Inge Broer

Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum

The phosphinothricin (Pt) N-acetyltransferase gene (pat) of Streptomyces viridochromogenes Tü494 is located on a 0.8-kb BglII fragment [Strauch et al., Gene 63 (1988) 65-74]. By sequencing a 1.3-kb BglII-SstII fragment, an open reading frame representing the pat gene was found. It encodes a polypeptide of 183 amino acids with an Mr of 20,621. The base composition of the pat gene is typical for Streptomyces [70.1 mol% (G + C) in total and 93.5 mol% (G + C) in the third position]. Translation of pat is initiated by a GTG codon which was identified by frameshift mutations in Escherichia coli as well as in Streptomyces lividans. Significant homology of the pat gene was found to the bialaphos-resistance gene (bar) of Streptomyces hygroscopicus [Thompson et al., EMBO J. 9 (1987) 2519-2523]. However, variations were detected in the 5-noncoding region of the two resistance genes which may reflect differences in regulation. Since Pt is a potent herbicide, the pat gene was modified and introduced into Nicotiana tabacum by Agrobacterium-mediated leaf-disc transformation. The GTG start codon of pat was replaced by ATG. Subsequently the modified pat-coding region was fused to the 35S promoter of the cauliflower mosaic virus. Transgenic plants could directly be selected on Pt-containing medium.

TRANSGENIC PLANTS CONTAINING THE PHOSPHINOTHRICIN-N-ACETYLTRANSFERASE GENE METABOLIZE THE HERBICIDE L-PHOSPHINOTHRICIN (GLUFOSINATE) DIFFERENTLY FROM UNTRANSFORMED PLANTS

Abstractl-Phosphinothricin (l-Pt)-resistant plants were constructed by introducing a modified phosphinothricin-N-acetyl-transferase gene (pat) via Agrobacterium-mediated gene transfer into tobacco (Nicotiana tabacum L), and via direct gene transfer into carrot (Daucus carota L). The metabolism of l-Pt was studied in these transgenic, Pt-resistant plants, as well as in the untransformed species. The degradation of l-Pt, 14C-labeled specifically at different C-atoms, was analysed by measuring the release of 14CO2 and by separating the labeled degradation products on thin-layer-chromatography plates. In untransformed tobacco and carrot plants, l-Pt was deaminated to form its corresponding oxo acid 4-methylphosphinico-2-oxo-butanoic acid (PPO), which subsequently was decarboxylated to form 3-methylphosphinico-propanoic acid (MPP). This compound was stable in plants. A third metabolite remained unidentified. The l-Pt was rapidly N-acetylated in herbicide-resistant tobacco and carrot plants, indicating that the degradation pathway of l-Pt into PPO and MPP was blocked. The N-acetylated product, l-N-acetyl-Pt remained stable with regard to degradation, but was found to exist in a second modified form. In addition, there was a pH-dependent, reversible change in the mobility of l-N-acetyl-Pt thin-layer during chromatography.

High frequency, heat treatment-induced inactivation of the phosphinothricin resistance gene in transgenic single cell suspension cultures of Medicago sativa

SummaryOne descendant of the Medicago sativa Ra-3 transformant T304 was analysed with respect to the somatic stability of the synthetic phosphinothricin-N-acetyltransferase (pat) gene which was used as a selective marker and was under the control of the 5′/3′ expression signals of the cauliflower mosaic virus (CaMV) gene VI. In order to quantify gene instability, we developed a system for culturing and regenerating individual cells. Single cell suspension cultures derived from T304 and the ancestral non-transgenic M. sativa cultivar Ra-3, were established. The cells were regenerated into monoclonal calli. In transgenic calli, the phosphinothricin (Pt)-resistance phenotype was retained after more than 2 months of non-selective growth. In contrast, up to 12% of the suspension culture cells grown under non-selective conditions and at constant temperature (25° C) lost the herbicide-resistance phenotype within 150 days. Surprisingly, a heat treatment (37° C), lasting for 10 days, during the culture period resulted in an almost complete (95%) loss of the Pt resistance of the suspension culture cells. However, the frequency of cell division was identical in cultures grown under normal and heat treatment conditions. A biochemical test revealed that no phosphinothricin-N-acetyltransferase activity was present in heat treated, Pt-sensitive cells. The resistance level of the Pt-sensitive transgenic cells was equivalent to that of the wild-type cells. A PCR analysis confirmed the presence of the pat gene in heat treated, Pt-sensitive cells. From these results it is concluded that the Pt resistance gene was heat-inactivated at a high frequency in the M. sativa suspension cultures.

A 200 bp region of the pea ENOD12 promoter is sufficient for nodule-specific and nod factor induced expression.

ENOD12 is one of the first nodulin genes expressed upon inoculation with Rhizobium and also purified Nod factors are able to induce ENOD12 expression. The ENOD12 gene family in pea (Pisum sativum) has two members. A cDNA clone representing PsENOD12A [26] and a PsENOD12B genomic clone [7] have been previously described. The isolation and characterization of a PsENOD12A genomic clone is presented in this paper. By using a Vicia hirsuta-Agrobacterium rhizogenes transformation system it is shown that both genes have a similar expression pattern in transgenic V. hirsuta root nodules. Promoter analyses of both PsENOD12 promoters showed that the 200 bp immediately upstream of the transcription start are sufficient to direct nodule-specific and Nod factor-induced gene expression.

The promoter of the Vicia faba L. VfENOD-GRP3 gene encoding a glycine-rich early nodulin mediates a predominant gene expression in the interzone II-III region of transgenic Vicia hirsuta root nodules

We recently reported on the broad bean gene VfENOD-GRP3 encoding a glycine-rich early nodulin. This gene was predominantly expressed in the interzone II–III region of Vicia faba root nodules. The VfENOD-GRP3 promoter contained several sequence motifs potentially involved in the regulation of gene expression. To investigate the molecular basis for the specific VfENOD-GRP3 expression, defined VfENOD-GRP3 promoter fragments were fused to an intron-containing gusAint gene. Agrobacterium rhizogenes ARqual strains carrying these fusions integrated into the TL DNA were used to generate hairy roots on Vicia hirsuta, which subsequently were nodulated. Histochemical analysis of transgenic nodules indicated that a strong gusAint expression in the interzone II–III region was mediated by the −1252/ + 10 VfENOD-GRP3 promoter region. This reporter gene expression in V. hirsuta was comparable to the location of VfENOD-GRP3 transcripts in V. faba nodules. An analysis of defined promoter fragments revealed that a strong gusAint expression in the interzone II–III region was also mediated by the −737/ + 10 promoter, whereas the −239/ + 10 promoter only mediated a weak gusAint expression in the interzone II–III region. Since the −239/ + 10 promoter fragment did not resemble published nodulin gene promoters, we propose that it contains new sequence motifs involved in mediating gene expression in the interzone II–III region of vicia nodules.

Stress inactivation of foreign genes in transgenic plants

Abstract The inactivation of endogenous plant genes during periods of stress or environmental changes has already been demonstrated. Although the expanding use of transgenic plants in scientific research has given hints for transgene inactivation caused by transinactivation, the first inactivation of transgene-encoded proteins caused by environmental changes was observed not before 1990. Transgenic Petunia hybrida plants, carrying the maize A1 cDNA under the control of the CaMV35S RNA promoter causing a salmon red flower phenotype, were released to the field. Environmental factors, possibly including heat, lead to a stable loss of flower pigmentation, accompanied by the methylation of the viral promoter. In the same year, the inactivation of the phosphinothricin resistance gene in single-cell cultures of a transgenic Medicago sativa line was observed in more than 90% of the cells after a 10-day heat treatment. In transgenic tobacco, different transgenes could be inactivated by a heat treatment (37°C), which did not lead to changes in the growth of the plant. These inactivations seem to be not correlated with methylation. The heat-induced inactivation of herbicide resistance genes led to sensitivity to the herbicide during the heat treatment. The transgene activity was regained after the temperature decreased to normal cultivation conditions. To date, heat-induced transgene inactivation has not been reported in the field, but it may give rise to problems if it does occur. Therefore, strategies to analyse the causes of heat-induced transgene inactivation and to circumvent the problem have to be developed.

TISSUE-SPECIFIC ACTIVITY AND LIGHT-DEPENDENT REGULATION OF A SOYBEAN RBCS PROMOTER IN TRANSGENIC TOBACCO PLANTS MONITORED WITH THE FIREFLY LUCIFERASE GENE

Abstract The spacial and temporal promoter activity of the soybean rbcS gene SRS1 in transgenic tobacco plants was investigated. A 5′-fragment of the gene was fused to the coding region of the North American firefly luciferase (luc), to function as a reporter gene. The hybrid gene was introduced into Nicotiana tabacum by Agrobacterium-mediated leaf disk transformation and transgenic plants were regenerated. In planta and in vitro luciferase assays demonstrated that the activity of the soybean rbcS promoter in the heterologous tobacco background not only remained organ-specific, but was also stimulated by light. This is the first report demonstrating the suitability of the firefly luciferase reporter gene to investigate the temporal expression pattern of an environmentally stimulated plant gene.

Identification of the Agrobacterium tumefaciens C58 T-DNA genes e and f and their impact on crown gall tumour formation

DNA sequence analysis of the 4.4 kilobases (kb) Eco RI fragment 14 from T-DNA of Agrobacterium tumefaciens C58 revealed three open reading frames. One of them (945 bp) was supposed to encode the transcript e, the function of which has not been identified to date. Furthermore, a so far undescribed open reading frame (1035 bp) was identified, located in the centre of the Eco RI fragment 14 and termed gene f. The third open reading frame encoded the carboxy-terminal part of the agrocinopine synthase (Acs). The gene e-encoded protein showed significant homologies to the gene products of the Agrobacterium rhizogenes rolB gene and the Agrobacterium tumefaciens gene 5. Both gene products are supposed to regulate the plants reaction on auxin. Depending on the plant species tested, Agrobacterium strains carrying mutations in gene e induced only small or almost no detectable crown gall tumours. According to these mutational studies and the protein homologies observed, the gene e product is suggested to be involved in tumour formation. Infection of several plant species with Agrobacterium carrying a mutated gene f, as well as expression of the gene f in transgenic tobacco plants did not lead to visible morphological changes. Therefore, in contrast to gene e, the gene f seems not to be essential for tumour formation. In order to study whether gene f is an active gene, its expression in agrobacteria and plants was monitored by translational lacZ fusion. In planta, the putative gene f-promoter mediates a tissue-specific expression pattern. Although gene f was expressed in free-living agrobacteria as well as in transgenic plants, the function of the f locus remained unclear. DNA homology studies with the f gene region revealed a mosaic-like DNA structure, indicating that this locus might be the result of genetic exchanges between different Agrobacterium strains during evolution.

The heat-treatment induced reduction of the pat gene encoded herbicide resistance in Nicotiana tabacum is influenced by the transgene sequence

Summary After 10 days of cultivation at 37 °C, the herbicide resistance encoded by the chimaeric pat 4l gene (coding region from Streptomyces viridochromogenes fused to the 823 bp CaMV35S promoter) was strongly reduced in all of the 27 independent transgenic Nicotiana tabacum SRI lines analyzed. This reversible reduction occurred in sterile and unsterile culture in the first and second generation and even when the overnight temperature was reduced to 24 °C. Neither the enzyme activity, the protein nor the pat 4l specific RNA could be detected in the heat treated plants, regardless of the number of copies and the hemior homozygous state. In contrast to this, the expression of the synthetic pat S coding region fused to the 534bp CaMV35S promoter and coding for essentially the same protein, was stable in heat treated plants. The exchange of the GC rich coding region of the pat 4l gene by the AT rich synthetic DNA fragment carrying the pat S coding region led to the stabilization of the specific RNA steady state level. However, the presence of the transgene-encoded protein at 37 °C could only be achieved by using specific 5′ and 3′ untranslated regions of the synthetic patS gene.

Heat-induced loss of transgene activity detected and analysed in several transgenic Nicotiana tabacum lines.

Heat treatment (37 °C) of various transgenic tobacco (Nicotiana tabacum) plants obtained from three different transformation experiments resulted in a temporary loss of transgene activity. During the heat treatment, almost no transgene-encoded protein is detectable. In this work, the influence of methylation on the observed reaction has been analysed applying two different approaches: Firstly, in vivo incubation with 5-azacytidine was carried out by various methods to induce a widespread demethylation in the plant genome. Secondly, Southern analysis with methylation-sensitive restriction endonucleases was performed using DNA isolated from heat-treated and non-heat-treated plants, respectively. With this method, the methylation status of single C residues could be examined. However, no evidence could be obtained that DNA methylation is involved in the heat-induced strong reduction of the transgene-encoded phenotype.