Combined expression of the Arabidopsis metallothionein MT2b and the heavy metal transporting ATPase HMA4 enhances cadmium tolerance and the root to shoot translocation of cadmium and zinc in tobacco

We expressed the AtMt2b and AtHMA4 genes under the 35S cauliflower mosaic virus promoter simultaneously in Nicotiana tabacum (SR1), using leaf disc transformation. A single AtMT2b tobacco T2 line was used for re-transformation with AtHMA4 to obtain the double transformant. Cadmium (Cd) and zinc (Zn) tolerance, uptake and translocation were measured in the double transformant, and compared to untransformed (‘wild type’) tobacco and single gene transformants. The double transformant exhibited enhanced Cd-tolerance, enhanced Cd and Zn root to shoot transport, but unaltered Zn tolerance and Cd and Zn uptake, compared with wild type.The single transformant lines did not show significant phenotypes. Our results suggest that the phenotypes of the double transformant are due to synergistic interaction between the transgenes. Except for Cd tolerance, the phenotypes were moderate for Cd and Zn root to shoot transport, which may be due to use of the 35S promotor, resulting in incorrect tissue-specificity.     

Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression

In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells [45, 46]. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5 regions were translationally fused with the -D-glucuronidase reporter gene (GUS). The GNT1 5 region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5 regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.Department of Plant Molecular Biology, Leiden University     

Quantitative determination of mosaic <Emphasis Type="Italic">GFP</Emphasis> gene expression in tobacco

A specific form of gene silencing that was observed visually as a mosaic distribution of fluorescent and non-fluorescent cells apparently dispersed at random within tissues was found in a few green fluorescent protein (GFP)-transformed tobacco lines. To characterize this event quantitatively, we studied flow cytometric measurements in GFP-expressing and -silenced cells in T₁ and T₂ progeny of four selected plants. The proportion of silenced cells varied considerably among the T₁ lines but with notable genotype differences. Mosaic expression was inherited into the T₂ generation in which the majority of progenies tested exhibited a level of silencing similar to that of their T₁ parental plants. However, in some T₂ progenies segregation, evident as a decrease or increase in the proportion of fluorescent cells, was observed. We discuss several factors, such as copy number, promoter activity or polyploidy, that may be the possible causes of the gene silencing, but none sufficiently explain the appearance of the mosaic distribution.     

Changes with time in the distribution of virus and PR protein around single local lesions of TMV infected tobacco

Summary ELISA was used to determine PR la protein and TMV accumulation in local necrotic lesions produced on salicylic acid and water sprayed Nicotiana tabacum cv Xanthi-nc leaves. The amount of PR la protein produced is the result of an interaction between the salicylic acid treatment and lesion growth. The implication of these observations for experiments investigating the relationship between PR proteins and resistance are discussed.The distribution of TMV and PR la protein in and around single local necrotic lesions up to 14 days after inoculation was measured by ELISA. The highest concentration of TMV was in the centre of the lesion and decreased rapidly with distance from the centre. In contrast there was very little PR la protein in the centre of the lesion, the largest amounts were just outside the centre, and the concentration then decreased with distance from the centre. This is the distribution that might be expected for a substance closely associated with the restriction of virus spread.     

Quantitative transient gene expression: Comparison of the promoters for maize polyubiquitin1, rice actin1, maize-derivedEmu andCaMV 35S in cells of barley,maize and tobacco

The particle gun approach was used for the quantification of promoter efficiency in a test system for transient gene expression. β-Glucuronidase was used as reporter gene for determining promotote strength. The variability inherent in this gene transfer system was considerably reduced by calculating a transformation efficiency factor given by the expression of a cotransferred second reporter gene (firefly luciferase). The calibration of β-glucuronidase activity by the transformation efficiency factor caused a lower statistical variance of the values and allowed reliable results to be obtained with a smaller set of repetitions. The CaMV 35S promoter (as a control) and the monocot-specific promoters for maize polyubiquitin1, rice actin 1 and the maize-derivedEmu were characterized and compared with respect to expression strength, as tested under identical conditions in suspension cell cultures of maize, barley and tobacco. Compared to the 35S promoter, the monocot-specific promoters show up to 15-fold higher expression in maize and barley but give only weak expression in tobacco. No expression was found for the rice actin 1 promoter in tobacco. The level of reporter gene expression is influenced by the osmotic potential in the agar medium. For theEmu promoter, the calibrated β-glucuronidase activities remained mearly constant at low sucrose concentrations. Above 8% sucrose, the calibrated activities increased steadily with increasing osmotic conditions, reaching a three-to four-fold higher level at the highest sucrose concentration (32%) as compared to the standard concentration (4% sucrose) in the medium.     

Apple polygalacturonase inhibiting protein1 expressed in transgenic tobacco inhibits polygalacturonases from fungal pathogens of apple and the anthracnose pathogen of lupins

Extracts from apple fruit (cultivar "Granny Smith") inhibited the cell-wall degrading polygalacturonase (PG) activity of Colletotrichum lupini, the causal agent of anthracnose on lupins, as well as Aspergillus niger PG. Southern blot analysis indicated that this cultivar of apple has a small gene family of polygalacturonase inhibiting proteins (pgips), and therefore heterologous expression in transgenic tobacco was used to identify the specific gene product responsible for the inhibitory activity. A previously isolated pgip gene, termed Mdpgip1, was introduced into tobacco (Nicotiana tabacum) by Agrobacterium-mediated transformation. The mature MdPGIP1 protein was purified to apparent homogeneity from tobacco leaves by high salt extraction, clarification by DEAE-Sepharose and cation exchange HPLC. Purified MdPGIP1 inhibited PGs from C. lupini and PGs from two economically important pathogens of apple trees, Botryosphaeria obtusa and Diaporthe ambigua. It did not inhibit the A. niger PG, which was in contrast to the apple fruit extract used in this study. We conclude that there are at least two active PGIPs expressed in apple, which differ in their charge properties and ability to inhibit A. niger PG.     

Use of a simple semiquantitative method for appraisal of green fluorescent protein gene expression in transgenic tobacco plants

We have applied a simple method for evaluation of gfp gene expression in plants using a CCD camera and computerized processing of images. Transgenic tobacco plants were obtained by Agrobacterium tumefaciens-mediated transfer of plasmid T-DNA bearing a m-gfp5-ER sequence governed by the 35S promoter together with the nptII selectable marker gene. Presence of the gfp gene in plants was confirmed by a polymerase chain reaction method. Mean brightness values measured using image analysis software showed differences between transgenic and control plants and suggest the possibility of rapid selection of transgenic individuals among regenerants and their progenies.     

Expression of phosphinothricin acetyltransferase from the root specific par promoter in transgenic tobacco plants is sufficient for herbicide tolerance

Summary The pat gene, coding for phosphinothricin acetyltransferase (PAT) from Streptomyces viridochromogenes, was cloned behind the par promoter of the hemoglobin gene from Parasponia andersonii, Introduction into tobacco (Nicotiana tabacum) resulted in predominantly root specific PAT expression. Application of 5 l/ha BASTA^® (herbicidal component: phosphinothricin) did not effect growth morphology and vigor of the plants. After application of 20 l/ha BASTA^® the plants showed herbicide damage. Nevertheless, they all recovered by forming new undamaged leaves and resumed full growth despite virtually non-detectable expression of the PAT enzyme in the leaves.Abbreviations BAP 6-benzylaminopurine - CaMV Cauliflower Mosaic Virus - IAA indole-3-acetic acid - kb kilobases - LB Luria-Bertani - MS Murashige and Skoog - par Parasponia andersonii - PAT phosphinothricin acetyltransferase - ppt phosphinothricin - TCA trichloric acid     

Phytase activity in tobacco (Nicotiana tabacum) root exudates is exhibited by a purple acid phosphatase

Phytases are enzymes that catalyze liberation of inorganic phosphates from phytate, the major organic phosphorus in soil. Tobacco (Nicotiana tabacum) responds to phosphorus starvation with an increase in extracellular phytase activity. By a three-step purification scheme, a phosphatase with phytase activity was purified 486-fold from tobacco root exudates to a specific activity of 6,028 nkat mg(-1) and an overall yield of 3%. SDS-PAGE revealed a single polypeptide of 64 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography suggested that the enzyme was a ca. 56 kDa monomeric protein. De novo sequencing by tandem mass spectrometry resulted in a tryptic peptide sequence that shares high homology with several plant purple acid phosphatases. The identity of the enzyme was further confirmed by molybdate-inhibition assay and cDNA cloning. The purified enzyme exhibited pH and temperature optima at 5.0-5.5 and 45 degrees C, respectively, and were found to have high affinities for both p-nitrophenyl phosphate (pNPP; K(m)=13.9 microM) and phytate (K(m)=14.7 microM), but a higher kcat for pNPP (2,056 s(-1)) than phytate (908 s(-1)). Although a broad specificity of the enzyme was observed for a range of physiological substrates in soil, maximum activity was achieved using mononucleotides as substrates. We conclude that the phytase activity in tobacco root exudates is exhibited by a purple acid phosphatase and its catalytic properties are pertinent to its role in mobilizing organic P in soil.     

cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

Distribution dynamics of the Tnt1 retrotransposon in tobacco

Retrotransposons contribute significantly to the size, organization and genetic diversity of plant genomes. Although many retrotransposon families have been reported in plants, to this day, the tobacco Tnt1 retrotransposon remains one of the few elements for which active transposition has been shown. Demonstration that Tnt1 activation can be induced by stress has lent support to the hypothesis that, under adverse conditions, transposition can be an important source of genetic variability. Here, we compared the insertion site preference of a collection of newly transposed and pre-existing Tnt1 copies identified in plants regenerated from protoplasts or tissue culture. We find that newly transposed Tnt1 copies are targeted within or close to host gene coding sequences and that the distribution of pre-existing insertions does not vary significantly from this trend. Therefore, in spite of their potential to disrupt neighboring genes, insertions within or near CDS are not preferentially removed with age. Elimination of Tnt1 insertions within or near coding sequences may be relaxed due to the polyploid nature of the tobacco genome. Tnt1 insertions within or near CDS are thus better tolerated and can putatively contribute to the diversification of tobacco gene function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.