Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant

An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.     

Ethanol-resistant mutants of Nicotiana plumbaginifolia are deficient in the expression of pollen and seed alcohol dehydrogenase activity

Summary Six independent mutant lines ofNicotiana plumbaginifolia resistant to ethanol, designated E3, E8, E101, E112, E144 and E251, were isolated as germinating seedlings on selective medium. In all cases, resistance to ethanol was conferred by a single recessive nuclear mutation at the same locus. Mutant seeds and pollen lacked detectable ADH activity, with the exception of E251 where a residual activity was detected. An antiserum directed againstArabidopsis thaliana ADH detected an ADH-related polypeptide of 44 kDa present in wild-type seeds and, to a lesser extent, in the seeds of the leaky mutant E251. No ADH-related polypeptide could be detected in seeds of the other mutants. However, all of them had a nearly normal level of ADH mRNA except one which did not synthesize any mRNA. These results suggest that these ethanol-resistant mutants are impaired in one of the structural genes coding for alcohol dehydrogenase. The corresponding locus has been designatedAdh1.Abbreviations ADH alcohol dehydrogenase - EMS ethyl methane-sulfonate - MTT dimethyl thiazol tetrazolium - NAD nicotinamide adenine dinucleotide - NBT nitro blue tetrazolium - p-cells protoplast-derived cells - PMS phenazine methosulfate - SDS sodium dodecyl sulfate     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.     

High frequency stable transformation ofArabidopsis thaliana by particle bombardment

Root explants ofArabidopsis thaliana ecotype C24 were bombarded with the plasmid pCH harboring the hygromycin phosphotransferase gene (hpt). A selection condition with post-bombardment culture of 3 days followed by culture with 20 mgl⁻¹ hygromycin gave the highest yield of transformants. More than 44% of explant clumps formed transformant shoots.     

Adh n5: A temperature-sensitive mutant at the Adh locus in Drosophila

Individuals of an alcohol dehydrogenase-negative strain of Drosophila melanogaster designated Adh ⁿ⁵ are resistant to ethanol poisoning at low but not at high temperatures. The basis for this behavior is that Adh ⁿ⁵ flies contain a mutant form of alcohol dehydrogenase which is less heat stable than that of wild-type flies. The mutation in Adh ⁿ⁵ maps at, or very close to, the presumptive structural locus and is not complemented by any of 11 other alcohol dehydrogenase-null mutants.This research was supported by Grant No. GM 18254 from the National Institutes of Health and Grant No. M55.2217 from the National Cancer Institute.Publication No. 768 from the Department of Biology, Johns Hopkins University.     

Identification of a tRNA isopentenyltransferase gene from Arabidopsis thaliana

The tRNA of most organisms contain modified adenines called cytokinins. Situated next to the anticodon, they have been shown to influence translational fidelity and efficiency. The enzyme that synthesizes cytokinins on pre-tRNA, tRNA isopentenyltransferase (EC 2.5.1.8), has been studied in micro-organisms like Escherichia coli and Saccharomyces cerevisiae, and the corresponding genes have been cloned. We here report the first cloning and functional characterization of a homologous gene from a plant, Arabidopsis thaliana. Expression in S. cerevisiae showed that the gene can complement the anti-suppressor phenotype of a mutant that lacks MOD5, the intrinsic tRNA isopentenyltransferase gene. This was accompanied by the reintroduction of isopentenyladenosine in the tRNA. The Arabidopsis gene is constitutively expressed in seedling tissues.     

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)⁺-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses.The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5 non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5 sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3 sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site.This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.Abbreviations AK aspartate kinase - HSDH homoserine dehydrogenase - ID intermediate domain - Tp transit peptide     

Genetic complementation of a floral homeotic mutation,apetala3, with an Arabidopsis thaliana gene homologous to DEFICIENS of Antirrhinum majus

Among the homeotic mutants with altered floral organs, two mutants of Arabidopsis thaliana, apetala3 and pistillata, and two mutants of Antirrhinum majus, deficiens and globosa, have a homeotic conversion of the floral organs in whorl 2 and 3, namely petals to sepals and stamens to carpels. We have isolated a homologue of the DEFICIENS gene from A. thaliana wild type and shown complete complementation of apetala3 mutation by introducing the isolated gene using Agrobacterium-mediated transformation. These results show that the APETALA3 is a homologue of DEFICIENS structurally and functionally. The 5-upstream region of APETALA3 contains three SRE-like sequence, where MADS box-containing proteins are assumed to bind and regulate expression in tissue-and stage-specific manner.     

Reduced intracellular content of methotrexate in an isolated MTX-resistant cell line of Nicotiana plumbaginifolia

Summary In plant cells methotrexate (MTX) may exert its toxic effect through several mechanisms, including inhibition of its target protein dihydrofolate reductase. Resistance based on a mechanism operating before MTX binds to proteins should confer protection to plant cells. A methotrexate-resistant cell line of Nicotiana plumbaginifolia was isolated by a stepwise selection procedure. This cell line survived in the presence of 10 M MTX which is 50–100 fold higher than the lethal dose for the wild type cells. Neither alteration in kinetic characteristics of dihydrofolate reductase, nor elevated binding capacity of ³H-MTX to target protein(s), were observed. However, in comparison with wild type cells, markedly lower amounts of intracellular ³H-MTX were found after the selected cell line was incubated with ³H-MTX, indicating that either reduced uptake or enhanced efflux of MTX is the major reason for MTX-resistance in this cell line.     

Cloning of Arabidopsis thaliana phosphatidylinositol synthase and functional expression in the yeast pis mutant

It is believed that phosphatidylinositol (PI) metabolism plays a central role in signalling pathways in both animals and higher plants. PI is synthesized from CDP-diacylglycerol (CDP-DG) and myo-inositol by phosphatidylinositol synthase (PI synthase, EC 2.7.8.11). Here we report the identification of a plant cDNA (AtPIS1) encoding a 26 kDa PI synthase from Arabidopsis thaliana. The plant enzyme as deduced from its cDNA sequence shares 35–41% identical amino acids with PI synthases from Saccharomyces cerevisiae and mammals. AtPIS1 functionally complements a mutant of S. cerevisiae with a lesion in PI synthase, and recombinant AtPIS1 protein present in yeast membranes strongly depends on the two principal substrates, myo-inositol and CDP-DG, and requires Mg²⁺ ions for full activity.     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.     

Effect of repeated DNA sequences on direct gene transfer in protoplasts of Nicotiana plumbaginifolia

Summary Highly repeated nuclear DNA sequences from leaves of Nicotiana plumbaginifolia were cloned in pBR322 and tested for their effect on direct gene transfer in protoplasts of the same organism. Protoplasts were prepared from suspension cultures and were incubated in the presence of the plasmid pHP23 carrying the kanamycin resistance gene APH(3)II and in the presence of the plasmids carrying the cloned sequence. DNA uptake was induced by a polyethyleneglycol (PEG) treatment. Out of the 22 tested clones, 3 significantly stimulated the frequency of appearance of transformed colonies. DNA was extracted from some of the kanamycin-resistant calli obtained by co-transformations. Dot-blots have shown that the stimulatory effect on transformation frequency is often accompanied by a consistent increase in integrated genes sequences.     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

A novel blue light- and abscisic acid-inducible gene of Arabidopsis thaliana encoding an intrinsic membrane protein

Continuous irradiation with blue light (400–500 nm) induces flower formation in plantlets of Arabidopsis thaliana (C24) while red light (600–700 nm) is ineffective. This observation started a search for genes that are activated by blue light and initiate the morphogenic programme leading to flower formation. Several genes were identified via their cDNAs. From these clone AthH2, with an open reading frame for a hydrophobic 30.5 kDa polypeptide, was selected for further characterization of the corresponding gene. From a genomic library a DNA fragment of about 6.4 kb was isolated, comprising the coding region as well as 5-upstream and 3-downstream flanking segments. The coding region is composed of four exons, which specify a polypeptide of 286 amino acids. Several potential regulatory elements were found between position –670 and –1140 including GA and ABA sequence motifs. The latter could account for the observed induction of the AthH2 gene by ABA. Southern blot analysis of Arabidopsis genomic DNA suggests that the AthH2 gene is encoded by a single-copy gene. Hydropathy plots and secondary structure analysis of the putative polypeptide predict six membrane-spanning domains implicating a function as transmembrane channel protein. It displays significant homology with the proteins TR7a of pea (82%) and RD 28 of A. thaliana (68%).     

High-frequency transformation ofArabidopsis thaliana byAgrobacterium tumefaciens

Incorporation of 5 mg/L silver thiosulphate into media for seed germination and callus induction, as used in the transformation protocol originally described by Valvekens et al. (1988), was found to increase the frequency of regeneration of transformants ofArabidopsis thaliana ecotypes C24 and Landsbergerecta by at least 10- to 100-fold. Other factors, such as density of the bacterial inoculation culture, density of the root explants and duration of bacteria-plant cocultivation period, were also found to influence the efficiency of recovery of transformants.     

Isolation of a poplar and anArabidopsis thaliana dihydrodipicolinate synthase cDNA clone

A poplar DHDPS cDNA clone has been isolated by functional rescue of thedapA-deficient AT997 mutant ofEscherichia coli. By sequence comparison between the poplar and maize DHDPS cDNAs, two oligonucleotides were designed to perform polymerase chain reaction (PCR) onArabidopsis thaliana genomic DNA. The PCR fragment was subsequently used to isolate anArabidopsis DHDPS genomic and cDNA clone.     

Isolation and biochemical analysis of ethyl methanesulfonate-induced alcohol dehydrogenase null mutants ofArabidopsis thaliana (L.) Heynh

Several mutants have been isolated at theArabidopsis thaliana (L.) Heynh. alcohol dehydrogenase (ADH) gene locus using allyl alcohol selection on ethyl methanesulfonate (EMS)-mutagenized seeds. Eleven mutants were isolated in theADH1-A electrophoretic allele, and 21 in theADH1-S allele. These null mutants are characterized by the absence of measurable ADH activity and genetic data showed that the mutations were confined to theADH1 gene locus ofArabidopsis. Eleven mutants in theADH1-A background were further characterized at the protein and mRNA level. These experiments revealed striking differences in the ADH protein and mRNA content. Some of the mutants did not synthesize any mRNA or ADH-like protein, whereas some of them had a nearly normal level of ADH protein and mRNA. Others had a very low level of both protein and mRNA. ADH null mutants differed physiologically from the wild type by their higher sensitivity to anaerobic treatment in plants and significantly reduced resistance to acetaldehyde in suspension cultures.This research was supported by the Geconcerteerde Onderzoeksactie, Grant 86/91–103, and the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL), Grant 4972A.