Maria Szwacka

Variable properties of transgenic cucumber plants containing the thaumatin II gene from Thaumatococcus daniellii

Thaumatin represents a unique class of the sweet-tasting plant proteins. Transgenic cucumber (Cucumis sativus L.) plants with stable integrated constructs consisting of the cauliflower mosaic virus 35S promoter and thaumatin II cDNA were produced. Transformed cucumber plants were obtained using Agrobacterium tumefaciens, with one, two or five integration sites in diploid cucumber and with inheritance confirmed by a 3:1 Mendelian ratio and normal morphologies and viable seeds. Inter- and intra-transformant variabilities in the expression of the thaumatin II gene were observed. The variability was independent of integrated copy number of the T-DNA. Variation in thaumatin II protein accumulation levels in the ripe fruits and the lack of correlation between protein and mRNA levels were observed, suggesting that thaumatin may be controlled at the levels of both transcription and translation. Transgenic fruits accumulating thaumatin II protein exhibited sweet phenotype and positive correation between thaumatin accumulation levels and sweet taste intensity was noticed. Thaumatin II belongs to the pathogenesis-related (PR) proteins family. Some of the T2 progeny plants expressing thaumatin II protein did not exhibit tolerance for pathogenic fungus Pseudoperonospora cubensis. These results, together with previously reported results, suggest no relationship between transgenic protein levels and the increased tolerance phenotype.

Aroma Evaluation of Transgenic, Thaumatin II‐Producing Cucumber Fruits

Fruits of transgenic cucumber lines expressing preprothaumatin II gene were evaluated concerning their aroma. Four homozygous lines, that is, 210 06, 212 01, 224 09, and 225 03 with different levels of transgene expression were selected. Recipient line cv. Borszczagowski, which was formed by inbred line of Cucumis sativus L., was used as a control. The experiment was carried out in a greenhouse and an outdoor experimental plot. The aroma of cucumber fruits was evaluated by GC/MS, as well as GC/MS/TOF in the distillates and by SPME. Irrespective of the isolation/separation technique used, the differences between aroma compounds in transgenic cucumbers and the control were quantitative, and not qualitative. Modified samples showed higher concentrations of volatiles, particularly of the main cucumber fruits odorant (E, Z)-2,6 nonadienal. Transgenic expression of the thaumatin II gene resulted not only in a sweeter taste of fruits in comparison with the control, but also higher aroma acceptability. This was shown by sensory profile analysis. Also electronic nose measurements differentiated between transgenic lines and the control.

Expression pattern of the pre-prothaumatin II gene under the control of the CaMV 35S promoter in transgenic cucumber [Cucumis sativus L.] flower buds and fruits

Thaumatin II is an extremely sweet-tasting protein produced by fruits of the West African shrubThaumatococcus daniellii Benth, so it can be used in biotechnology to improve the tastes of various plant products. This study is concerned with the spatial and temporal aspects of expression of the 35S-pre-prothaumatin II chimeric gene in flower buds and fruits of transgenic cucumber (Cucumis sativus L.) line 225. The activity of the 35S promoter in organs of line 225 was compared with its activity in 2 other transgenic lines. The accumulation of recombinant thaumatin varied spatially in flower bud tissues of transgenic lines. We found that these differences in the spatial accumulation of transgenic protein concerned the ovary of female buds and the perianth of male buds. In contrast to flower parts, recombinant thaumatin was found in nearly all parts of the young fruit from the transgenic plants. The pre-prothaumatin II gene expression was detected at a very early developmental stage in male buds, and its pattern was rather conserved as the buds aged. The expression of the transgene was also detected in vascular tissues of examined organs but was undetectable in pollen grains, in agreement with the generally held view that the CaMV 35S promoter is virtually silent in pollen. Immunocytochemical analyses of sections of control organs revealed endogenous homolog(s) of thaumatin when using polyclonal antisera, but not when using monoclonal antibodies for recombinant thaumatin detection in transgenic cucumber.

Tobacco PR-2d promoter is induced in transgenic cucumber in response to biotic and abiotic stimuli

The PR-2d promoter/uidA (GUS) gene construct was introduced into the cucumber (Cucumis sativus L.) genome and several transgenic lines were produced. Activation of the PR-2d promoter was investigated in these plants in response to inoculation with fungal pathogens and after salicylic acid (SA) or cold treatments. Treatment with exogenous SA increased GUS activity 2 to 11 fold over that of the control. Endogenous SA and its conjugate salicylic acid glucoside (SAG) rose in parallel after inoculation with the fungal pathogen Pseudoperonospora cubensis, with SAG becoming the predominant form. The free SA levels increased 15 fold above the basal level at 5 dpi and preceded the induction of the PR-2d promoter by five days, which occurred at 10 dpi with a 12 fold increase over the control. Inoculation with another fungal pathogen, Erysiphe polyphage, increased GUS activity 4 to 44 fold over that of the control. During normal development of flowers in the cucumber, the PR-2d/uidA gene expressed in the floral organs was similar to that of the primary host. In addition, we present the first evidence that the PR-2d promoter was induced (624 fold) under cold stress. We demonstrate that in the heterologous state the gene construct was expressed according to the signalling pattern of the native species and was stably transmitted to progeny over four generations.

Leaf morphology and anatomy of transgenic cucumber lines tolerant to downy mildew.

The objective of the present paper was to investigate the reason of increased tolerance to the pathogenic fungus Pseudoperonospora cubensis found in transgenic cucumber (Cucumis sativus L.) lines 210 and 212 bearing 35S:cDNA preprothaumatin II gene construct. The tolerance investigation was accomplished by comparing the morphological and anatomical structure of plant leaves. The results obtained demonstrate that leaves of both lines exhibited some anatomical and morphological characteristics (e.g. wax load and composition, cuticle ultrastructure, ultrastructure of secondary wall, arrangement of mesophylll cells) which may be responsible for enhanced tolerance.

The role of thaumatin II in cucumber resistance against Tetranychus urticae Koch: laboratory and greenhouse evaluation.

The role of thaumatin II in cucumber resistance against Tetranychus urticae Koch: laboratory and greenhouse evaluation The role of thaumatin II in the resistance of transgenic cucumber plants of the T6 generation against the two-spotted spider mite (TSSM) (Tetranychus urticae C. L. Koch 1836, Acari: Tetranychidae) was determined in greenhouse experiments evaluating: (1) mite behaviour towards cucumber lines (T224 09, T212 01, T210 06) differing in the level of thaumatin II expression, (2) mite reproductive capacity, (3) mite colonisation ability, and (4) mite leaf damage. Additionally, using Western blot, the presence of thaumatin II in leaves of transgenic cucumber plants of the T6 generation was analysed before and after TSSM feeding. Even plants with a relatively high leaf thaumatin II level (T224 09 and T212 01) were equally well accepted by TSSM in a ‘free-choice’ test, which excludes thaumatin II as an important factor in the process of host plant acceptance. However, a reduced fecundity of mite females and a lower, than in the control, number of mites found in the period of permanent feeding on thaumatin-rich plants, indicate a putative role of thaumatin II in the plant-mite interactions. The lack of a clear relationship between the level of thaumatin II and plant resistance to TSSM may be a sign that thaumatin II is not a direct factor involved in antibiosis. In response to TSSM, the thaumatin II content decreases, increases, or remains unchanged in mite-infested leaves, which excludes thaumatin II as being directly involved in induced defence.

Thaumatin expression in transgenic cucumber plants

Thaumatin is produced in the African plant known Thaumatococcus daniellii as a family of five or more proteins [Higginbotham, Hough, 1977]. Thaumatins are nearly 100000 times sweeter than sucrose on a molar basis [Iyengar et al., 1979]. Taumatins: I and II are the most abundant in fruit, and the cDNA for thaumatin II has been cloned and sequenced [Edens et al., 1982]. So thaumatin is a potentially interesting target for genetic engineering studies and we have recently begun work in this direction developing Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) with thaumatin II gene. We used an efficient plant regeneration procedure described by Burza and Malepszy [1995]. Transformation was carried out using the binary plasmid pRUR528s bearing sense construct of T. daniellii thaumatin II cDNA under the control of constitutive promoter 35S CaMV [Szwacka et al., 1996]. Transformation status was confirmed by PCR amplification of nptII gene. Genomic DNA hybridization experiments proved that independent primary transformants carried one to three copies of the expression cassette. Here, we present data on thaumatin mRNA transcription in independent primary transformants (Fig. 1A). We observed marked differences in the level of thaumatin transcript. Western blot analysis (Fig. 1B) indicated the presence of mature thaumatin (22 kDa) [Vos de, 1985] in five from fifteen analysed primary transformants. In two of them (no. 214 and no. 224) high level of thaumatin mRNA correlated with high level of mature thaumatin (Fig. 1A and 1B).

Biological significance, computational analysis, and applications of plant microRNAs

AbstractmicroRNA molecules belong to a class of small non-coding RNAs composed of 21–24 nucleotides and have been identified in most eukaryotes. These small RNA molecules can either transcriptionally or post-transcriptionally regulate expression of their target messenger RNAs. Access to the latest RNA-profiling technologies (e.g. high-throughput sequencing) in combination with computational analysis has contributed to rapid development in the field of miRNA research. Species-specific and highly conserved miRNAs’ control in plants biological processes. Nevertheless, regulatory functions of plant miRNAs have not been still fully understood. Hence, one of the major challenges in plant miRNA research is to find out their regulatory activities that may create an opportunity to develop new strategies for improving crops. This paper provides an overview of the current knowledge concerning the mechanisms related to plant gene regulation via miRNAs. Moreover, it includes an updated overview on the bioinformatic approaches that are available for identification of new miRNAs and their targets. It also includes some specific data on key functions of plant miRNAs to show potential impact of such small RNA molecules on diverse biological processes and their biotechnological significance. Current challenges and future perspectives have also been highlighted.

Nutritive and Dietetic Value of Genetically-Modified Tomatoes Expressing Thaumatin Gene

Abstract Genetically-modified (GM) tomatoes, carrying thaumatin gene encoding sweet-tasting protein may be a component of diet with high sensory values, constituting a valuable source of nutrients and substances with a health-promoting role. Good utilization and a lack of the effect on animal growth, value of hematological parameters, concentration of immunoglobulins and most of chemical blood parameters of laboratory rats were demonstrated in the nutritional studies on fruits of tomato GM plants. The biological response of the rats receiving GMO or its isogenic equivalent in the diet was recognized as similar. However, the unfavourable effect of the diets containing addition of tomatoes with the recombined thaumatin on the degree of oxidative degradation of DNA of rats liver was recorded. At the same time, the discussed dietary component had no effect on values of the remaining parameters of the oxidative status of tissue of the above mentioned organ and its histological image