The Antimicrobial Effects of Myrosinase Hydrolysis Products Derived from Glucosinolates Isolated from Lepidium draba.

Lepidium draba (hoary cress) is a perennial plant belonging to the Brassicaceae family that produces two dominant glucosinolates (GLSs): glucoraphanin (GRN) and sinalbin (SBN). They represent the stored form, which is converted upon the myrosinase (Myr) hydrolysis activity to active compounds, mainly isothiocyanates (ITCs) such as sulforaphane (SFN) or p-hydroxybenzyl isothiocyanate (pHBITC). Research on ITCs that have proven anticancer, antimicrobial, and chemoprotective properties is usually conducted with pure commercially available compounds. However, these are chemically reactive, making it difficult to use them directly for preventive purposes in dietary supplements. Efforts are currently being made to prepare dietary supplements enriched with GLS and/or Myr. In this study, we report a simple but efficient chromatographic procedure for the isolation and purification of GLSs from MeOH extract from hoary cress based on a combination of ion exchange and gel permeation chromatography on DEAE-Sephadex A-25 and Sephadex LH-20. To obtain the Myr required for efficient hydrolysis of GLSs into antibacterial ITCs, we developed a rapid method for its extraction from the seeds of Lepidium sativum (garden cress). The yields of GLSs were 22.9 ± 1.2 mg GRN (purity 96%) and 10.4 ± 1.1 mg SBN (purity 92%) from 1 g of dry plant material. Both purified GLSs were used as substrates for the Myr. Analysis of the composition of hydrolysis products (HPs) revealed differences in their hydrolysis rates and in the degree of conversion from GLSs to individual ITCs catalyzed by Myr. When GRNs were cleaved, SFNs were formed in an equimolar ratio, but the formation of pHBITCs was only half that of cleaved SBNs. The decrease in pHBITC content is due to its instability compared to SFN. While SFN is stable in aqueous media during the measurement, pHBITC undergoes non-enzymatic hydrolysis to p-hydroxybenzyl alcohol and thiocyanate ions. Testing of the antimicrobial effects of the HPs formed from GRN by Myr under premix or in situ conditions showed inhibition of the growth of model prokaryotic and eukaryotic microorganisms. This observation could serve as the jumping-off point for the design of a two-component mixture, based on purified GLSs and Myr that is, usable in food or the pharmaceutical industry in the future.

Effect of chronic radiation on the flax (Linum usitatissimum L.) genome grown for six consecutive generations in the radioactive Chernobyl area.

The growth of plants under chronic radiation stress in the Chernobyl area may cause changes in the genome of plants. To assess the extent of genetic and epigenetic changes in nuclear DNA, seeds of the annual crop flax (Linum usitatissimum L.) of the Kyivskyi variety, sown 21 years after the accident and grown for six generations in radioactive (RAD) and remediated (REM) fields were analysed. Flaxseed used for sowing first generation, which served as a reference (REF), was also analysed. The AFLP (Amplified Fragment Length Polymorphism) revealed a higher number of specific EcoRI-MseI loci (3.4-fold) in pooled flaxseed samples harvested from the RAD field compared with the REM field, indicating a link between the mutation process in the flax genome and the ongoing adaptation process. MSAP (Methylation-Sensitive Amplified Polymorphism) detecting EcoRI-MspI and EcoRI-HpaII loci in flax nuclear DNA genome showed no significant differences in methylation level, reaching about 33% in each of the groups studied. On the other hand, significant changes in the DNA methylation pattern of flaxseed samples harvested from the RAD field compared with controls were detected. Pairwise FST comparison revealed within both, EcoRI-MspI and transformed methylation-Sensitive data sets more than a 3-fold increase of genetic divergence in the RAD field compared with both controls. These results indicate that the nuclear genome of flax exposed to chronic radiation for six generations has more mutations and uses DNA methylation as one of the adaptation mechanisms for sustainability under adverse conditions.

Basic ß-1,3-Glucanase from Drosera binata Exhibits Antifungal Potential in Transgenic Tobacco Plants.

The basic ß-1,3-glucanase of the carnivorous plant Drosera binata was tested as a purified protein, as well as under the control of a double CaMV35S promoter in transgenic tobacco for its capability to inhibit the growth of Trichoderma viride, Rhizoctonia solani, Alternaria solani, and Fusarium poae in an in-vitro assay. The purified protein inhibited tested phytopathogens but not the saprophytic fungus T. viride. Out of the analysed transgenic plants, lines 13, 16, 19, and 22 exhibited high DbGluc1 transcript abundance normalised to the actin transcript. Because of DbGluc1 transgene expression, lines 13 and 16 showed a 1.7-fold increase and lines 19 and 22 showed more than a 2-fold increase in total ß-1,3-glucanase activity compared to the non-transgenic control. In accordance with the purified ß-1,3-glucanase in-vitro antifungal assay, crude protein extracts of lines 19 and 22 significantly inhibited the growth of phytopathogens (14-34%). Further analyses revealed that the complementary action of transgenic ß-1,3-glucanase and 20% higher activity of endogenous chitinase(s) in these lines were crucial for maximising the antifungal efficiency of crude protein extracts.

Biochemical and antifungal characteristics of recombinant class I chitinase from Drosera rotundifolia.

DrChit is class I chitinase involved in the digestion of insect prey of Drosera rotundifolia plants. Herein, we cloned the DrChit-S open reading frame lacking the 5'- sequence coding signal peptide into the pET32a vector and its derivate lacking the thioredoxin tag. After DrChit-S + Trx and DrChit-S-Trx overexpression in Escherichia coli cells and purification on Ni-NTA agarose, both enzymes exhibited maximum activity at pH 6.0 and 38 °C. Surprisingly, the DrChit -S + Trx exerted double enzyme activity and improved all kinetic parameters for FITC-chitin substrate degradation resulting in catalytic efficiency three times higher (46.2 mM-1. s-1) than DrChit-S-Trx (13.63 mM-1. s-1). The 3D-structure of DrChit-S + Trx revealed different spatial arrangement of the three tyrosine residues in chitin-binding site, while their aromatic rings showed better stacking geometry for CH/π interactions with the carbohydrate substrate. In contrast, there were no significant differences between both enzymes when the effect of metal ions and their antifungal potential were tested. Quantitative in vitro assays showed growth suppression of Fusarium poae (40%), Trichoderma viride (43.8%), and Alternaria solani (52.6%) but not Rhizoctonia solani (sp.). Our study indicates that sundew chitinase has potential in biotechnology either for degradation of chitin to oligomers applicable in medicine or for plant defense fortification.

Expression of Drosera rotundifolia Chitinase in Transgenic Tobacco Plants Enhanced Their Antifungal Potential.

In this study, a chitinase gene (DrChit) that plays a role in the carnivorous processes of Drosera rotundifolia L. was isolated from genomic DNA, linked to a double CaMV35S promoter and nos terminator in a pBinPlus plant binary vector, and used for Agrobacterium-mediated transformation of tobacco. RT-qPCR revealed that within 14 transgenic lines analysed in detail, 57% had DrChit transcript abundance comparable to or lower than level of a reference actin gene transcript. In contrast, the transgenic lines 9 and 14 exhibited 72 and 152 times higher expression level than actin. The protein extracts of these two lines exhibited five and eight times higher chitinolytic activity than non-transgenic controls when measured in a fluorimetric assay with FITC-chitin. Finally, the growth of Trichoderma viride was obviously suppressed when the pathogen was exposed to 100 µg of crude protein extract isolated from line 9 and line 14, with the area of mycelium growth reaching only 56.4% and 45.2%, of non-transgenic control, respectively. This is the first time a chitinase from a carnivorous plant with substrate specificity for long chitin polymers was tested in a transgenic plant with the aim of exploring its antifungal potential.

Structural and functional characterisation of a class I endochitinase of the carnivorous sundew (Drosera rotundifolia L.).

MAIN CONCLUSION: Chitinase gene from the carnivorous plant, Drosera rotundifolia , was cloned and functionally characterised. Plant chitinases are believed to play an important role in the developmental and physiological processes and in responses to biotic and abiotic stress. In addition, there is growing evidence that carnivorous plants can use them to digest insect prey. In this study, a full-length genomic clone consisting of the 1665-bp chitinase gene (gDrChit) and adjacent promoter region of the 698 bp in length were isolated from Drosera rotundifolia L. using degenerate PCR and a genome-walking approach. The corresponding coding sequence of chitinase gene (DrChit) was obtained following RNA isolation from the leaves of aseptically grown in vitro plants, cDNA synthesis with a gene-specific primer and PCR amplification. The open reading frame of cDNA clone consisted of 978 nucleotides and encoded 325 amino acid residues. Sequence analysis indicated that DrChit belongs to the class I group of plant chitinases. Phylogenetic analysis within the Caryophyllales class I chitinases demonstrated a significant evolutionary relatedness of DrChit with clade Ib, which contains the extracellular orthologues that play a role in carnivory. Comparative expression analysis revealed that the DrChit is expressed predominantly in tentacles and is up-regulated by treatment with inducers that mimick insect prey. Enzymatic activity of rDrChit protein expressed in Escherichia coli was confirmed and purified protein exhibited a long oligomer-specific endochitinase activity on glycol-chitin and FITC-chitin. The isolation and expression profile of a chitinase gene from D. rotundifolia has not been reported so far. The obtained results support the role of specific chitinases in digestive processes in carnivorous plant species.

The pollen- and embryo-specific Arabidopsis DLL promoter bears good potential for application in marker-free Cre/loxP self-excision strategy.

KEY MESSAGE: Marker-free transgenic plants can be generated with high efficiency by using the Cre/ lox P self-excision system controlled by the pollen- and embryo-specific Arabidopsis DLL promoter. In this work, we aimed to study the feasibility of using the pollen- and embryo-specific DLL promoter of the At4g16160 gene from Arabidopsis thaliana in a Cre/loxP self-excision strategy. A Cre/loxP self-excision cassette controlled by the DLL promoter was introduced into the tobacco genome via Agrobacterium-mediated transformation. No evidence for premature activation of the Cre/loxP system was observed in primary transformants. The efficiency of nptII removal during pollen and embryo development was investigated in transgenic T1 progenies derived from eight self- and four cross-pollinated T0 lines, respectively. Segregation and rooting assays were performed to select recombined T1 plants. Molecular analyses of these plants confirmed the excision event in all analysed T0 lines and marker-free transgenic T1 plants were obtained with efficiency of up to 96.2%. The Arabidopsis DLL promoter appears to be a strong candidate to drive Cre-mediated recombination not only in tobacco as a model plant, but also in other plant species.