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PubMed1992

The function of vacuolar beta-1,3-glucanase investigated by antisense transformation. Susceptibility of transgenic Nicotiana sylvestris plants to Cercospora nicotianae infection.

Vacuolar class I beta-1,3-glucanases (EC 3.2.1.39) are believed to be important in the induced defense reaction of plants to fungal infection. We used antisense transformation to test this hypothesis and to identify other possible physiological functions of this enzyme. Nicotiana sylvestris plants were transformed with antisense constructions containing the region from position 27 to 608 of the coding sequence of the basic, vacuolar beta-1,3-glucanase gene GLA of tobacco regulated by cauliflower mosaic virus 35S RNA expression signals. Plants homozygous for this transgene showed a marked, ca. 20-fold reduction in the constitutive expression of class I beta-1,3-glucanase antigen in their leaves. RNA blot analysis indicated that the antisense plants expressed low levels of the sense transcript of the host beta-1,3-glucanase gene and the antisense transcript of the transgene. Immune blot analysis of plant extracts indicated that only expression of the N. sylvestris homologue of class I tobacco beta-1,3-glucanase and not the acidic, class II isoforms of the enzyme was blocked in the antisense plants. Class I isoforms of beta-1,3-glucanase and chitinase were coordinately induced in leaves of untransformed and empty-vector-transformed N. sylvestris plants treated with ethylene or infected with the fungal leaf pathogen Cercospora nicotianae. In antisense plants, chitinase but not beta-1,3-glucanase was induced under these conditions indicating that antisense transformation effectively blocks constitutive as well as induced expression of class I beta-1,3-glucanase. Under greenhouse conditions, antisense plants developed normally and were fertile. The plants did not exhibit increased susceptibility to C. nicotianae infection. These results suggest that expression of the beta-1,3-glucanase isoform blocked by antisense transformation is not necessary for 'housekeeping' functions of N. sylvestris nor defense against the fungal pathogen tested.

Blotting, Northern↗

PubMed1993

Transgene copy number can be positively or negatively associated with transgene expression.

Two different types of T-DNA insert were found in tobacco plants transformed with Agrobacterium tumefaciens. High-expressing (H) types had one copy of the T-DNA at a locus and produced high expression of the transgene uidA, as measured by uidA RNA levels and beta-glucuronidase activity; low-expressing (L) types had inverted repeats of the T-DNA at a locus and produced low uidA expression. H-types from different transformants acted additively, and cross-fertilization between two different homozygous transformants with H-type inserts produced F1 plants with GUS activity that equalled the parents and individual F2 plants with 50%, 100%, 150% and 200% of parental values. However, the L-type inserts worked in trans to suppress uidA expression from H-type inserts when both were present in the same genome. Hence when a transformant homozygous for the L-type insert was crossed to one homozygous for the H-type, all plants in the F1 and F2 generations with both types of insert had low GUS activity while F2 segregants that only had the H-type inserts had high GUS activity again. Suppression of the H-type gene was associated with increased methylation of the insert. Particle acceleration was used to introduce further copies of uidA into tissues of the transformants. Regardless of the promoter used, those plants with endogenous L-type inserts showed none of the distinct loci of GUS activity readily visible in material with no inserts, showing that L-type inserts could suppress not only the uidA expression of genomic homologues, but also of copies added in vitro.

Agrobacterium tumefaciens↗

PubMed1996

Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter.

The cassava vein mosaic virus (CVMV) is a double stranded DNA virus which infects cassava plants (Manihot esculenta Crantz) and has been characterized as a plant pararetrovirus belonging to the caulimovirus subgroup. Two DNA fragments, CVP1 of 388 nucleotides from position -368 to +20 and CVP2 of 511 nucleotides from position -443 to +72, were isolated from the viral genome and fused to the uidA reporter gene to test promoter expression. The transcription start site of the viral promoter was determined using RNA isolated from transgenic plants containing the CVMV promoter:uidA fusion gene. Both promoter fragments were able to cause high levels of gene expression in protoplasts isolated from cassava and tobacco cell suspensions. The expression pattern of the CVMV promoters was analyzed in transgenic tobacco and rice plants, and revealed that the GUS staining pattern was similar for each construct and in both plants. The two promoter fragments were active in all plant organs tested and in a variety of cell types, suggesting a near constitutive pattern of expression. In both tobacco and rice plants, GUS activity was highest in vascular elements, in leaf mesophyll cells, and in root tips.

Base Sequence↗

PubMed1995

Features of the hmg 1 subfamily of genes encoding HMG-CoA reductase in potato.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the beta-glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R2 pollen of R1 progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.

Amino Acid Sequence↗

PubMed1993

A complex ensemble of cis-regulatory elements controls the expression of a Vicia faba non-storage seed protein gene.

We have identified cis-regulatory elements within the 5'-upstream region of a Vicia faba non-storage seed protein gene, called usp, by studying the expression of usp-promoter deletion fragments fused to reporter genes in transgenic tobacco seeds. 0.4 kb of usp upstream sequence contain at least six, but probably more, distinct cis-regulatory elements which are responsible for seemingly all quantitative, spatial and temporal aspects of expression. Expression-increasing and -decreasing elements are interspersed and include an AT-rich sequence, a G-box element and a CATGCATG motif. The latter acts as a negative element in contrast to what has been found for the same motif in legumin- and vicilin-type seed storage protein gene promoters. Seed specificity of expression is mainly determined by the -68/+51 region which confers, however, only very low levels of expression. The data support the combinatorial model of promoter function.

Base Sequence↗

PubMed1992

Characterization of the gene encoding the plastid-located glutamine synthetase of Phaseolus vulgaris: regulation of beta-glucuronidase gene fusions in transgenic tobacco.

The gln-delta gene, which encodes the plastid-located glutamine synthetase of Phaseolus vulgaris, was cloned and its promoter region was sequenced. Primer extension analysis was used to map the two major transcription initiation sites which are about 90 nucleotides apart. A fusion of 2.3 kb of the upstream region of the gln-delta gene to the reporter gene uidA encoding beta-glucuronidase was shown to be expressed in the chlorophyllous cell types of leaves and stems and in the root meristem region of transgenic tobacco. Analysis of a series of three 5' promoter deletion fusions revealed the presence of a region essential for promoter activity between -786 and -327 and regions involved in tissue-specific regulation and light regulation between -786 and +43.

Base Sequence↗

PubMed1993

Haploid and diploid expression of a Brassica campestris anther-specific gene promoter in Arabidopsis and tobacco.

The anther-specific cDNA clone Bcp1 from Brassica campestris is expressed in both the haploid pollen and diploid tapetum, as shown by in situ hybridization. We have isolated Bgp1, a genomic clone homologous to Bcp1. The coding region and extensive 5' flanking sequences of Bgp1 have been sequenced, and the coding region shows 88% identity with Bcp1. RNA gel blot analysis confirmed the expression of Bgp1-specific transcripts in B. campestris pollen. A 767 bp 5' DNA fragment was fused to the reporter gene beta-glucuronidase (gus) and introduced into both Arabidopsis thaliana and Nicotiana tabacum by transformation. This 5' fragment directed high-level expression in the pollen and tapetum of transgenic Arabidopsis. In transgenic tobacco however, the same construct was expressed only in pollen. A series of 5' deletion constructs has been created and used to transform A. thaliana to analyse the 5' region of Bgp1. The results indicate that Bgp1 expression in the tapetum and pollen of Arabidopsis requires the presence of different 5' DNA sequences.

Amino Acid Sequence↗

PubMed1991

The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts.

A mutant Gin recombinase of the phage Mu DNA inversion system was successfully expressed in Arabidopsis thaliana and tobacco protoplasts. Site-specific recombination was monitored both physically and biologically with the help of a recombination assay system in which expression of a beta-glucuronidase (gus) gene requires Gin-mediated recombination. We demonstrate that the wild-type Gin protein is not able to promote recombination in plant protoplasts, presumably because plant cells do not contain a protein that can substitute for the Escherichia coli FIS protein needed for full activity of wild-type Gin in E. coli. A FIS-independent Gin mutant protein on the other hand was efficient in promoting recombination on recombination substrates introduced transiently and on substrates stably integrated into the plant genome. We discuss the various advantages this system can provide for genetic manipulation of plant cells.

Bacteriophage mu↗

PubMed1994

Foreign DNA sequences are received by a wild-type strain of Aspergillus niger after co-culture with transgenic higher plants.

Different transgenic plants of Brassica napus, Brassica nigra, Datura innoxia and Vicia narbonensis expressing the hph gene under the control of the 35s promoter were co-cultivated with mycelial material of Aspergillus niger in microcosms under sterile conditions. A significantly higher number of hygromycin B-resistant colonies of re-isolated fungi was obtained if compared with co-cultures with non-transgenic plants. The hph gene and other foreign sequences could be detected in some of the resistant strains only for a short time after selection, indicating a rapid loss of foreign DNA. A more stable transgenic strain was obtained after co-culture with transgenic plants of D. innoxia including a high number of hph copies in their genome. DNA with detected pUC sequences was prepared to transform E. coli DH5 alpha. One of the recovered plasmids is shown to include pieces of the plant-transforming vector and a foreign sequence. The 35s-regulated expression of genes is studied in A. niger.

Aspergillus niger↗

PubMed1994

Nucleotide sequence of the tryptophan decarboxylase gene of Catharanthus roseus and expression of tdc-gusA gene fusions in Nicotiana tabacum.

The enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) converts tryptophan into tryptamine. In Catharanthus roseus and other plants capable of producing terpenoid indole alkaloids (TIAs) TDC links primary metabolism to the secondary metabolic pathway involved in the biosynthesis of these compounds. The accumulation of tdc mRNA in C. roseus cells is developmentally regulated and transcriptionally influenced by elicitors (induction) and auxins (repression). Here we report that TDC is encoded by a single copy gene in the C. roseus genome. No introns were observed upon isolation and sequencing of this gene. To study gene expression controlled by the tdc promoter, a 2 kb promoter fragment and a number of 5' deleted promoter derivatives were joined in translational fusion to a beta-D-glucuronidase reporter gene (gusA). Expression of the chimaeric constructs was monitored in stably transformed tobacco plants and in transiently transfected tobacco protoplasts. Histochemical and fluorimetric analysis of transgenic plants revealed that 1938 bp of the tdc promoter (with respect to the translational start codon) give rise to GUS activity in roots, stems and leaves. No tissue or cell type specificity was noted. Promoter deletions up to nucleotide -398 directed lower levels of gusA expression but conferred the same pattern of staining for GUS activity as the -1938 construct. Further deletion of the tdc promoter up to nucleotide -232 resulted in drastically reduced GUS activity levels and loss of GUS staining in all parts of the transgenic plants. In contrast to stable transformation, the -232 tdc-gusA construct gave rise to GUS activity levels comparable to those of the -398 construct in an assay system for transient expression in protoplasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

The endosperm-specific expression of a rice prolamin chimaeric gene in transgenic tobacco plants.

The 5' upstream region (-680 to +40), containing the potential promoter and complete signal peptide coding sequence of the rice seed storage prolamin gene was amplified in vitro using the polymerase chain reaction from the genome of Chinese rice cultivar Zhonghua 8. The physical map and DNA sequence analysis show strong homology with the 5' flanking region of the rice prolamin gene published by Kim and Okita (1988a). No change in the signal peptide coding sequence and a long leader sequence with several small open reading frames were found. The chimaeric gene containing the 5' flanking region of the prolamin gene (-680 to -18) was transcriptionally fused with the beta-glucuronidase (GUS) reporter gene and the fusion junction was confirmed by both physical mapping and DNA sequence analysis. The resultant chimaeric gene was used to transform tobacco explants, using the Ti binary system of Agrobacterium tumefaciens LBA4404. Three transgenic tobacco plants with as many as 20 copies of the chimaeric GUS gene (confirmed by dot and Southern hybridization) were analysed further. Histochemical analysis revealed GUS activity in the endosperm tissue of tobacco seed at the developmental stage about 20 days after flowering (DAF). No GUS activity was found in leaves, stems, roots and flowers of the transgenic tobacco plants. Therefore, we conclude that the 5' upstream region from -680 to -18 was sufficient to confer the endosperm-specific expression of the rice prolamin gene.

Base Sequence↗

PubMed1996

Repeated sequences from the Arabidopsis thaliana genome function as enhancers in transgenic tobacco.

Sixteen segments of Arabidopsis thaliana DNA that function as enhancers in transgenic tobacco plants were isolated using the pROA97 enhancer cloning vehicle and library transformation of Nicotiana tabacum. The sequences were compared for AT content, homology, repeated motifs, and expression pattern in transgenic N. tabacum. The sequences were average with respect to the AT content of A. thaliana DNA. They could be placed into seven homology groups. Five of the sequences are single-copy sequences. The remaining eleven sequences represent two homology groups. Homology Group I contains seven sequences with minor differences. Homology Group II contains four sequences with minor differences. Two repeated motifs were identified (5'-CCTCT-3' and 5'-AAGGAT-3'). Both repeated motifs are found in other plant enhancers, and in the promoter region of the cauliflower mosaic virus 35S gene. In the 35S gene TATA region, the motifs can form two alternative stem-loop structures. The TATATAA sequence is located in the loop region of both stem-loop structures.

Arabidopsis↗

PubMed1996

Evidence that the plastid signal and light operate via the same cis-acting elements in the promoters of nuclear genes for plastid proteins.

Nuclear-encoded genes for proteins of the photosynthetic machinery represent a particular subset of genes. Their expression is cooperatively stimulated by discrete factors including the developmental stage of plastids and light. We have analyzed in transgenic tobacco the plastid- and light-dependent expression of a series of 5' promoter deletions of various nuclear genes from spinach, of fusions of defined promoter segments with the 90-bp 35S RNA CaMV minimal promoter, as well as with mutations in sequences with homologies to characterized cis-elements, to address the question of whether the plastid signal and light operate via the same or different cis-acting elements. In none of the 160 different transgenic lines (representing 32 promoter constructs from seven genes) analyzed, could significant differences be identified in the responses to the two regulatory pathways. The data are compatible with the idea that both signals control the expression of nuclear genes for plastid proteins via the same cis-acting elements.

Gene Expression Regulation, Plant↗

PubMed2000

Leaf-specific overexpression of plastidic glutamine synthetase stimulates the growth of transgenic tobacco seedlings.

The impact of increased plastidic glutamine synthetase (GS-2; EC 6.1. 3.2) activity on foliar amino-acid levels and on biomass production was examined in transgenic tobacco. For that, tobacco was transformed via Agrobacterium tumefaciens with a binary vector containing a tobacco GS-2 cDNA downstream of the leaf-specific soybean ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promotor. Two transgenic tobacco lines with 15- to 18-fold higher foliar GS-2 transcript levels than the wild type were obtained. The GS-2 protein pools and the specific GS-2 activities were, however, only 2- to 2.3-fold higher in the leaves of the transgenic plants than in the leaves of the wild type. This discrepancy may reflect a post-transcriptional control of GS-2 protein accumulation. The increased GS-2 activity was correlated with a decrease in the leaf ammonium pool (3.7-fold) and an increase in the levels of some free amino acids, including glutamate (2. 5-fold) and glutamine (2.3-fold). The accumulation of soluble protein per unit fresh weight, however, remained unchanged. This result indicates that a process downstream of the synthesis of the primary organic products of N-assimilation is limiting leaf protein accumulation. Nevertheless, the overexpression of GS-2 stimulated the growth rate of the transgenic tobacco seedlings which, consequently, were larger (20-30% on a fresh-weight basis) than wild-type seedlings grown under identical conditions. This result suggests that GS-2 is the rate-limiting enzyme during biomass production in tobacco seedlings. The requirement for glutamate as the ammonium acceptor in the reaction catalysed by GS-2 may imply that there is co-regulation of GS-2 and ferredoxin dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) gene expression. Increased leaf GS-2 activity had, however, no influence on the foliar Fd-GOGAT protein abundance. This result suggests that in tobacco leaves, more Fd-GOGAT is present than required to meet the demands of primary ammonium assimilation and that there is no strong interdependence between GS-2 and Fd-GOGAT protein expression.

Amino Acid Oxidoreductases↗

PubMed2003

Genetic transformation of the figwort, Scrophularia buergeriana Miq., an Oriental medicinal plant.

Scrophularia buergeriana Miq. (figwort) contains a diverse group of bioactive natural products and is used to treat a variety of ailments, including fever, constipation, neuritis, and laryngitis. A transformation protocol was established for S. buergeriana using Agrobacterium tumefaciens. Kanamycin-resistant plants were regenerated from leaf explants co-cultivated with A. tumefaciens strain GV3101. The shoot regeneration medium was supplemented with 2 mg l(-1) 6-benzylaminopurine and 70 mg l(-1) putrescine to improve the efficiency of organogenesis. Detection of the neomycin phosphotransferase gene, the presence of high levels of beta-glucuronidase (GUS) transcripts and enzyme activity, and the histochemical localization of GUS confirmed the genetic transformation of S. buergeriana. This work demonstrates the potential of using A. tumefaciens to efficiently transfer foreign genes into a commercially and culturally important Oriental medicinal plant.

Agrobacterium tumefaciens↗

PubMed2006

Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures.

Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A(4)GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B(5) medium. Initially, 42 independent hairy root clones were maintained and seven clones belongs to different category were evaluated for growth, morphology, integration and expression of Ri T-DNA genes, and alkaloid contents in dry root samples. On the basis of total root elongation, lateral root density and biomass accumulation on solid media, hairy root clones were separated into three categories. PCR and Southern hybridization analysis revealed both left and right T-DNA integration in the root clones and RT-PCR analysis confirmed the expression of hairy root inducible gene. GUS assay was also performed to confirm the integration of left T-DNA. The accumulation of considerable amounts of the root-specific secoiridoid glucosides gentiopicroside was observed in GM1 (T +/L and T +/R) and the GM2 (T +/L and T -/R DNA) type clones in considerably higher amount whether as two T -/L but T +/R callus-type clones (GM3) accumulated much less or only very negligible amounts of gentiopicroside. Out of four media composition the 1/2 MS + B(5) vitamin media was found most suitable. We found that initial establishment of root cultures largely depends on root:media ratio. Maximum growth rate was recorded in 1:50 root:media ratio. The maximum biomass in terms of fresh weight (33-fold) was achieved in 1/2 MS + B(5) media composition after 35 days in comparison to sixfold increase in control. The biomass increase was most abundant maximum from 15 to 30 days. Influence of A. rhizogenes strains and Ri plasmid of hairy root induction, the possible role of the T(L)-DNA and T(R)-DNA genes on growth pattern of hairy root, initial root inoculum:media ratio and effect of media composition is discussed.

DNA, Bacterial↗

PubMed2001

Cytoplasmic kinase and phosphatase activities can induce PsaF gene expression in the absence of functional plastids: evidence that phosphorylation/dephosphorylation events are involved in interorganellar crosstalk.

PsaF is a nuclear gene for subunit III of the reaction center of photosystem I, and its expression is stimulated by cytokinins and light, when monitored at the mRNA level or at the level of GUS activity directed by chimeric promoter::uidA gene fusions in transgenic tobacco. These inductive effects can be mimicked by pertussis toxin, serotonin, phorbol acetate myristate or Ca2+, suggesting the involvement of heterotrimeric G proteins, phospholipids and Ca2+-dependent processes. Both breakdown products of the phosphatidylinositol cycle, inositol triphosphate (IP3) and diacylglycerol (or its homolog phorbol myristate acetate, PMA) appear to be involved. The IP3-dependent pathway requires kinase activity, and the signal operates via a 42-bp Ca2+-responsive element located between positions -220 and -178, while the PMA-dependent pathway requires phosphatase activity and a binding element that lies further upstream in the promoter. The effects of G proteins, phospholipids and Ca2+ on GUS gene expression are restricted to tissues with functional plastids, while modulation of phosphatase and kinase activities activates the responsive PsaF promoter regions even in photobleached material. Thus, activation of kinases and phosphatases can bypass the plastid-mediated inhibition of PsaF gene expression in tobacco seedlings. One cytoplasmic target which reflects the functional state of the plastids is protein kinase C. The enzyme can be efficiently phosphorylated in protein extracts from seedlings in which plastid function is impaired, but not in extracts from green tissue.

Calcium↗

PubMed1998

Multiple elements of the S2-RNase promoter from potato (Solanum tuberosum L.) are required for cell type-specific expression in transgenic potato and tobacco.

A functional analysis of the promoter of the S2-RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S2-RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S2 promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on the transformant, the predominant one being characterized by expression in the stigma and the transmitting tract of the style, whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S1-RNase and S2-RNase promoters, termed motif and motif III, are located in a fragment of the S2 promoter extending from position of -200 to bp -100, and motif II, located between by -498 and -480, was identified on the basis of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for pollen-specific expression. Two submotifs, A and B, were identified with the motif I. Both were essential for expression in the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence to motif I, it was not sufficient to confer-either pollen- or pistil-specific expression. However, deletion of motif III abolished pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs may be needed to specify cell type-specific expression. In transgenic potato the S2-RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than in tobacco, with most plants also exhibiting GUS expression in other issues.

Enhancer Elements, Genetic↗