Dynamics of IAA and cytokinins in flower tissues of transgenic tobacco mutant plants with mutant phenotype

A comparative analysis of IAA and total cytokinin contents at various developmental stages of floral morphogenesis (I–XII) in transgenic tobacco (Nicotiana tabacum L.) plants with mutant phenotypes was performed. Disturbances in flower structure were correlated with changes in the content and dynamics of these phytohormones. In plants with the phenotype “wavy corolla”, IAA content increased by three times as compared with control at stage VIII; in plants with the phenotype “longostyly”, IAA content increased gradually from VIII to XII stage and sharply (by nine times) at stage XII. In plants with the phenotype “wavy corolla”, total content of cytokinins was lower than in control at all developmental stages (except for stage I), whereas their dynamics coincided with that in control flowers. In plants with the phenotype “longostyly”, cytokinin content was twice lower than in control at early developmental stages, whereas it oscillated at later stages, approaching to control value at stage XII. It is supposed that differences observed could result from T-DNA insertion into plant own genes, which led to changes in the content and dynamics of phytohormones in developing flowers and production of anomalies in the flower structure.     

Expression of mutant patatin protein in transgenic tobacco plants: role of glycans and intracellular location.

The influence of N-glycosylation and subcellular compartmentation on various characteristics of a vacuolar glycoprotein is described. One member of the patatin gene family was investigated as a model system. Different glycosylation mutants obtained by destroying the consensus site Asn-X-Ser/Thr by oligonucleotide-directed mutagenesis were expressed in leaves of transgenic tobacco plants under the control of a light-inducible promoter. The various patatin glycomutants retained their properties in comparison with the wild-type protein with respect to protein stability, subcellular compartmentation, enzymatic activity, and various physicochemical properties studied showing the N-glycosylation not to be essential for any of these characteristics. To test the importance of the cotranslational transport and the subcellular (vacuolar) location for the properties of the patatin protein, another mutant was constructed in which the signal peptide was deleted, leading to its synthesis and accumulation in the cytosol. Biochemical analysis of this protein in comparison with its vacuolar form again revealed no significant differences with respect to its enzymatic activity or its stability in normal vegetative cells. During seed development, however, the cytoplasmic form was more stable than the vacuolar form, indicating the appearance of proteases specific for the protein bodies of developing seeds.     

Production of herbicide-resistant transgenic maize plants using electroporation of seed-derived embryos

The improvement of commercial maize lines via biotechnological approaches is limited by the lack of a transformation system that is tissue culture free. In this paper, the development of a genetic transformation system is presented using electroporation for gene delivery and seed-derived embryo as the gene target. Plasmid DNA (pBARGUS), which contained the selectablebar gene for resistance to the herbicide Basta and the screenablegus gene, was delivered into enzymatically wounded mature maize embryos via electroporation. Transformed plants were identified by their ability to grow on a selective medium containing 30 mg/L of phosphinothricin. Southern hybridization, plant resistance to the application of Basta, GUS expression, and segregation analysis indicated that a functionalbar gene had integrated into the maize genome and was inherited in a mendelian fashion by the progeny.     

Expression of tandem gene fusions in transgenic tobacco plants.

We have studied the expression of four sets of tandem gene fusions in transgenic tobacco plants. This was to determine if the problem of between-transformant variability in expression of introduced genes could be overcome by using a linked reference gene as a co-ordinately expressed control. Tandem gene fusions containing identical 5' flanking regions (SSU301-ocs with either SSU301-cat or SSU301-SSU911) were not co-ordinately expressed in the transgenic tobacco plants whereas the tandem gene fusions containing similar but not identical 5' flanking regions (SSU301-ocs with SSU911-cat or SSU911-SSU301) were co-ordinately expressed. The lack of co-ordinate expression of some of the tandem gene fusions appears to be partially explained by absence of the corresponding genomic DNA segments in the transgenic plants.     

N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants

Since plants are emerging as an important system for the expression of recombinant glycoproteins, especially those intended for therapeutic purposes, it is important to scrutinize to what extent glycans harbored by mammalian glycoproteins produced in transgenic plants differ from their natural counterpart. We report here the first detailed analysis of the glycosylation of a functional mammalian glycoprotein expressed in a transgenic plant. The structures of the N-linked glycans attached to the heavy chains of the monoclonal antibody Guy's 13 produced in transgenic tobacco plants (plantibody Guy's 13) were identified and compared to those found in the corresponding IgG1 of murine origin. Both N-glycosylation sites located on the heavy chain of the plantibody Guy's 13 are N-glycosylated as in mouse. However, the number of Guy's 13 glycoforms is higher in the plant than in the mammalian expression system. Despite the high structural diversity of the plantibody N-glycans, glycosylation appears to be sufficient for the production of a soluble and biologically active IgG in the plant system. In addition to high-mannose-type N-glycans, 60% of the oligosaccharides N-linked to the plantibody have beta(1, 2)-xylose and alpha(1, 3)-fucose residues linked to the core Man3GlcNAc2. These plant-specific oligosaccharide structures are not a limitation to the use of plantibody Guy's 13 for topical immunotherapy. However, their immunogenicity may raise concerns for systemic applications of plantibodies in human.     

Expression of a thermostable bacterial cellulase in transgenic tobacco plants

The bacterial gene of the thermostable endo-beta-1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased business and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.     

Cross protection in transgenic tobacco plants expressing a mild strain of tobacco mosaic virus

Summary Cross protection of plant viruses is a phenomenon in which plants infected with one strain of a virus are protected from the effects of superinfection by other related strains. Recently, we have succeeded in the introduction and expression of a cDNA copy of the tobacco mosaic virus (TMV) genomic RNA in transgenic tobacco plants. Using this system, we introduced a cDNA copy of a mild strain of TMV into tobacco plants. The transgenic plants did not develop any severe symptoms upon inoculation with a virulent TMV strain, indicating that these transgenic plants were cross protected against TMV infection. The system described here can be a useful model system to study the mechanism(s) of cross protection.     

Congruent evidence from alpha-tubulin and beta-tubulin gene phylogenies for a zygomycete origin of microsporidia

The origin of microsporidia and the evolutionary relationships among the major lineages of fungi have been examined by molecular phylogeny using alpha-tubulin and beta-tubulin. Chytrids, basidiomycetes, ascomycetes, and microsporidia were all recovered with high support, and the zygomycetes were consistently paraphyletic. The microsporidia were found to branch within zygomycetes, and showed relationships with members of the Entomophthorales and Zoopagales. This provides support for the microsporidia having evolved from within the fungi, however, the tubulin genes are difficult to interpret unambiguously since fungal and microsporidian tubulins are very divergent. Rapid evolutionary rates a characteristic of practically all microsporidian genes studied, so determining their evolutionary history will never be completely free of such difficulties. While the tubulin phylogenies do not provide a decisive conclusion, they do further narrow the probable origin of microsporidia to a zygomycete-like ancestor.     

Pathogen resistance of transgenic tobacco plants producing caffeine

Caffeine (1,3,7-trimethylxanthine) is a typical purine alkaloid, and produced by a variety of plants such as coffee and tea. Its physiological function, however, is not completely understood, but chemical defense against pathogens and herbivores, and allelopathic effects against competing plant species have been proposed. Previously, we constructed transgenic tobacco plants, which produced caffeine up to 5 microg per gram fresh weight of leaves, and showed them to repel caterpillars of tobacco cutworms (Spodoptera litura). In the present study, we found that these transgenic plants constitutively expressed defense-related genes encoding pathogenesis-related (PR)-1a and proteinase inhibitor II under non-stressed conditions. We also found that they were highly resistant against pathogens, tobacco mosaic virus and Pseudomonas syringae. Expression of PR-1a and PR-2 was higher in transgenic plants than in wild-type plants during infection. Exogenously applied caffeine to wild-type tobacco leaves exhibited the similar resistant activity. These results suggested that caffeine stimulated endogenous defense system of host plants through directly or indirectly activating gene expression. This assumption is essentially consistent with the idea of chemical defense, in which caffeine may act as one of signaling molecules to activate defense response. It is thus conceivable that the effect of caffeine is bifunctional; direct interference with pest metabolic pathways, and activation of host defense systems.     

Aluminum borate whisker-mediated production of transgenic tobacco plants

An aluminum borate whiskers-mediated transformation system for calluses of tobacco (Nicotiana tabacum, cv. SR-1) has been developed. A total of 50 small pieces of calluses were vigorously agitated in a liquid medium containing aluminum borate whiskers, pBI221 plasmid carrying the -glucuronidase (GUS) gene, and pBI222 plasmid carrying the hygromycin phosphotransferase (HPT) gene. After treatment, calluses were cultured to select for hygromycin resistance, and three resistant calluses were obtained. Adventitious shoots were produced from each hygromycin-resistant callus and were transferred to rooting medium. A total of three plantlets obtained from each hygromycin-resistant callus were acclimatized and established in soil. Polymerase chain reaction analysis revealed that all the plantlets were cotransformed with both the GUS and HPT genes. Detached leaves of transgenic individuals showed clear hygromycin resistance when cultured in liquid medium. Histochemical assay for GUS revealed that one of these transgenic plants expressed the GUS gene, indicating coexpression of foreign genes.     

Altered microtubule dynamics by expression of modified alpha-tubulin protein causes right-handed helical growth in transgenic Arabidopsis plants

The proper organization of cortical microtubule arrays is essential for anisotropic growth in plants but how distinct array patterns are formed is not understood. Here, we report a relationship between microtubule dynamics and array organization using transgenic plants expressing modified tubulins. When green fluorescent protein (GFP) or a hemaglutinin epitope tag was fused to the N-terminus of tubulins and expressed in Arabidopsis plants, these tubulins were incorporated into microtubules along with endogenous tubulins. Plants expressing the modified beta-tubulins were phenotypically normal and possessed transversely oriented cortical arrays in the epidermal cells of the root elongation zone; however, the expression of modified alpha-tubulins caused right-handed helical growth, increased trichome branching, and a shallow left-handed (S-form) helical array organization. In cells expressing the modified alpha-tubulins, microtubule dynamicity was suppressed and polymerization was promoted, and GFP-EB1 (End Binding 1) labeled larger regions of the microtubule end more frequently, when compared with control cells. We propose that the N-terminal appendage introduced into alpha-tubulin inhibits GTP hydrolysis, thus producing polymerization-prone microtubules with an extended GTP cap. Consistent with this interpretation, plants expressing an alpha-tubulin mutated in the GTPase-activating domain exhibited similar microtubule properties, with regard to dynamics and the localization of GFP-EB1, and showed right-handed helical growth.     

Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin.

Overexpression of alpha- and beta-tubulin genes in Saccharomyces cerevisiae, separately or together, leads to accumulation of large excesses of each of the polypeptides and arrest of cell division. However, other consequences of overexpression of these genes differ in several ways. As shown previously (D. Burke, P. Gasdaska, and L. Hartwell, Mol. Cell. Biol. 9:1049-1059, 1989), overexpression of beta-tubulin leads, at early times, to loss of microtubule structures and loss of viability. Eventually, the excess beta-tubulin forms abnormal structures. We show here that, in contrast, overexpression of alpha-tubulin led to none of these phenotypes and in fact could suppress each of the phenotypes associated with beta-tubulin accumulation. Truncated forms of beta-tubulin that were not competent to carry out microtubule functions also failed to elicit the beta-tubulin-specific phenotypes when overexpressed. The data support the hypothesis that beta-tubulin in excess over alpha-tubulin is uniquely toxic, perhaps because it interferes with normal microtubule assembly.     

The expression of a heat-inducible chimeric gene in transgenic tobacco plants

Summary A chimeric gene under the control of the hsp70 promoter of Drosophila is heat regulated in roots, stems and leaves, but not in pollen of transgenic tobacco plants. For these and other parameters, it behaves similarly to plant heat-shock genes.     

Expression of a functional human adenosine deaminase in transgenic tobacco plants

An inherited disorder, adenosine deaminase deficiency is a form of severe combined immunodeficiency, which is ultimately caused by an absence of adenosine deaminase (ADA), a key enzyme of the purine salvage pathway. The absence of ADA-activity in sufferers eventually results in a dysfunctional immune system due to the build-up of toxic metabolites. To date, this has been treated with mixed success, using PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. It is likely, however, that an enzyme replacement therapy protocol based on recombinant human ADA would be a more effective treatment for this disease. Therefore, as a preliminary step to produce biologically active human ADA in transgenic tobacco plants a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and both human and TMV 5′ UTR control regions. Plant vector expression constructs have been used to transform tobacco plants via Agrobacterium-mediated transformation. Genomic DNA, RNA and protein blot analyses have demonstrated the integration of the cDNA construct into the plant nuclear genome and the expression of recombinant ADA mRNA and protein in transgenic tobacco leaves. Western blot analysis has also revealed that human and recombinant ADA have a similar size of approximately 41 kDa. ADA-specific activities of between 0.001 and 0.003 units per mg total soluble protein were measured in crude extracts isolated from transformed tobacco plant leaves.