonstrated by enzyme activity tests and Southern blot analysis of R1 plants.

Acetyltransferases↗

Transgenic cereal (tritordeum) plants obtained at high efficiency by microprojectile bombardment of inflorescence tissue.

Transgenic cereal plants expressing the beta-glucuronidase (uidA) and neomycin phosphotransferase (neo) genes were obtained via microprojectile bombardment of immature inflorescence tissue of tritordeum (the fertile Hordeum x Triticum amphiploid, HchHchAABB). A total of 17 independent transgenic plants were recovered from 32 bombardments (on average four inflorescences per shot). Of the bombardment and culture parameters tested, explant preculture had the most influence on stable transformation frequency. The uidA and neo genes were supplied on two separate plasmids (co-transformation) and 88% of the transgenic plants recovered expressed both genes. Southern analysis confirmed the results of histochemical GUS and NPT II assays. Transgenic plants were grown to maturity and flowered and set seed. Pollen from four T0 GUS+ plants analysed showed GUS activity and T1 seedlings derived from one of the transgenic plants showed a segregation of 2.75:1 (GUS+:GUS-) for uidA gene activity.

Culture Techniques↗

Representative cDNA libraries from few plant cells.

A reverse transcriptase/polymerase chain reaction (RT/PCR) method for the construction of representative cDNA libraries originating from few isolated cells is described. Poly(A)+ RNA was extracted from an average of 100 maize cells and reverse transcribed into sscDNA. The sscDNA was dG-tailed at its 3' end and amplified during a two-step PCR reaction. The generated PCR products were analysed and the majority < or = 2 kbp were full-size cDNAs. A fraction of the amplified cDNA from 128 isolated maize egg cells was cloned into the lambda Uni-ZAP XR vector and a primary library of 6.8 x 10(6) p.f.u. was obtained. The average insert size is 860 bp. It was further determined, that 0.31% of the clones hybridized to a cytosolic GAPDH probe. It is thought that, with this method, the first cDNA library of egg cells in higher plants was generated.

Base Sequence↗