Bernd Reiss

An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics.

The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics. Here we provide details about the establishment of the material, demonstrate the populations functionality and discuss results from quality control studies. T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T1 plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders. After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed. Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.

A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts

A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [gamma-32P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.

Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana.

A recessive pale mutation, designated as cs, was identified by transferred‐DNA (T‐DNA)‐mediated insertional mutagenesis in Arabidopsis thaliana. The pale mutation, cosegregating with the hygromycin resistance marker of the T‐DNA, was mapped to the position of the ch‐42 (chlorata) locus on chromosome 4. Lack of genetic complementation between cs and ch‐42 mutants indicated allelism. Plant boundaries of the T‐DNA insert rescued from the pale mutant were used as probes for the isolation of genomic and full‐length cDNA clones of the wild‐type cs gene. Transformation of the pale mutant with T‐DNA vectors carrying these clones resulted in a normal green phenotype, thus demonstrating positive complementation of the T‐DNA induced mutation. DNA sequence comparison of the cs mutant and its wild‐type allele revealed that the T‐DNA insertion occurred 11 bp upstream of the stop codon. A fusion protein, seven amino acids longer than its wild‐type counterpart of Mr 46,251, is therefore synthesized in the pale mutant. Transcript analysis during dark‐light transition, in vitro protein transport assay, and the absence of DNA sequence homology between cs and known genes indicates that the light regulated expression of the cs gene results in the synthesis of a novel chloroplast protein.

The Chromatin Assembly Factor Subunit FASCIATA1 Is Involved in Homologous Recombination in Plants

DNA replication in cycling eukaryotic cells necessitates the reestablishment of chromatin after nucleosome redistribution from the parental to the two daughter DNA strands. Chromatin assembly factor 1 (CAF-1), a heterotrimeric complex consisting of three subunits (p150/p60/p48), is one of the replication-coupled assembly factors involved in the reconstitution of S-phase chromatin. CAF-1 is required in vitro for nucleosome assembly onto newly replicated chromatin in human cells and Arabidopsis thaliana, and defects in yeast (Saccharomyces cerevisiae) affect DNA damage repair processes, predominantly those involved in genome stability. However, in vivo chromatin defects of caf-1 mutants in higher eukaryotes are poorly characterized. Here, we show that fasciata1-4 (fas1-4), a new allele of the Arabidopsis fas1 mutant defective in the p150 subunit of CAF-1, has a severe developmental phenotype, reduced heterochromatin content, and a more open conformation of euchromatin. Most importantly, homologous recombination (HR), a process involved in maintaining genome stability, is increased dramatically in fas1-4, as indicated by a 96-fold stimulation of intrachromosomal HR. Together with the open conformation of chromatin and the nearly normal expression levels of HR genes in the mutant, this result suggests that chromatin is a major factor restricting HR in plants.

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Protein fusions with the kanamycin resistance gene from transposon Tn5.

The gene for the neomycin phosphotransferase II (NPT II) from transposon Tn5 was fused at the amino or carboxy terminus to foreign DNA sequences coding for 3‐300 amino acids and the properties of the fused proteins were investigated. All amino‐terminal fusions examined conferred kanamycin resistance to their host cell, but profound differences in their enzymatic activity and stability were detected. Short additions to the amino terminus of the NPT II resulted in highly enzymatically active fusion proteins whereas long amino‐terminal fusions often had to be proteolytically degraded to release active proteins. Fusions at the carboxy‐terminal end of the NPT II protein did not always induce kanamycin resistance and their enzymatic activity depended more stringently on the nature of the junction sequence.

The importance of the transit peptide and the transported protein for protein import into chloroplasts

SummaryWe compared the transport in vitro of fusion proteins of neomycin phosphotransferase II (NPTII) with either the transit peptide of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase or the transit peptide and the 23 aminoterminal amino acids of the mature small subunit. The results showed that the transit peptide is sufficient for import of NPTII. However, transport of the fusion protein consisting of the transit peptide linked directly to NPTII was very inefficient. In contrast, the fusion protein containing a part of the mature SSU was imported with an efficiency comparable to that of the authentic SSU precursor. We conclude from these results that other features of the precursor protein in addition to the transit peptide are important for transport into chloroplasts. In order to identify functional regions in the transit peptide, we analyzed the transport of mutant fusion proteins. We found that the transport of fusion proteins with large deletions in the aminoterminal, or central part was drastically reduced. In contrast, duplication of a part of the transit peptide led to a marked increase in transport.

The PAC protein affects the maturation of specific chloroplast mRNAs in Arabidopsis thaliana.

Abstract The pale cress (pac) mutation arrests chloroplast development at an early stage in Arabidopsis thaliana and leads to a white phenotype. Chlorophyll fluorescence measurements demonstrated that the photosynthetic apparatus was impaired. The mutation did not reduce transcription of nuclear genes with photosynthetic function. However, distinct chloroplast-encoded transcripts were affected. The mutation mainly changed the maturation pattern, but the abundance of specific transcripts was also reduced. The defects observed imply a specific role for PAC in chloroplast mRNA maturation. PAC is encoded by a nuclear gene and is transported into the chloroplast. Therefore PAC may be one of the nucleus-encoded factors that function in plastid mRNA maturation and accumulation.

Homologous recombination and gene targeting in plant cells

Gene targeting has become an indispensable tool for functional genomics in yeast and mouse; however, this tool is still missing in plants. This review discusses the gene targeting problem in plants in the context of general knowledge on recombination and gene targeting. An overview on the history of gene targeting is followed by a general introduction to genetic recombination of bacteria, yeast, and vertebrates. This abridged discussion serves as a guide to the following sections, which cover plant-specific aspects of recombination assay systems, the mechanism of recombination, plant recombination genes, the relationship of recombination to the environment, approaches to stimulate homologous recombination and gene targeting, and a description of two plant systems, the moss Physcomitrella patens and the chloroplast, that naturally have high efficiencies of gene targeting. The review concludes with a discussion of alternatives to gene targeting.

Characterization of the activity of a plastid-targeted green fluorescent protein in Arabidopsis

In Arabidopsis thaliana the PALE CRESS (PAC) gene product is required for both chloroplast and cell differentiation. Transgenic Arabidopsis plants expressing a translational fusion of the N-terminal part of the PAC protein harboring the complete plastid-targeting sequence and the green fluorescent protein (GFP) exhibit high GFP fluorescence. Detailed analyses based on confocal imaging of various tissues and cell types revealed that the PAC-GFP fusion protein accumulates in chloroplasts of mature stomatal guard cells. The GFP fluorescence within the guard cell chloroplasts is not evenly distributed and appears to be concentrated in suborganellar regions. GFP localization studies demonstrate that thin tubular projections emanating from chloroplasts and etioplasts often connect the organelles with each other. Furthermore, imaging of non-green and etiolated tissue further revealed that GFP fluorescence is present in proplastids, etioplasts, chromoplasts, and amyloplasts. Even photobleaching of carotenoid-free plastids does not affect PAC-GFP accumulation in the organelles of the guard cells indicating that the protein translocation machinery is functional in all types of plastids. The specific accumulation of GFP in guard cell chloroplasts, their tubular connections, the translocation of the precursor polypeptide into the different types of organelles, as well as the use of a plastid-targeted GFP protein as a versatile marker is discussed in the context of previously described observations.