Agrobacterium T-DNA integration in Arabidopsis is correlated with DNA sequence compositions that occur frequently in gene promoter regions

Mobile insertion elements such as transposons and T-DNA generate useful genetic variation and are important tools for functional genomics studies in plants and animals. The spectrum of mutations obtained in different systems can be highly influenced by target site preferences inherent in the mechanism of DNA integration. We investigated the target site preferences of Agrobacterium T-DNA insertions in the chromosomes of the model plant Arabidopsis thaliana. The relative frequencies of insertions in genic and intergenic regions of the genome were calculated and DNA composition features associated with the insertion site flanking sequences were identified. Insertion frequencies across the genome indicate that T-strand integration is suppressed near centromeres and rDNA loci, progressively increases towards telomeres, and is highly correlated with gene density. At the gene level, T-DNA integration events show a statistically significant preference for insertion in the 5 and 3 flanking regions of protein coding sequences as well as the promoter region of RNA polymerase I transcribed rRNA gene repeats. The increased insertion frequencies in 5 upstream regions compared to coding sequences are positively correlated with gene expression activity and DNA sequence composition. Analysis of the relationship between DNA sequence composition and gene activity further demonstrates that DNA sequences with high CG-skew ratios are consistently correlated with T-DNA insertion site preference and high gene expression. The results demonstrate genomic and gene-specific preferences for T-strand integration and suggest that DNA sequences with a pronounced transition in CG- and AT-skew ratios are preferred targets for T-DNA integration.Electronic Supplementary Material Supplementary material is available for this article at .This revised version was published online in March 2005 with corrections to Dr. Tatarinovas name.     

Nucleotide sequences of nuclear tRNACys genes from Nicotiana and Arabidopsis and expression in HeLa cell extract

We have recently characterized Nicotiana cytoplasmic (cyt) tRNA_GCA ^Cys as novel UGA suppressor tRNA. Here we have isolated its corresponding (NtC1) and a variant (NtC2) gene from a genomic library of Nicotiana rustica. Both tRNA^Cys genes are efficiently transcribed in HeLa cell nuclear extract and yield mature cyt tRNAs^Cys. Sequence analysis of the upstream region of the RAD51 single-copy gene of the Arabidopsis thaliana genome revealed a cluster of three tRNA^Cys genes which have the same polarity and comprise highly similar flanking sequences. Of the three Arabidopsis tRNA^Cys genes only one (i.e. AtC2) appears to code for a functional gene which exhibits an almost identical nucleotide sequence to NtC1. These are the first sequenced nuclear tDNAs^Cys of plant origin.     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

Characterization of a chlorate-hypersensitive,high nitrate reductase Arabidopsis thaliana mutant

Summary A population of A. thaliana, produced by self-fertilization of ethylmethane sulfonate treated plants, was exposed to chlorate in the watering solution, and plants showing early susceptibility symptoms were rescued. Among the progeny lines of these plants five were shown to be repeatably chlorate-hypersusceptible. One of these lines (designated C-4) possessed elevated activity of nitrate reductase (NR). The NR activity of mutant C-4 was higher than that of normal plants throughout the life cycle. Nitrite reductase and glutamine synthetase activities of C-4 were normal, as were chlorate uptake rate and tissue nitrate content. The elevated NR activity apparently was responsible for the chlorate hypersusceptibility of C-4. Inheritance studies of NR indicated that the elevated activity of C-4 was probably controlled by a single recessive allele.     

Alcohol-inducible gene expression in transgenic Populus

We tested the efficiency and optimized the conditions for controlled alcohol-inducible transgene expression in Populus using gus as a reporter gene. Specificity of induction, efficiency in different organs, effect of three chemical inducers, and induction methods were tested using up to 10 independent transgenic events generated in two different Populus genotypes. The optimal inducer concentration and the duration of induction period were determined in dose–response and in time–course experiments. Under in vitro conditions, β-glucuronidase (GUS) induction was efficient both in the aerial parts and in the roots of regenerated plantlets. Among the chemical inducers tested, ethanol was the most effective activator with no apparent phytotoxicity when concentrations were at or below 2%. After 5 days of treatment, fluorometrically-determined the GUS activity could be detected when inducing with ethanol at concentrations as low as 0.5%. Prolonged induction by ethanol vapors significantly increased the GUS activity in leaves from both the tissue culture plants and greenhouse-grown plants.Electronic Supplementary Material Supplementary material is available for this article at     

The myrosinase gene family in Arabidopsis thaliana: gene organization,expression and evolution

Myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1.) is in Brassicaceae species such as Brassica napus and Sinapis alba encoded by two differentially expressed gene families, MA and MB, consisting of about 4 and 10 genes, respectively. Southern blot analysis showed that Arabidopsis thaliana contains three myrosinase genes. These genes were isolated from a genomic library and two of them, TGG1 and TGG2, were sequenced. They were found to be located in an inverted mode with their 3 ends 4.4 kb apart. Their organization was highly conserved with 12 exons and 11 short introns. Comparison of nucleotide sequences of TGG1 and TGG2 exons revealed an overall 75% similarity. In contrast, the overall nucleotide sequence similarity in introns was only 42%. In intron 1 the unusual 5 splice border GC was used. Phylogenetic analyses using both distance matrix and parsimony programs suggested that the Arabidopsis genes could not be grouped with either MA or MB genes. Consequently, these two gene families arose only after Arabidopsis had diverged from the other Brassicaceae species. In situ hybridization experiments showed that TGG1 and TGG2 expressing cells are present in leaf, sepal, petal, and gynoecium. In developing seeds, a few cells reacting with the TGG1 probe, but not with the TGG2 probe, were found indicating a partly different expression of these genes.     

Extension-growth responses and expression of flavonoid biosynthesis genes in the Arabidopsis hy4 mutant

The hy4 mutant of Arabidopsis thaliana(L.) Heynh. was previously shown to be impaired in the suppression of hypocotyl extension specifically by blue light. We report here that hy4 is altered in a range of blue-light-mediated extension-growth responses in various organs in seedlings and mature plants: it shows greater length of bolted stems, increased petiole extension and increased leaf width and area in blue light compared to the wild type. The hy4 mutant shows decreased cotyledon expansion in both red and blue light compared to the wild type. Anthocyanin formation and the expression of several flavonoid biosynthesis genes is stimulated by blue light in the wild type but to a much lower extent in hy4. The results indicate that the HY4 gene product is concerned with the perception of blue light in a range of extension-growth and gene-expression responses in Arabidopsis.Abbreviations DFR dihydroflavonol reductase - CHS chalcone synthase - CHI chalcone isomerase We thank the UK Agricultural and Food Research Council for supporting this work through the award of a research grant to G.I.J. We are grateful to Robert Brown for excellent technical assistance and Drs B.W. Shirley (Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, USA), C.D. Silflow (Department of Genetics and Cell Biology, University of Minnesota, St. Paul, USA) and I.E. Somssich (Department of Biochemistry, Max-Planck-Institut, Köln, Germany) for providing plasmid DNA.     

The second intron of AGAMOUS drives carpel- and stamen-specific expression sufficient to induce complete sterility in Arabidopsis

Gene containment technologies that prevent transgene dispersal through pollen, fruit and seed are in immediate demand to address concerns of gene flow from transgenic crops into wild species or close relatives. In this study, we isolated the enhancer element of Arabidopsis AGAMOUS that drives gene expression specifically in stamens and carpels. By fusing this AG enhancer to a minimal 35S promoter fragment, two tissue-specific promoters, fAGIP and rAGIP in forward and reverse orientations, respectively, were created and fused to the GUS reporter. Transgenic Arabidopsis plants harboring either fAGIP::GUS or rAGIP::GUS displayed similar GUS expression specifically in carpel and stamen tissues and their primordial cells. To test their utility for engineering sterility, the promoters were fused to the Diphtheria toxin A (DT-A) gene coding for a ribosome inactivating protein as well as the Barnase gene coding for an extracellular ribonuclease, and tested for tissue-specific ablation. Over 89% of AGIP::DT-A and 68% of AGIP::Barnase transgenic plants displayed specific and precise ablation of stamens and carpels and are completely sterile. These transgenic plants showed normal vegetative development with prolonged vegetative growth. To evaluate the stability of the sterile phenotype, 16 AGIP::DT-A lines underwent two consecutive cutback generations and showed no reversion of the floral phenotype. This study demonstrates a simple, precise and efficient approach to achieve absolute sterility through irreversible ablation of both male and female floral organs. This approach should have a practical application for transgene containment in ornamental, landscaping, and woody species, whose seeds and fruits are of no economic value.     

Expression of three <Emphasis Type="Italic">Arabidopsis</Emphasis> cytokinin oxidase/dehydrogenase promoter::GUS chimeric constructs in tobacco: response to developmental and biotic factors

Expression patterns of three Arabidopsis thaliana cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions were investigated in tobacco plants. While cytokinin oxidase/dehydrogenase promoter 2 showed no expression in tobacco, the cytokinin oxidase/dehydrogenase promoters 3 and 4 were active in various tissues throughout development of the tobacco. Recently, the 1452 bp promoter region of AtCKX3 was reported as almost inactive in Arabidopsis. In contrast, the 1627 bp DNA fragment preceding the AtCKX3 coding region drove expression of the reporter GUS gene in various tobacco tissues. The promoter was mainly expressed in tobacco leaves and roots during early stages of development but also later in young flower buds as well as in pollen grains. The construct was particularly active before (hypocotyl region) and during (vascular system) lateral root initiation, supporting the idea of an inhibitory role of active cytokinins in the process of root initiation. The cytokinin oxidase/dehydrogenase promoter 4::GUS fusion in tobacco was shown to share some common (but weaker) expression patterns with promoter 3, namely in the leaves and pollen, but also conferred specific expression in tobacco root cap cells and trichomes. In addition, the response of cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions to infection with the leafy gall-forming bacteria Rhodococcus fascians was examined. While an avirulent strain of R. fascians did not induce expression of any of the cytokinin oxidase/dehydrogenase promoters, the cytokinin oxidase/dehydrogenase promoter 3::GUS fusion was specifically induced at the site of infection when plants were challenged with a virulent strain of R. fascians, providing a possible explanation for the lack of significantly elevated cytokinin concentrations in tissues infected with virulent strains of R. fascians.This revised version was published online in August 2005 with some black and white figures replaced by coloured figures.     

Isolation,expression, and evolution of the gene encoding mitochondrial elongation factor Tu in Arabidopsis thaliana

We have characterized a second nuclear gene (tufM) in Arabidopsis thaliana that encodes a eubacterial-like protein synthesis elongation factor Tu (EF-Tu). This gene does not closely resemble the previously described Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes. However, the predicted amino acid sequence includes an N-terminal extension which resembles an organellar targeting sequence and shares three unique sequence elements with mitochondrial EF-Tu's, from Saccharomyces cerevisiae and Homo sapiens, suggesting that this gene encodes the Arabidopsis mitochondrial EF-Tu. Consistent with this interpretation, the gene is expressed at a higher level in flowers than in leaves. Phylogenetic analysis confirms the mitochondrial character of the sequence and indicates that the human, yeast, and Arabidopsis tufM genes have undergone considerably more sequence divergence than their cytoplasmic counterparts, perhaps reflecting a cross-compartmental acceleration of gene evolution for components of the mitochondrial translation apparatus. As previously observed for tufA, the tufM gene is present in one copy in Arabidopsis but in several copies in other species of crucifers.     

The seasonal activity and the effect of mechanical bending and wounding on the PtCOMT promoter in Betula pendula Roth

In this study, 900-bp (signed as p including nucleotides –1 to –886) and partly deleted (signed as dp including nucleotides –1 to –414) COMT (caffeate/5-hydroxyferulate O-methyltransferase) promoters from Populus tremuloides Michx. were fused to the GUS reporter gene, and the tissue-specific expression patterns of the promoters were determined in Betula pendula Roth along the growing season, and as a response to mechanical bending and wounding. The main activity of the PtCOMTp- and PtCOMTdp-promoters, determined by the histochemical GUS assay, was found in the developing xylem of stems during the 8th–13th week and in the developing xylem of roots in the 13th week of the growing season. The GUS expression patterns did not differ among the xylem cell types. The PtCOMT promoter-induced GUS expression observed in phloem fibres suggests a need for PtCOMT expression and thus syringyl (S) lignin synthesis in fibre lignification. However, the PtCOMTdp-promoter induced GUS expression in stem trichomes, which may contribute to the biosynthesis of phenylpropanoid pathway-derived compounds other than lignin. Finally, a strong GUS expression was induced by the PtCOMT promoters in response to mechanical stem bending but not to wounding. The lack of major differences between the PtCOMTp- and PtCOMTdp-promoters suggests that the deleted promoter sequence (including nucleotides −415 to −886) did not contain a significant regulatory element contributing to the GUS expression in young B. pendula trees.     

Structural and functional characterization of AtPTR3, a stress-induced peptide transporter of Arabidopsis

A T-DNA tagged mutant line of Arabidopsis thaliana, produced with a promoter trap vector carrying a promoterless gus (uidA) as a reporter gene, showed GUS induction in response to mechanical wounding. Cloning of the chromosomal DNA flanking the T-DNA revealed that the insert had caused a knockout mutation in a PTR-type peptide transporter gene named At5g46050 in GenBank, here renamed AtPTR3. The gene and the deduced protein were characterized by molecular modelling and bioinformatics. Molecular modelling of the protein with fold recognition identified 12 transmembrane spanning regions and a large loop between the sixth and seventh helices. The structure of AtPTR3 resembled the other PTR-type transporters of plants and transporters in the major facilitator superfamily. Computer analysis of the AtPTR3 promoter suggested its expression in roots, leaves and seeds, complex hormonal regulation and induction by abiotic and biotic stresses. The computer-based hypotheses were tested experimentally by exposing the mutant plants to amino acids and several stress treatments. The AtPTR3 gene was induced by the amino acids histidine, leucine and phenylalanine in cotyledons and lower leaves, whereas a strong induction was obtained in the whole plant upon exposure to salt. Furthermore, the germination frequency of the mutant line was reduced on salt-containing media, suggesting that the AtPTR3 protein is involved in stress tolerance in seeds during germination.Figure a Induction of AtPTR3 gene by amino acids. GUS staining of line 9 plants eight hours after induction with amino acids. Control indicates plant treated with water. His, Leu and Phe indicate plants treated with 10 mM amino acids histidine, leucine or phenylalanine, respectively. b Induction of AtPTR3 gene by salt. GUS staining of line 9 plants grown on MS medium on different salt concentrations: Control indicates plant grown on MS medium and 100 mM, 120 mM and 140 mM indicate plants grown on MS medium supplemented with the indicated NaCl concentrations. Size of the plants grown on salt medium has been magnified. c Germination frequency of Atptr3 knockout mutant line is reduced on salt medium. Atptr3 knockout mutant (9) and wild type C24 (WT) sown on MS medium (Control) and MS medium supplemented with salt (140 mM NaCl).      

Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgenic tobacco

The β-glucosidase gene of maize (ZmGLU1) was suggested to hydrolyze cytokinin-conjugate and release free cytokinin during plant growth and development. A clone containing the upstream region of ZmGLU1 was isolated and sequenced from a maize genomic library. The full-length ZmGLU1 promoter and a series of its 5′ deletions were fused to the beta-glucuronidase (GUS) reporter gene and transferred into tobacco. The GUS activity of transgenic plants was assayed at various developmental stages. The results showed that ZmGLU1 promoter-driven GUS gene had the highest expression level in the roots and that the expression of GUS gene declined during seed maturation and down to the lowest level in mature seeds. The ZmGLU1 promoter-driven GUS expression increased during seed germination, reaching a peak on day 11. The results also showed that this promoter could be inhibited by 6-BA, trans-zeatin, and NAA, but was not affected by GA₃, ABA, SA, cold, salt, drought, and submergence treatments. The histochemical staining revealed that GUS activity was located in vigorous cell division zones with dominant staining associated with vascular tissues. Deletion analysis showed that the promoter contained a putative leaf-specific and stem-specific negative regulative element and two putative enhancers.