Jutta Schulze

The Molybdenum Cofactor Biosynthetic Protein Cnx1 Complements Molybdate-Repairable Mutants, Transfers Molybdenum to the Metal Binding Pterin, and Is Associated with the Cytoskeleton

Molybdenum (Mo) plays an essential role in the active site of all eukaryotic Mo-containing enzymes. In plants, Mo enzymes are important for nitrate assimilation, phytohormone synthesis, and purine catabolism. Mo is bound to a unique metal binding pterin (molybdopterin [MPT]), thereby forming the active Mo cofactor (Moco), which is highly conserved in eukaryotes, eubacteria, and archaebacteria. Here, we describe the function of the two-domain protein Cnx1 from Arabidopsis in the final step of Moco biosynthesis. Cnx1 is constitutively expressed in all organs and in plants grown on different nitrogen sources. Mo-repairable cnxA mutants from Nicotiana plumbaginifolia accumulate MPT and show altered Cnx1 expression. Transformation of cnxA mutants and the corresponding Arabidopsis chl-6 mutant with cnx1 cDNA resulted in functional reconstitution of their Moco deficiency. We also identified a point mutation in the Cnx1 E domain of Arabidopsis chl-6 that causes the molybdate-repairable phenotype. Recombinant Cnx1 protein is capable of synthesizing Moco. The G domain binds and activates MPT, whereas the E domain is essential for activating Mo. In addition, Cnx1 binds to the cytoskeleton in the same way that its mammalian homolog gephyrin does in neuronal cells, which suggests a hypothetical model for anchoring the Moco-synthetic machinery by Cnx1 in plant cells.

A Novel Role for Arabidopsis Mitochondrial ABC Transporter ATM3 in Molybdenum Cofactor Biosynthesis

A mitochondrial ATP binding cassette transporter in Arabidopsis is found to play an important role in molybdenum cofactor biosynthesis. This transporter is involved in exporting cyclic pyranopterin monophosphate from the mitochondrion to the cytosol, where it is converted to molybdenum cofactor in a three-step process. The molybdenum cofactor (Moco) is a prosthetic group required by a number of enzymes, such as nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Its biosynthesis in eukaryotes can be divided into four steps, of which the last three are proposed to occur in the cytosol. Here, we report that the mitochondrial ABC transporter ATM3, previously implicated in the maturation of extramitochondrial iron-sulfur proteins, has a crucial role also in Moco biosynthesis. In ATM3 insertion mutants of Arabidopsis thaliana, the activities of nitrate reductase and sulfite oxidase were decreased to ∼50%, whereas the activities of xanthine dehydrogenase and aldehyde oxidase, whose activities also depend on iron-sulfur clusters, were virtually undetectable. Moreover, atm3 mutants accumulated cyclic pyranopterin monophosphate, the first intermediate of Moco biosynthesis, but showed decreased amounts of Moco. Specific antibodies against the Moco biosynthesis proteins CNX2 and CNX3 showed that the first step of Moco biosynthesis is localized in the mitochondrial matrix. Together with the observation that cyclic pyranopterin monophosphate accumulated in purified mitochondria, particularly in atm3 mutants, our data suggest that mitochondria and the ABC transporter ATM3 have a novel role in the biosynthesis of Moco.

Fertile transgenic barley of different cultivars obtained by adjustment of bombardment conditions to tissue response

Abstract Fertile transgenic barley ( Hordeum vulgare L.) plants of five cultivars were obtained by an improved transformation protocol using different particle delivery devices. Varieties of barley which are hardly amenable for genetic transformation due to low tissue culture response were stably transformed by optimizing transformation parameters for each genotype separately according to their tissue culture response. Scutella of immature zygotic embryos were transformed with the plasmid pAHC25 carrying the selectable marker gene bar and the reporter gene uidA . Gene delivery was optimized using the uidA -expression as transient marker for the transformation efficiency and taking into account the maintenance of the regeneration capacity after the bombardment. After selection on media with bialaphos as selective agent, 40 independently transformed plants were regenerated and analyzed for functional expression of the bar and uidA gene. Molecular analysis by Southern hybridization confirmed the integration of both genes into the nuclear genome of the plants. Thirty-five T 0 -plants of five barley cultivars reached maturity so far. Analysis of the progeny confirmed the inheritance of the marker genes.

Integration of Bioinformatics and Synthetic Promoters Leads to the Discovery of Novel Elicitor-Responsive cis-Regulatory Sequences in Arabidopsis

A combination of bioinformatic tools, high-throughput gene expression profiles, and the use of synthetic promoters is a powerful approach to discover and evaluate novel cis-sequences in response to specific stimuli. With Arabidopsis (Arabidopsis thaliana) microarray data annotated to the PathoPlant database, 732 different queries with a focus on fungal and oomycete pathogens were performed, leading to 510 up-regulated gene groups. Using the binding site estimation suite of tools, BEST, 407 conserved sequence motifs were identified in promoter regions of these coregulated gene sets. Motif similarities were determined with STAMP, classifying the 407 sequence motifs into 37 families. A comparative analysis of these 37 families with the AthaMap, PLACE, and AGRIS databases revealed similarities to known cis-elements but also led to the discovery of cis-sequences not yet implicated in pathogen response. Using a parsley (Petroselinum crispum) protoplast system and a modified reporter gene vector with an internal transformation control, 25 elicitor-responsive cis-sequences from 10 different motif families were identified. Many of the elicitor-responsive cis-sequences also drive reporter gene expression in an Agrobacterium tumefaciens infection assay in Nicotiana benthamiana. This work significantly increases the number of known elicitor-responsive cis-sequences and demonstrates the successful integration of a diverse set of bioinformatic resources combined with synthetic promoter analysis for data mining and functional screening in plant-pathogen interaction.

Biolistic transformation of cucumber using embryogenic suspension cultures: long-term expression of reporter genes

The generation of transgenic cucumber (Cucumis sativus L.) plants was achieved by biolistic transformation of a highly embryogenic cell suspension culture using the nptII and uidA gene. Functional expression of the genes in transgenic plants was determined by neomycin phosphotransferase and β-glucuronidase enzyme assays. Southern analysis of DNA isolated from kanamycin-resistant plants confirmed stable integration of the genes as well as multicopy integration and rearrangements. A study of gene expression showed activity of the uidA gene in plants regenerated from kanamycin-resistant calli about one year after bombardment, indicating a high stability of the nonselectable gene.

An improved protocol for eliminating endogenous β-glucuronidase background in barley

Abstract Scutella of immature barley embryos were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into the scutella by biolistic transformation. The amount of intrinsic activity was strongly genotype dependent and could be detected with both the histochemical and fluorimetrical assay. This interfering activity could not be eliminated by performing the assay at pH 7.0 nor using additional methanol in the incubation buffer. Only pretreatment of the bombarded scutella for 60 min at 55°C followed by staining at the same temperature in a modified buffer was found to completely suppress barley-endogenous β-glucuronidase. In addition to the spots of strong GUS activity, these improved conditions made it possible to observe high numbers of smaller GUS-spots that became visible on the white scutellu, being no longer masked by a previous non-specific blue staining pattern. Further, this protocol can be used for unequivocally differentiating between transgenic and non-transgenic barley plants.

A rapid and sensitive method to evaluate genotype specific tolerance to phosphinothricin-based selective agents in cereal transformation

Summary Isolated scutella from immature embryos of barley and wheat, commonly used as targets for genetic transformation, revealed a considerable variation in susceptibility towards bialaphos and glufosinate (phosphinothricin) among a range of cultivars investigated. Using the pH indicator chlorophenol-red, a rapid assay was established to determine within two days genotype specific basal level of tolerance based on spectrophotometric determination of colour change in the medium. A strong correlation was observed using the response of scutella incubated for 3 weeks on callus induction medium containing different amounts of bialaphos with their ability to change colour of chlorophenol-red in the medium within two days. Thus it can be concluded that the amount of bialaphos which is needed by a given genotype to achieve an absorbance of 0.5 at 575 nm, represents a tight selection condition for the appropiate genotype. The colour change of the medium caused by phosphinothricin based agents after exposure of expiants results from the interaction between acidification due to metabolic processes and rise of pH caused by ammonia accumulation due to inhibition of glutamine synthetase.

Sexual Transfer of Transgene (Bar) into Non-Transformed Wheat Genotypes and Cell-Level Selection of the Marker Gene in Microspore- and Anther Culture

Fertile transgenic wheat (Triticum aestivum L.) plants have been produced during the past few years (Vasil et. al 1993, Weeks et al. 1993, Becker et al., Nehra et. al 1994). At the end of 20th century the fundamental discoveries made in the field of molecular biology and cell and tissue culture have initiated a biological revolution in plant breeding and agricultural production. The first genetically engineered food crops have been introduced into the market in some countries (Nehra et al 1995), but several questions have been under discussion. The fate of transgene is an up-to-date question in plant genetic and breeding research.