Dragana Božić

Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes.

Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split.

Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae : Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis

Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.

Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast

Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.

Combined metabolome and transcriptome profiling provides new insights into diterpene biosynthesis in S. pomifera glandular trichomes.

BackgroundSalvia diterpenes have been found to have health promoting properties. Among them, carnosic acid and carnosol, tanshinones and sclareol are well known for their cardiovascular, antitumor, antiinflammatory and antioxidant activities. However, many of these compounds are not available at a constant supply and developing biotechnological methods for their production could provide a sustainable alternative. The transcriptome of S.pomifera glandular trichomes was analysed aiming to identify genes that could be used in the engineering of synthetic microbial systems.ResultsIn the present study, a thorough metabolite analysis of S. pomifera leaves led to the isolation and structure elucidation of carnosic acid-family metabolites including one new natural product. These labdane diterpenes seem to be synthesized through miltiradiene and ferruginol. Transcriptomic analysis of the glandular trichomes from the S. pomifera leaves revealed two genes likely involved in miltiradiene synthesis. Their products were identified and the corresponding enzymes were characterized as copalyl diphosphate synthase (SpCDS) and miltiradiene synthase (SpMilS). In addition, several CYP-encoding transcripts were identified providing a valuable resource for the identification of the biosynthetic mechanism responsible for the production of carnosic acid-family metabolites in S. pomifera.ConclusionsOur work has uncovered the key enzymes involved in miltiradiene biosynthesis in S. pomifera leaf glandular trichomes. The transcriptomic dataset obtained provides a valuable tool for the identification of the CYPs involved in the synthesis of carnosic acid-family metabolites.

Phytosociological alliances in the vegetation of arable fields in the northwestern Balkan Peninsula

A stratified dataset of 2426 relevés of weed vegetation of arable fields of the Balkan Peninsula was analysed by cluster analysis . The major division in species composition was associated with the type of crop . This accords with the syntaxonomical and ecological pattern already detected for southeast Europe and is in conflict with the Central European classification that has appeared in recent years . Clusters resulting from numerical classification reproduced the majority of traditionally recognized phytosociological alliances (Oxalidion, Panico-Setarion and Eragrostion are associated with root crops, while Scleranthion, Caucalidion and Galeopsion with cereals) . Galeopsion was grouped with some Caucalidion relevés, which is not surprising since both consist of weed communities from cereals . Vernal communities form a separate cluster and indicate a special community type, which has been treated in some classification systems as a phenological aspect .

Antimicrobial, antioxidant and anti-inflammatory activity of young shoots of the smoke tree, Cotinus coggygria Scop

In this study, we investigated the antimicrobial activity of the young shoots of the smoke tree, Cotinus coggygria Scop., Anacardiaceae. The acetone extract and the derived ethyl acetate fraction effectively inhibited the growth of Gram‐positive and Gram‐negative bacteria (MIC 25–200 µg/ml), while the chloroform fraction showed pronounced activity against the yeast Candida albicans (MIC 3.12 µg/ml). The ethyl acetate fraction exhibited a significant ferric‐reducing ability (10.7 mmol Fe2+/g extract), a very high DPPH radical scavenging activity (SC50 = 1.7 µg/ml) and inhibition of lipid peroxidation (IC50 = 41.8 µg/ml). High amounts of total phenolics (929.8 mg/g), tannins (833.8 mg/g) and flavonoids (35.5 mg/g) were determined in the ethyl acetate fraction, which also exerted significant anti‐inflammatory (76.7%) and cytotoxic effects (IC50 = 15.6 µg/ml). Copyright

Effect of plant growth promoting rhizobacteria on Ambrosia rtemisiifolia L. seed germination

SUMMARY Soil bacteria are able either to stimulate or inhibit seed germination. If seed germination is stimulated, the seedlings of weed species emerge more uniformly, so that they could be killed in the next step of weed control. This investigation focused on testing the germination of Ambrosia artemisiifolia L. on several media: Pseudomonas fluorescens (B1), Azotobacter chroococcum (B2), Bacillus licheniformis (B3), B. pumilus (B4), B. amyloliquefaciens (B5). In control, seeds germinated in water. Seed germination varied depending on bacterial media. Germination was inhibited by bacterial treatments B1 and B3, treatments B2 and B4 stimulated germination, while germination in treatment B5 was similar to control.

Alien plant species and factors of invasiveness of anthropogenic vegetation in the Northwestern Balkans — a phytosociological approach

We studied the anthropogenic vegetation of the Northwest Balkans in order to determine its susceptibility to invasion by alien plant species. We compiled a dataset of 3089 vegetation plots sampled between 1939 and 2009, recording a set of variables for each sample plot in order to determine which factors have the most effect on a habitat’s vulnerability to invaders. We calculated the proportion of native species, archaeophytes and neophytes for each plot. We used regression tree models to determine the site conditions of the most invaded anthropogenic habitats. The sample plots contained an average of 12.7% alien plant species, with a low proportion of archaeophytes (4.3%) and 8.4% neophytes. Local habitat conditions proved to have the largest effect, rather than climatic variables or propagule pressure. The proportion of archaeophytes follows a different pattern than that seen in central and northern Europe, indicating that macroecological factors are more important. Neophytes show a similar distribution to other European locations.

Temperature effects on Cuscuta campestris Yunk. seed germination.

Studies of biological characteristics of seeds and conditions for their germination have a major importance for planning and executing rational measures of weed control. The aim of this study was to investigate the effect of different temperatures on germination of C. campestris seeds. Three treatments (T1- storage at room temperature; T2 - exposure to 4°C for 30 days; T3 - scarification by concentrated sulphuric acid) differing in manipulation with seeds before germination were tested at different temperatures (5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C). Germinated seeds were counted daily for ten days and the length of seedlings was measured on the last day. The results showed that differences in germination of C. campestris seeds were very prominent between temperatures, as well as between treatments T1, T2 and T3. Seeds failed to germinate at 5°C and 45°C in all treatments (T1, T2, T3). Germination ranged from 6.25 at 10°C to 96.88%, the highest percentage, achieved at 30°C. [Projekat Ministarstva nauke Republike Srbije, br.III 46008 i br. TR 31043]

Quantification of biofilm formation on silicone intranasal splints: An in vitro study

OBJECTIVES Biofilms are associated with persistent infections and resistant to conventional therapeutic strategies. The aim of this study was to investigate the quantity of biofilm produced on silicone intranasal splints. METHODS Quantity of biofilm formation on silicone splints (SS) was tested on 15 strains of Staphylococcus aureus and Moraxella catarrhalis, respectively. Antimicrobial susceptibility testing was performed in accordance with European Committee on Antimicrobial Susceptibility Testing recommendations. RESULTS All tested strains formed different amounts of biofilm on SS: 66.7% S. aureus and 93.3% M. catarrhalis were weak biofilm producers and 33.3% S. aureus and 6.7% M. catarrhalis were moderate biofilm producers. S. aureus formed significantly higher quantity of biofilm compared with M. catarrhalis (p < 0.05). Multidrug resistant S. aureus produced significantly higher amount of biofilm compared with non-multidrug resistant strains (p < 0.05). CONCLUSION Quantity of biofilm on SS is highly dependent on bacterial species and their resistance patterns. Future studies are needed to ascertain another therapeutic option for prophylaxis prior to SS placement.