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Brassicaceae family counts around 350 genera and 3500 species [1], including crops of great economic importance, such as Brassica oleracea and B. rapa, which represent a significant part of human and animal diet [2]. The vegetables from this family have diverse morphological forms, such as leaves that form a head of cabbage, non- heading kale, collard greens and Chinese kale, an enlarged stem of kohlrabi, axillary buds of brussels sprouts, immature inflorescences of cauliflower and broccoli. Broccoli (Brassica oleracea botrytis var. cymosa) has a characteristic miniature tree-like appearance with a lot of elongated lateral shoots starting at the maturation of the terminal inflorescence [3]. What about the broccoli organism from the inside? Using a light microscope, we captured the structure of its tissues, cells, cell walls, tubes, vessels and pits. It is just as attractive and picturesque on the inside as it is on the outside.

Due to the high demand for healthy food, various biofortification strategies are developed to improve the quality of food. A promising biofortification technique used to improve the content of healthy phytochemicals in plant food is the interspecific metabolite transfer. It relies on the transfer of specialized metabolites from donor plant extract to acceptor plant. In our study, broccoli (Brassica oleracea var. italica) seedlings were used as an acceptor plant, and as a donor of specialized metabolites, water extract of tea (Camellia sinensis) was used. To test the possibility of enhancing the transfer from donor plant extract through the acceptor plant cell membrane, known membrane permeabilizers, EDTA and Tween, were used in a combination with donor plant extract. Changes in the content of phenolic compounds in broccoli seedlings after the interspecific metabolite transfer were evaluated using spectrophotometric assays and high- performance liquid chromatography (HPLC). A 19% increase in total phenolic content compared to the control was observed in seedlings treated with tea extract in combination with EDTA. In seedlings treated with tea extract in combination with EDTA and Tween a 28% and 27% increase in catechin content was observed, respectively. Furthermore, using HPLC, an increase in vitamin C, flavonoid quercetin and alkaloid caffeine content was observed. Treated seedlings’ extracts also displayed an increase in antioxidant capacity (AC). AC measured by DPPH revealed an increase in seedlings treated with tea and EDTA, AC measured by ABTS revealed an increase in seedlings treated with tea in combination with EDTA or Tween, and AC measured by FRAP revealed an increase in all treated samples, compared to control. It was also revealed that seedlings treated with tea and tea in combination with EDTA possess a higher pancreatic lipase inhibitory activity, suggesting their anti-obesity potential. Results point to interspecific metabolite transfer being a promising and eco-friendly biofortification strategy to improve the phytochemical content and bioactivity of broccoli seedlings.

Exposure of plants to extreme temperatures present challenges for global food security because it could cause changes in metabolite profile. Such changes could have a significant impact on the nutritional and health properties of plants, and indirectly (via plant-based-diet) on animal health. This particularly applies to the widely cultivated and consumed Brassicaceae family, including crops of great economical importance, such as Brassica oleracea and B. rapa, which present a major part of human diet. The aim of this preliminary study was to differentiate high temperature (HT) susceptible from resistable metabolic parameters of broccoli (Brassica oleracea L. convar. botrytis) seedlings before and during in vitro simulated human digestion. Methods included in vitro simulated human digestion, spectrophotometric quantification of total phenolics, flavonoids, flavonols, anthocyanins, hydroxycinnamic acids and antioxidant capacity by the methods ABTS, FRAP and DPPH. Using a RP-HPLC method, vitamin C, kaempferol, quercetin, tiliroside, gallic, p-coumaric, benzoic, caffeic, ferulic and sinapic acid were identified and quantified. Before enzymatic digestion, the highest number of parameters were resistable to HT, however total phenolics, flavonols, benzoic acid and kaempferol were increased, while ABTS, vitamin C, caffeic and ferulic acid were decreased. Both before and after digestion, benzoic acid was increased, total flavonoids resistable, while vitamin C and caffeic acid reduced. During digestion, total hydroxycinnamic acids, flavonols, benzoic and sinapic acid were increased, total phenolics and flavonoids resistable, and caffeic acid was decreased. The results indicate plasticity of brocolli seedlings metabolism under the HT influence, and provide a basis for further research of this effect.

The high demand for healthy food in recent years led to an increasing need for highly bioactive plant materials. One simple method to improve the nutritional properties of plants could be the treatment of plants of known bioactive potential (acceptor) with extracts of different plant species (donors) that contain additional high bioactive compounds, so-called interspecific source-sink phytochemical transfer. The aim of the present study was to evaluate the effect of interspecific transfer of metabolites on plant phenolic and vitamin C profile to improve their gastrointestinal bioavailability, hypoglycemic potential, and antioxidant capacity as well as cytotoxicity. Additionally, it was tested whether there is an effect of phytochemical transfer on the expression of marker genes in phenolic biosynthetic pathways. Chinese cabbage (Brassica rapa ssp. pekinensis) sprouts were chosen as a model of plant acceptor, while the inflorescences of St John`s-wort (Hypericum perforatum) and chamomile (Matricaria chamomilla), leaves of rose (Rosa sp.) and shoots of black bryony (Tamus communis) were used as plant donors. Even though the chamomile extract increased the highest number of individually identified compounds in Chinese cabbage sprouts, that was not crucial for the improvement of their bioactivity. Rather, black bryony application induced more biopotential parameters in the sprouts such as significantly improved bioavailability of vitamin C, kaempferol and total phenolics both before and during gastrointestinal digestion of sprouts, increased hypoglycemic activity of sprouts, and their antioxidant capacity during digestion. For an increased cytotoxicity against MCF cells, St. John`s-wort, rose and chamomille extracts could be applied to Chinese cabbage sprouts. The results suggest that interspecific phytochemical transfer could enhance the phytochemical profile and the bioactive properties of the acceptor plant.

With this project we want to address the chain interactions between temperature shift, plant biochemical traits and mammals (fed with those plants) physiology, employing targeted specific metabolomics approach for plant analyses, biosafety and bioactivity tests on mammals, and statistical data analyses and modelling in order to construct a model showing a tentative pattern of environment temperature indirect (through plant diet) effect on mammal physiology. Our strategy is to identify metabolites of plants that have been altered by high temperature (HT), to quantify the intensity of changes, and to correlate those with changes in the physiological parameters of mice fed by these plants. As a plant representative we will use Brassica oleracea botrytis var. cymosa (broccoli) seedlings and mature plants, and as a mammal representative a mouse strain Swiss Webster albino will be used. Additionally, for in vitro biosafety analyses the cell culture of human hepatocellular carcinoma (HepG2) and mouse embryonal fibroblasts (MEF) will be used. This type of research would serve as a basis (case study) for future studies in this field (other plant species and growth stages, additional biochemical and physiological traits in plants and animals), which we have in our plan for a longer period. The results of this project will further clarify whether the phytochemical ability of plant to adapt to HT (simulating global warming) will be crucial for its nutritional value and biological effects in mammals, i.e. mammal health status.

The high demand for healthy food in recent years led to an increasing need for highly bioactive plant materials. One simple method to improve the nutritional properties of plants could be the treatment of plants of known bioactive potential (acceptor) with extracts of different plant species (donors) that contain additional high bioactive compounds, socalled interspecies source-sink phytochemical transfer. The aim of the present study was to evaluate the effect of interspecies transfer of metabolites on plant phenolic and vitamin C profile to improve their gastrointestinal bioavailability, hypoglycemic potential, and antioxidant capacity as well as cytotoxicity. Additionally, it was tested whether there is an effect of phytochemical transfer on the expression of marker genes in phenolic biosynthetic pathways. Chinese cabbage (Brassica rapa ssp. pekinensis) sprouts were chosen as a model of plant acceptor, while the inflorescences of St. John`s-wort (Hypericum perforatum) and chamomile (Matricaria chamomilla), leaves of rose (Rosa sp.) and shoots of black bryony (Tamus communis) were used as plant donors. Even though the chamomile extract increased the highest number of individually identified compounds in Chinese cabbage sprouts, that was not crucial for the improvement of their bioactivity. Rather, black bryony application induced more biopotential parameters in the sprouts such as significantly improved bioavailability of vitamin C, kaempferol and total phenolics both before and during gastrointestinal digestion of sprouts, increased hypoglycemic activity of sprouts, and their antioxidant capacity during digestion. For an increased cytotoxicity against MCF cells, St. John`s-wort, rose and chamomille extracts could be applied to Chinese cabbage sprouts. The results suggest that interspecies phytochemical transfer could enhance the phytochemical profile and the bioactive properties of the acceptor plant.

Methodological approach for determination of phenolic compounds and antioxidant capacity of honey differs between research papers. Extraction process is an important step in the evaluation of total phenolics and antioxidant capacity, but most of the available data on phenolic composition of honey originate from spectrophotometric methods performed without extraction, directly on fresh honey samples, often with different solvents. In this study we screened for the possible differences in total phenolics and antioxidant capacity results depending on the extraction type to which the honey was subjected. Experiments were performed using honey of Castanea sativa, Tillia spp. and Robinia pseudoacacia species, in triplicates for each honey sample - (a) fresh honey dissolved in water, (b) fresh honey dissolved in ethanol and (c) ethanol extract prepared using column chromatography. In most cases, the results acquired with ethanol extract obtained by column chromatography had significantly higher readings (for example C. sativa: ABTS (42.1±2.1%), DPPH (48.6±0.7%), FRAP (91.9±0.4, total phenolics (160.1±12.9 mg galic acid equivalents/100 g of honey)) then the results acquired with both dilutions of fresh honey samples (C. sativa: dissolved in water - ABTS (24.2±0.4%), DPPH (24.6±1.5%), FRAP (81.3±0.2%), total phenolics (67.1±5.0 mg galic acid equivalents/100 g of honey) ; C. sativa dissolved in ethanol - ABTS (16.2±4.8%), DPPH (3.0±0.2%), FRAP (78.5±0.7%), total phenolics (44.1±9.2 mg galic acid equivalents/100 g of honey). Among the fresh honey samples, no strict tendency could be observed, in some cases water was better solvent, but in another ethanol was better (for example total phenolics in R. pseudoacacia were 14.9±4.2% for water dissolved honey, and 49.7±17.6% for ethanol dissolved honey, while FRAP was 44.7±0.1% for water dissolved honey, and 30.2±1.8% for ethanol dissolved honey). Furthermore, total phenolic content and antioxidant capacity results do not completely match between fresh honey samples and those extracted using column chromatography. These results imply that omission of extraction step using column chromatography reduces the sensitivity and specificity of spectrophotometric methods for assessment of total phenolics and antioxidant capacity of honey.

Roses (genus Rosa L.) leaves and petals are used in food and cosmetic industry due to the bioactive specialized metabolites they contain, however there are no comparative data on the qualitative and quantitative content of those metabolites in the cultivars of different colors. 13. HBK 213 13th CBC Photosynthetic pigments, flavonoids and phenolic acids play a key role in photosynthesis, metabolism and plant protection against biotic and abiotic stress, and are main bioactive components of roses. Therefore, we screened for these components in leaves and petals of three differently coloured cultivars collected in the Botanical Garden in Zagreb with the aim of comparing their bioactive potential. The results revealed that more bioactive components were present in leaves than in petals of roses. Roses (both leaves and petals) of different colours showed a specialisation in bioactive components representation. The results showed that in cultivars of different colours a trade-off between amount of photosynthetic pigments, flavonoids and phenolic acids, especially in leaves, exists.

Phenolic compounds are among the major contributors to the antioxidant properties of honey, but their content is not thoroughly assessed when legal quality checks are performed. For this reason, there is no standardized methodological approach, and the data on the phenolic composition of many types of honey are still missing. In scope of this work we investigated the content of phenolic compounds and the antioxidant capacity of three types of honey – acacia (Robinia pseudoacacia L.), chestnut (Castanea sativa Mill.) and lime-tree (Tilia spp.) honey that originate from Croatia and Germany. Spectrophotometric methods for the determination of total phenolic compounds and antioxidant capacity, as well as high performance liquid chromatography analysis of flavonoids were performed. The Croatian honey samples had higher antioxidant capacity and quantitative content of total phenolics than the same botanical type of German honey samples, except for lime-tree whose amount of total phenolics did not differ significantly. According to its botanical origin, the chestnut honey had a higher antioxidant capacity (the only exception was ABTS measurement which did not yield significant difference between Croatian chestnut and acacia honey) and quantitative content of total phenolic compounds than the acacia or the lime-tree honey. The results imply that both the botanical and the geographical origin influence the final quality of honey in terms of its phenolic compounds and antioxidant capacity

The influence of salicylic acid (SA) applied in the range 10-1000 µM on the qualitative and quantitative profile of flavonoids and phenolic acids in seedlings, and leaves of healthy and Plasmodiophora brassicae-infected Brassica rapa ssp. pekinensis (Chinese cabagge) plants was investigated using a HPLC method. In seedlings the treatment induced, in a dose dependent manner, the synthesis of caffeic, chlorogenic, ferulic and sinapic acid, and kaempferol. In healthy plants SA treatment caused an increase in the phenolic acids concentration of identified compounds, but not in a dose dependent manner ; and a decrease in kaempferol levels. Upon infection, the amount of phenolic compounds was increased compared to healthy plants, however the effect of SA on the phenolic profile of infected plants was variable and no unequivocal conclusion could be drawn. None of the SA concentrations was sufficient to reduce disease symptoms. This result indicates specific responses of Chinese cabagge, on the level of flavonoids and phenolic acids, to SA depending on developmental stage and health condition.

The aim of this research was to develop a bryophyte-virus system which could serve for pathophysiology analysis in nonvascular plants. Physcomitrella patens, a moss with sequenced genome, was chosen as a host plant, and a satellite-associated Cucumber mosaic virus (CMVsat), which has a wide range of hosts, as a virus model. P. patens infection with CMVsat was confirmed using an ELISA method. Both, plants on agar medium and on soil were successfully infected. Two time points were tested – 5 and 10 dai, where the concentration of CMVsat was higher at the later time point. Plants on agar medium were easier to work with, because there was no time consuming soil washouts, however medium was prone to secondary infection (bacteria and mold) and therefore later time points were difficult to achieve. The morphological differences between infected and control colonies could be seen - infected colonies were dark green, control were light green. A HPLC analysis revealed that infected P. patens contained higher amounts of polyphenolics than controls. The established moss-virus system could serve for comparative studies of defense responses against viruses in nonvascular and vascular plants.

The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana which is characterised by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. P. brassicae seems to suppress plant defence reactions, but knowledge of how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease and show that P. brassicae possesses a methyltransferase (PbBSMT) that is involved in SA methylation. The accumulation of SA and emission of methylated SA (MeSA) were observed in P. brassicae-infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the Atbsmt1 mutant indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. The PbBSMT protein has a predicted signal sequence indicating that it might be secreted into the host cells. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses we confirmed that PbBSMT can methylate SA, benzoic and anthranilic acids. Taken together, our results suggest that PbBSMT participates in metabolising SA in Arabidopsis.

The aim of this study was to i) investigate the content of inorganic (macro- and microelements) and organic compounds (total phenolics, total flavonoids and total tannins) and ii) evaluate the antioxidant activities of total phenolics and total tannins in S. domestica L. bark, fruits (separately exocarp and mesocarp) and seeds using phytochemical analyses. Results revealed all the tested service tree samples were rich in potassium. Bark, among the tested samples, was the best source of calcium and zinc, while seeds were the best source of magnesium. Extracts of immature exocarp and bark contained the highest amounts of total phenolics. Ripening significantly decreased the amount of phenolics in fruits (both exocarp and mesocarp). Exocarp was significantly richer in total flavonoids than mesocarp, bark or seeds. The highest antioxidant activity showed phenolics from immature exocarp and then from bark. The antioxidant activity of phenolics and tannins from exocarp, but not from mesocarp, was significantly decreased after ripening. Based on the data we conclude that service tree bark and seeds are significantly better sources of macro- and microelements than fruits, while exocarp and bark are better sources of total phenols and flavonoids. We suggest that under-utilized species Sorbus domestica L. could be used as a great source of mineral elements and antioxidants important for human diet.

Qualitative and quantitative reversed-phase high performance liquid chromatography analyses of phenolics in water and ethanolic extracts of Croatian wild edible plants asparagus (Asparagus acutifolius L.), butcher’s broom (Ruscus aculeatus L.) and black bryony (Tamus communis L.) were conducted. Black bryony contained the highest amount of phenolics, followed by wild asparagus and butcher`s broom. Flavonol glycosides were the most abundant phenolics in black bryony and wild asparagus, in butcher`s broom phenolic acids were prevalent. Water extracts were subjected to two-phase (gastric and duodenal) in vitro digestion with human enzymes in order to compare the gastrointestinal stability of major phenolics in the selected plants extracts. Phenolic acids in all the tested water extracts were totally digested already after in vitro gastric digestion phase. Among flavonoids, two main kaempferol-glycosides from black bryony extract were best preserved (50% of each) after duodenal digestion. In wild asparagus extract approximately 20% of quercetin-3-O-rutinoside and isorhamnetin-3-O-rutinoside remained stable, and in the butcher`s broom extract no flavonoids could be detected upon duodenal digestion. Accordingly, among the tested plants black bryony would be the best source of natural antioxidants both because it contains the highest amount of flavonol glycosides, and because the highest percentage of this plant phenolics was preserved after in vitro gastrointestinal digestion.

Quercetin (Q) is a plant polyphenolic from the group of flavonoids with strong antioxidative, antiviral and antibacterial activities, yet its ability of long-distance movement through a plant is quite limited. Moreover, the mobility of exogenously applied Q through an Arabidopsis thaliana plant is minimal or not possible at all. Recently, it was discovered that biological properties of free flavonoids can be improved by their chelation with metals. In last years it was proven that a rare-earth metal europium (Eu(III)) is naturally present in some plants, though in small quantities and, more interesting, can affect their physiological processes. Accordingly, we tested whether Eu(III) could bind to flavonoid Q, and if so would and how affect the transport ability of Q through Arabidopsis. Our in vitro results showed that Eu(III) binds to Q, and at the ratio rQ/Eu(III) = 0.2 complex stoichiometry 1/1 (Q/Eu(III)) reaches its maximal concentration with ˃ 95% of the total concentration of UV/Vis absorbing species in the solution. This complex stoichiometry was therefore used in further in vivo examinations, and the results showed that the long-distance transport of Q through Arabidopsis root could be stimulated by complexation with Eu(III). During transport, the Q/Eu(III) complex got degraded and therefore enabled efficient, slow, release of protective metabolite at the distal site, offering enhanced Q delivery. The spectrophotometric data suggested, one of possible reasons of Q/Eu(III) degradation could be the interaction of the complex with double stranded RNAs present in Arabidopsis. Since Q itself has a very limited transportation ability, yet positively affects plant defence responses, its improved long-distance transportation could lead to a better protection of plant tissues. Accordingly, our results suggest that the rare-earth element Eu(III) can improve the biological relevance of protective biomolecule Q.

Auxin levels and the regulation of reversible auxin conjugation as a mechanism of auxin homeostasis has been investigated in Chinese cabagge (Brassica rapa L. ssp. pekinensis) upon biotic stress caused by Cucumber mosaic virus containing satellite RNA (CMVsat) infection and abiotic stress (salt stress, drought) as well as tretments with stress hormones (SA, JA, ABA). To detect the physiological state of plants and their ability to integrate given stress conditions, a set of biochemical markers (glutathione level, antioxidant enzymes activity, carbonyl content) were analysed. Endogenous free and total IAA, and ABA were analysed by GC-MS. The levels of auxin were significantly affected by biotic and abiotic stresses as well as treatments with stress hormones. All stress conditions caused perturbation of IAA levels with a tendency of increasing the IAA conjugate level. Among treatments with stress hormones, JA caused the most significant increase of free and conjugated IAA in comparison to others. Endogenous ABA was significantly higher under both abiotic stress conditions in comparison to the control showing a positive correlation with auxin levels. Results of gene expression experiments (qRT-PCR) showed an increased transcript level of auxin amidohydrolase BrIAR3 upon abiotic stress as well as after treatment with JA. The results implicated an important role of auxin and interactions with other stress hormones in the plant stress response.

Phenolic derivatives have many important roles in plants, yet mechanisms of their transport and all biological activities are still not elucidated. In scope of this dissertation, the transport abilities and certain biological activities of two types of phenolic derivatives (selected flavonoids and salicylic acid) were investigated. For research purposes, spectrophotometric methods were used, fluorescence microscopy, gass chromatography coupled with mass spectrometry, high performance liquid chromatography, and quantitative real time polymerase chain reaction. It was found that the complexation of quercetin with europium improves the long distance transport ability of quercetin. The involvement of this complex in certain aspects of Arabidopsis pathological states was further tested. Satellite-associated Cucumber mosaic virus (CMVsat) double stranded RNAs were found to specifically interact with this complex by degrading it into free quercetin. It was observed that the complex could moderate symptom development in Plasmodiophora brassicae-infected Arabidopsis. Another flavonoid, kaempferol, was shown to be involved in Arabidopsis thaliana-CMVsat interaction. Intriguing, in scope of this dissertation it was found that some compounds from flavonoid biosynthetic pathway are needed for CMVsat accumulation in Arabidopsis. Regarding salicylic acid, it was found that in Arabidopsis infected with P. brassicae it is synthesized from chorismate, and long distance transported in methylated form (as methyl-salicylate). The quantitative PCR analysis revealed that the expression of gene for isochorismate synthase and the expression of gene for salicylic acid methyltransferase were induced upon infection with P. brassicae. In Arabidopsis infected with CMVsat, salicylic acid is also transported as methyl-salicylate, but the transport rate and accumulation of salicylic acid are quite different than in Arabidopsis infected with P. brassicae. Biljke proizvode različite sekundarne metabolite. Sekundarne metabolite se u početku smatralo tek nusproduktima primarnog metabolizma (metabolizma ugljikohidrata, proteina, aminokiselina i lipida) koji se nakupljaju u biljnoj stanici zbog nedostatka djelotvornog sustava za njihovo izlučivanje. Međutim, uskoro je otkriveno da iako nisu neophodni za preživljavanje biljke, sekundarni metaboliti su joj potrebni za normalno funkcioniranje te joj mogu omogućiti bolju prilagodbu na novonastale uvjete. Sastav sekundarnih metabolita u biljci je kompleksan, ovisi o vrsti tkiva i organa te o stadiju razvoja biljke, razlikuje se između pojedinačnih biljaka i populacija (Paiva, 2000; Pichersky i Gang, 2000; Lattanzio, 2003; Osbourn, 2003, Wink, 2003). Sekundarne metabolite općenito dijelimo na fitoanticipine (konstitutivni, sintetiziraju se tijekom normalnog razvoja biljnih tkiva) i fitoaleksine (sintetiziraju se pod utjecajem nekog oblika stresa). Jednu od najvažnijih i najzastupljenijih skupina sekundarnih metabolita čine fenolni spojevi. U biljkama fenolni spojevi bivaju sintetizirani iz primarnih metabolita (prvenstveno aminokiselina i ugljikohidrata) procesima metilacije, hidroksilacije i glikozilacije (Korkina, 2006) kao produkti biosintetskog puta šikiminske kiseline i metabolizma fenilpropanoida. Biljkama fenolni spojevi služe za pigmentaciju, rast, razmnožavanje, signaliziranje, različite obrambene odgovore spram biotičkog i abiotičkog stresa te mnoge druge procese (Weisshaar i Jenkins, 1998; Winkel-Shirley, 2001; Forkmann i Martens, 2001). Zbog njihovih iznimnih svojstava, fenolni spojevi čine skupinu najistraživanijih biljnih bioaktivnih tvari te ih se koristi kao modelne spojeve u različitim područjima istraživanja. U skupinu fenolnih spojeva pripada nekoliko tisuća kemijskih spojeva, od kojih preko 10000 čine flavonoidi (Tahara, 2007). Među fenolnim spojevima najviše su istraživani flavonoidi i salicilna kiselina zbog njihovih iznimnih pozitivnih bioloških učinaka u biljnom organizmu. Flavonoidi su fenolni spojevi širokog spektra pozitivnih bioloških učinaka. Nijedna druga skupina sekundarnih metabolita ne pokazuje toliko učinaka na rast i razvoj biljnog organizma. Flavonoidi djeluju kao snažni antioksidansi te štite biljku od slobodnih radikala (Rice-Evans i sur., 1997; Shahidi i Wanasundara, 1992). Oni utječu na rast i razvoj biljnog organizma regulirajući polarni transport auksina (Fischer i sur., 1997; Murphy i sur., 2000; Brown i sur., 2001; Buer i sur., 2004; Rusak i sur., 2010). Flavonoidi djeluju kao signalne molekule među organizmima, stanicama te unutar stanica (Eckardt, 2006, Moscatiello i sur., 2010; Hassan i Mathesius, 2012). Oni štite biljku od UV zračenja, biotičkog i abiotičkog stresa (Reuber i sur., 1996; Shirley, 1996; Rozema i sur., 1997; Treutter, 2006). U proteklih deset godina dokazano je da se neke pozitivne biološke učinke flavonoida može dodatno pojačati njihovim vezivanjem s određenim metalima, npr. flavonoid-metal kompleksi znatno učinkovitije štite biljku od slobodnih radikala u usporedbi s flavonoidima od kojih potječu (Kostyuk i sur., 2001). Štoviše, sposobnost flavonoida da keliraju metale odgovorne za sintezu kisikovih i dušikovih reaktivnih spojeva također doprinosi njihovim antioksidativnim biološkim učincima. Osim njihova boljeg antioksidativnog djelovanja, kompleksi flavonoida s metalima su hidrofilniji i time bolje topivi u vodenim otopinama od čistih flavonoida (Afanas'ev i sur., 1989). Nadalje, kompleksi flavonoida s metalima učinkovitije uklanjaju superoksidne ione od čistih flavonoida (Kostyuk i sur., 2004). Također, dokazano je da se kompleks flavonoida kvercetina s lantanom znatno jače veže za dvolančane RNA molekule virusa mozaika krastavca s pridruženim satelitom u usporedbi sa slobodnim kvercetinom, što je potaknulo pretpostavku da bi vezivanje flavonoida s metalima moglo pojačati njihovo antiviralno djelovanje (Rusak i sur., 2009). Za sintezu kompleksa flavonoid-metal često se koriste rijetki zemljani metali zbog male toksičnosti ovih kompleksa te zbog činjenice da ti metali ne pokazuju kumulativni učinak u in vivo uvjetima. Iako jedan od najmanje zastupljenih rijetkih zemljanih metala, od 2002. godine do danas potvrđena je prisutnost europija u staničnoj membrani, citoplazmi i mitohondrijima određenih biljnih vrsta (McKenna i sur., 2002; Gao i sur., 2003; Fellows i sur., 2003). Svojstvo fluorescencije lantanida (jedne od skupina rijetkih zemljanih metala), prije svega europija, iznimno je korisno u istraživanjima njihovih kompleksa s biomolekulama (Mundoma and Greenbaum, 2003). Tian i sur. su 2003. i 2004. godine dokazali određene biološke učinke egzogeno apliciranog europija na korijen graška (Lathyrus sativus L.). Europij, naime, može aktivirati proteolitičke enzime te može pojačati aktivnost Na+/K+-ATPase mijenjanjem omjera Na+/K+. Nadalje, Zeng i sur. su 2003. godine dokazali da europij može poticati prijenos Ca2+ kroz plazmatsku membranu te tako utjecati na razvoj biljnog organizma. Yang i sur. su 2005. godine objavili da mala koncentracija europija (konkretno 0.1 μg mL-1) može potaknuti rast kalusa i sintezu flavonoida u vrste Glycyrrhiza uralensis L. Nedavno je objavljeno da je antitumorsko djelovanje kompleksa kvercetina s europijem znatno jače od onog čistog kvercetina (Fan i sur., 2012). U sklopu ove disertacije sintetiziran je kompleks kvercetin-europij u biološkom puferu te je spektrofotometrijskim metodama dokazano da isti različito reagira s jednolančanim i dvolančanim biološkim RNA molekulama. Dokazano je da dvolančane RNA molekule virusa mozaika krastavca s pridruženim satelitom specifično reagiraju s ovim kompleksom razgrađujući ga na slobodni kvercetin. Metodom fluorescencijske mikroskopije potvrđeno je da kompleks poboljšava sposobnost prijenosa kvercetina kroz korijen vrste Arabidopsis thaliana. Nadalje, primijećeno je da kompleks može ublažiti razvoj simptoma u Arabidopsisu inficiranom protistom Plasmodiophora brassicae. Osim kvercetina, drugi flavonoid čije je biološko djelovanje istraživano u sklopu ove disertacije jest kempferol. Rezultati su pokazali da kempferol sudjeluje u obrambenom odgovoru Arabidopsisa spram virusa mozaika krastavca s pridruženim satelitom. Naime, metodom tekućinske kromatografije visoke moći razlučivanja potvrđeno je da je u inficiranim Arabidopsisima znatno manja količina kempferola nego u odgovarajućim kontrolnim zdravim biljkama. Očito je da se kempferol troši u nekom od obrambenih mehanizama zaražene biljke domaćina, što je u skladu s već objavljenim rezultatima o antifitoviralnom djelovanju pojedinih flavonoida (Rusak i sur., 1997). S druge strane, u sklopu disertacije testiran je i suprotan proces, učinak sadržaja endogenih flavonoida na sposobnost razmnožavanja virusa mozaika krastavca s pridruženim satelitom u Arabidopsisu. U tu svrhu korištene su određene TRANSPARENT TESTA (tt) mutante Arabidopsisa koje sadrže mutacije u genima odgovornim za sintezu pojedinih enzima flavonoidnog biosintetskog puta (Koornneef i sur., 1982; Koornneef, 1990; Shirley i sur., 1995). Izrazito zanimljivo, utvrđeno je da se virus mozaika krastavca s pridruženim satelitom ne razmnožava u Arabidopsisu koji sadrži mutaciju u genu odgovornom za sintezu prvog enzima flavonoidnog biosintetskog puta (halkon sintaze (CHS)) te stoga ne posjeduje niti jedan flavonoid. Prema tome, dok neki flavonoidi imaju antifitoviralno djelovanje (Rusak i sur., 1997), neki od spojeva iz flavonoidnog biosintetskog puta su, upravo suprotno, potrebni za umnažanje virusa mozaika krastavca s pridruženim satelitom u Arabidopsisu. Nadalje, u sklopu ove disertacije testiran je učinak određenih endogenih i egzogeno apliciranih flavonoida na razvoj kalusnog tkiva Arabidopsisa. Dokazano je da endogeni naringenin u tt6 mutanti spriječava klijanje sjemenki Arabidopsisa. Također, primijećeno je da egzogeno aplicirani kempferol utječe na razvoj kalusa znatnije nego kvercetin ili naringenin. Posebno je taj učinak izražen kod sjemenki koje već u sebi sadrže endogeni kempferol, što upućuje na pretpostavku da je prijenos određenog egzogeno apliciranog flavonoida učinkovitiji kroz ona tkiva koja su sposobna isti i sama sintetizirati. Uz flavonoide, salicilna kiselina je jedan od najvažnijih fenolnih spojeva koji sudjeluju u obrambenim procesima biljnog organizma. Zanimljivo, iako je salicilna kiselina sama po sebi citotoksična i nema sposobnost prijenosa na veće udaljenosti kroz biljku (Vernooij i sur., 1994; Peer i sur., 2007), derivatizacijom iste (glukozilacijom, metilacijom, konjugacijom s aminokiselinama, sulfonacijom ili hidroksilacijom) unutar biljne stanice nastaju derivati koji nisu citotoksični te koji omogućavaju prijenos salicilne kiseline na veće udaljenosti (Vernooij i sur., 1994; Peer i sur., 2007) i njeno skladištenje (Dean i sur., 2003; 2005; Dean i Mills, 2004). Budući su prijenos i biosintetski put salicilne kiseline specifični za svaki biljka-patogen sustav, u sklopu ove disertacije proučavan je mehanizam prijenosa salicilne kiseline kroz Arabidopsis inficiran lokalnim patogenom (protist Plasmodiophora brassicae koji obitava u korijenu Arabidopsisa) te kroz Arabidopsis inficiran sustavnim patogenom (virus mozaika krastavca s pridruženim satelitom koji se od mjesta infekcije sustavno širi na novonastale dijelove Arabidopsisa). Utvrđeno je da se u oba slučaja salicilna kiselina prenosi na veće udaljenosti u obliku metil-salicilata. Međutim, učinci ovih dvaju patogena na intenzitet prijenosa i količinu salicilne kiseline u Arabidopsisu je znatno različit. Ovaj rezultat ukazuje na činjenicu da u istoj biljci domaćinu različiti patogeni imaju različit učinak na prijenos i skladištenje glavnog obrambenog spoja biljke. Također, u sklopu disertacije proučen je biosintetski put salicilne kiseline u P. brassicae-inficiranom Arabidopsisu. Dokazano je da se salicilna kiselina u P. brassicae-inficiranom Arabidopsisu primarno sintetizira iz korizmata, no u najvišem stupnju infekcije, kada je biljkama za obrambene reakcije potrebna najveća količina salicilne kiseline, manji dio iste može se sintetizirati, uz korizmat, čak i iz fenilalanina. Ovaj rezultat u skladu je s rezultatom koji su objavili Dempsey i sur. 2011. godine; oni su naime zaključili da Arabidopsis pod utjecajem stresa sintetizira salicilnu kiselinu primarno iz korizmata, dok fenilalanin također može biti korišten kao supstrat ali u značajno manjoj mjeri. Nadalje, za potrebe disertacije, proučen je učinak infekcije Arabidopsisa protistom P. brassicae na ekspresiju određenih gena povezanih s procesima prijenosa i biosinteze salicilne kiseline. Metodom lančane reakcije polimeraze u stvarnom vremenu (Real-Time PCR) utvrđeno je da je ekspresija gena koji kodira protein za metilaciju salicilne kiseline (At02174493_g1 označen kao SAMT1) povećana nakon infekcije Arabidopsisa spomenutim patogenom. Ovaj rezultat je u skladu s prethodno dokazanom činjenicom da se salicilna kiselina prvo mora metilirati kako bi se učinkovitije mogla prenositi kroz P. brassicae-inficirani Arabidopsis. Ekspresija gena koji kodira protein izokorizmat-sintazu (At02286260_g1 označen kao ICS1) također je povećana nakon infekcije ovim patogenom i taj je rezultat u skladu s prethodno dokazanom činjenicom da se u ovom sustavu salicilna kiselina sintetizira primarno iz korizmata. Ekspresija gena odgovornog za glukozilaciju salicilne kiseline (At02327040_gH označen kao SAGT) u dotičnom biljka-patogen sustavu ostaje za sada nerazjašnjena budući je normalizacija ekspresije ovog gena u odnosu na dvije odabrane endogene kontrole (At02200102_s1 označen kao F-box i At02183416_g1 označen kao YLS8) dala oprečne rezultate. Dobiveni rezultati u sklopu ove disertacije upućuju na moguće nove biološke učinke kompleksa kvercetin-europij te kempferola. Također, dokazano je da je metil-salicilat prijenosni oblik salicilne kiseline u P. brassicae- i CMVsat-inficiranom Arabidopsisu, uz naglasak da je intenzitet prijenosa i količina salicilne kiseline, s obzirom na kontrolne neinficirane biljke, između ova dva sustava značajno različit. Salicilna kiselina se u P. brassicae-inficiranom Arabidopsisu primarno sintetizira iz korizmata, no u najvišem stupnju infekcije, kada je biljkama za obrambene reakcije potrebna najveća količina salicilne kiseline, manji dio iste može se sintetizirati, uz korizmat, čak i iz fenilalanina. Ekspresija gena koji kodira protein za metilaciju salicilne kiseline i gena koji kodira protein izokorizmat-sintazu je nakon infekcije ovim patogenom povećana.

Salicylic acid (SA) is a defense hormone required for the induction of plant resistance to pathogens. SA biosynthesis pathways and the type of its mobile form associated with induced resistance are plantpathogen specific. The clubroot disease is one of the most devastating diseases affecting all the members within the plant family Brassicaceae by causing serious losses of vegetable crops worldwide. In this study we wanted to investigate 1. which pathway of SA biosynthesis is implicated in the clubroot plantpathogen interaction and 2. whether methyl-salicylate (MeSA) is a mobile form of SA in this system. We have chosen Arabidopsis thaliana as a host organism for Plasmodiophora brassicae – the causal agent of clubroot, because the infection process is fast and the resistance of A. thaliana to P. brassicae is conferred by a small number of genes. Using isotope feeding and GC-MS analysis, we monitored the SA biosynthesis pathway as well as transport and metabolism of exogenously applied deuterated SA and its derivative, deuterated MeSA, through the whole plant of healthy and P. brassicae-infected A. thaliana. The results showed that in A. thaliana clubroots 1. SA is synthesized from chorismate and 2. MeSA is the preferred mobile form of SA.

The mobile signals for systemic acquired resistance (SAR) in plants are plant-pathogen specific. It is known that salicylic acid (SA) enables the establishment of SAR in plants infected by biotrophic pathogens, but the nature of the long-distance mobile signal for SAR depends on the plant-pathogen system. The clubroot disease is one of the most devastating diseases affecting all the members within the plant family Brassicaceae by causing serious losses of vegetable crops worldwide. In this study, we wanted to investigate whether methyl-salicylate (MeSA) is a mobile form of SA implicated in this plant-pathogen interaction. We have chosen Arabidopsis thaliana as a host organism for Plasmodiophora brassicae – the causal agent of clubroot, because the infection process is fast and the resistance of Arabidopsis to P. brassicae is conferred by a small number of genes. Using a GC-MS method, we monitored the transport and the metabolism of exogenously applied deuterated salicylic acid and its derivative, deuterated methyl-salicylate, through the whole plant of healthy and P. brassicae-infected Arabidopsis. The results showed that MeSA is a mobile form of SA in Arabidopsis clubroots.